Science, Faculty of
Microbiology and Immunology, Department of
DSpace
UBCV
Jackson, Jennie Lynne
Master of Science - MSc
University of British Columbia
Translation is an important step in the flow of genetic information from DNA, to RNA, to protein. Yet, translational fidelity is not always maintained. One example of a recoding event is programmed ribosomal frameshifts (PRF). PRF is mainly utilized by viruses, yeast, or bacteria, where the outcome of a shifted reading frame may result in carboxy-terminally extended fusion proteins or mRNA degradation. In this way, PRF is a form of translational control. The regulation of cytokine receptors, including at the translational level, is valuable to understand due to their importance in immune cell signaling. Interleukin-7 receptor α (IL-7Rα) in particular, which has major roles in proper immune system development and function, is known to be tightly regulated. IL-7Rα has well-documented examples of transcriptional and post-translational regulation, but post-transcriptional and translational regulation remain areas requiring further study. Indeed, IL-7Rα, as well as IL-2Rγ – the other subunit of the heterodimeric IL-7 receptor – were computationally predicted to contain -1 PRF motifs in their mRNA. Initial in vitro testing with reporter constructs expressed in HeLa cells was promising, but HeLa cells are not the best model for intrinsic IL-7Rα signaling pathways. Therefore, I sought to test the putative IL-7Rα PRF motif in a physiologically relevant context. I hypothesized that translational regulation of the cytokine receptor IL-7Rα via -1 PRF modulated its expression in human T cells. Using flow cytometry, I developed a single cell level assay so that -1 PRF could be compared between different T cell subsets, such as naïve, effector, or memory, within the same sample. Through various techniques, I also characterized impaired and/or abnormal Renilla luciferase expression by our IL-7Rα PRF dual luciferase reporter plasmid. Overall, despite being unable to resolve whether IL-7Rα expression is regulated by -1 PRF in human T cells, the flow cytometry PRF assay remains an adaptable tool to study PRF at the single cell level.
ASSESSMENT OF POTENTIAL -1 PROGRAMMED RIBOSOMAL FRAMESHIFTING BY THE HUMAN CYTOKINE RECEPTOR IL-7Rα by Jennie Lynne Jackson B.Sc., The University of British Columbia, 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Microbiology and Immunology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2025 © Jennie Lynne Jackson, 2025 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Assessment of potential -1 programmed ribosomal frameshifting by the human cytokine receptor IL-7Rα submitted by Jennie Lynne Jackson in partial fulfilment of the requirements for the degree of Master of Science in Microbiology and Immunology Examining Committee: Dr. Ninan Abraham, Professor, Department of Microbiology and Immunology, Department of Zoology, UBC Supervisor Dr. Eric Jan, Professor, Department of Biochemistry and Molecular Biology, UBC Supervisory Committee Member Dr. Maria Tokuyama, Assistant Professor, Department of Microbiology and Immunology, UBC Additional Examiner Additional Supervisory Committee Members: Dr. François Jean, Professor, Department of Microbiology and Immunology, UBC Supervisory Committee Member iii Abstract Translation is an important step in the flow of genetic information from DNA, to RNA, to protein. Yet, translational fidelity is not always maintained. One example of a recoding event is programmed ribosomal frameshifts (PRF). PRF is mainly utilized by viruses, yeast, or bacteria, where the outcome of a shifted reading frame may result in carboxy-terminally extended fusion proteins or mRNA degradation. In this way, PRF is a form of translational control. The regulation of cytokine receptors, including at the translational level, is valuable to understand due to their importance in immune cell signaling. Interleukin-7 receptor α (IL-7Rα) in particular, which has major roles in proper immune system development and function, is known to be tightly regulated. IL-7Rα has well-documented examples of transcriptional and post-translational regulation, but post-transcriptional and translational regulation remain areas requiring further study. Indeed, IL-7Rα, as well as IL-2Rγ – the other subunit of the heterodimeric IL-7 receptor – were computationally predicted to contain -1 PRF motifs in their mRNA. Initial in vitro testing with reporter constructs expressed in HeLa cells was promising, but HeLa cells are not the best model for intrinsic IL-7Rα signaling pathways. Therefore, I sought to test the putative IL-7Rα PRF motif in a physiologically relevant context. I hypothesized that translational regulation of the cytokine receptor IL-7Rα via -1 PRF modulated its expression in human T cells. Using flow cytometry, I developed a single cell level assay so that -1 PRF could be compared between different T cell subsets, such as naïve, effector, or memory, within the same sample. Through various techniques, I also characterized impaired and/or abnormal Renilla luciferase expression by our IL-7Rα PRF dual luciferase reporter plasmid. Overall, despite being unable to resolve iv whether IL-7Rα expression is regulated by -1 PRF in human T cells, the flow cytometry PRF assay remains an adaptable tool to study PRF at the single cell level. v Lay Summary Immune cells, like T cells, can communicate with other cells by sending and receiving signaling molecules called cytokines. When a cytokine binds to its receptor on the cell’s surface, it sets off a molecular chain reaction that allows the cell to respond to its environment. Therefore, altering receptor expression on cells could affect their ability to receive certain signals. I tried to determine if programmed ribosomal frameshifting (PRF), a type of control of molecular synthesis in cells, affected the amount of interleukin-7 receptor α (IL-7Rα) expressed on T cells. IL-7Rα is important for proper immune cell development, but different types of T cells have differing amounts of IL-7Rα on their surface. I developed a tool that can measure PRF between T cell subpopulations. Problems I found with the IL-7Rα PRF reporter prevented a definitive answer to my question, but the tool I developed can be optimized for future experiments. vi Preface The research presented in this thesis was designed collaboratively by me together with Dr. Ninan Abraham. All experiments were performed (excluding Figure 3.14 and Appendix C) and analyzed by me, with general guidance/teaching from Dr. Jung Hee Seo and Dr. Etienne Melese. The experiments for Figure 3.14 and Appendix C were executed alongside Yihang Chen, from UBC’s Jan lab. In chapter 3.7, DNA sequencing was done by the UBC Sequencing and Bioinformatics Consortium. All experiments involving human blood collection were conducted in accordance with UBC Research Ethics Board guidelines (protocol H17-02607). The thesis was written by me, with editorial help from Dr. Ninan Abraham. Figure 1.1 and Table 1.1 are used under fair dealings in the Copyright Act. There was no use of Generative AI tools in this thesis. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Abbreviations ................................................................................................................. xiv Acknowledgements .................................................................................................................. xviii Dedication ................................................................................................................................... xix Chapter 1: Introduction ................................................................................................................1 1.1 Protein synthesis and its regulation................................................................................. 1 1.1.1 Fundamentals of translation ........................................................................................ 1 1.1.2 Control of translation .................................................................................................. 3 1.1.3 Programmed ribosomal frameshifts are a type of recoding event .............................. 4 1.1.4 Elongation fidelity and the -1 PRF motif .................................................................... 6 1.1.5 -1 PRF is a form of translational control .................................................................... 7 1.2 Interleukin-7 and its receptor .......................................................................................... 7 1.2.1 Role in immune system ............................................................................................... 7 1.2.2 Translational regulation of cytokines and their receptors ........................................... 9 1.2.3 IL-7Rα expression ....................................................................................................... 9 1.3 -1 PRF and IL-7R.......................................................................................................... 11 viii 1.4 Hypothesis and research aims ....................................................................................... 13 Chapter 2: Materials and Methods ............................................................................................14 2.1 Tissue culture and cell isolation .................................................................................... 14 2.1.1 HeLa .......................................................................................................................... 14 2.1.2 293T .......................................................................................................................... 14 2.1.3 Primary CD8+ T cells ................................................................................................ 14 2.1.3.1 Human blood donation criteria ......................................................................... 14 2.1.3.2 Primary CD8+ T cell isolation ........................................................................... 15 2.1.3.3 Primary CD8+ T cell culture ............................................................................. 15 2.2 Plasmid preparation ...................................................................................................... 16 2.2.1 Plasmid stocks ........................................................................................................... 16 2.2.2 Subcloning the IL-7Rα PRF plasmid ........................................................................ 18 2.3 Plasmid transfection ...................................................................................................... 18 2.3.1 Nucleofection ............................................................................................................ 18 2.3.2 Transfection .............................................................................................................. 19 2.4 Analysis of ribosome frameshifts ................................................................................. 19 2.4.1 Protein collection, quantification, and analysis ........................................................ 19 2.4.1.1 Dual luciferase enzyme activity ........................................................................ 20 2.4.1.2 Western blot ...................................................................................................... 20 2.4.2 Flow cytometry analysis ........................................................................................... 21 2.4.3 RT-qPCR analysis ..................................................................................................... 21 2.4.4 In vitro synthesis ....................................................................................................... 24 Chapter 3: Results........................................................................................................................25 ix 3.1 Plasmid design .............................................................................................................. 25 3.2 Plasmid delivery via nucleofection ............................................................................... 27 3.3 Optimization of anti-Rluc and anti-Fluc flow cytometry antibodies ............................ 29 3.3.1 Testing different fixation and permeabilization methods ......................................... 29 3.3.2 Testing flow cytometry antibodies together ............................................................. 32 3.3.3 Anti-Fluc titrations .................................................................................................... 34 3.3.4 Anti-Rluc titrations ................................................................................................... 36 3.4 Detecting control plasmids using flow cytometry ........................................................ 38 3.5 Flow cytometry detection of PRF ................................................................................. 40 3.6 Examining potential cell line differences in PRF ......................................................... 42 3.7 Investigating potential plasmid mutations .................................................................... 44 3.8 Rluc transcript levels of reporter plasmids ................................................................... 46 3.9 Evaluating Rluc protein expression .............................................................................. 48 3.10 24 hours versus 48 hours post-transfection timepoint comparison ............................... 52 3.11 In vitro transcription and translation of reporter plasmids ............................................ 55 Chapter 4: Conclusion .................................................................................................................58 4.1 Summary of results ....................................................................................................... 58 4.2 Overall discussion ......................................................................................................... 59 4.3 Clinical significance...................................................................................................... 65 4.4 Future directions ........................................................................................................... 65 References .....................................................................................................................................67 Appendices ....................................................................................................................................72 Appendix A DNA sequences of the dual luciferase reporter plasmids .................................... 72 x A.1 In-frame control plasmid (175f) ................................................................................ 72 A.2 Out-of-frame control plasmid (175e) ........................................................................ 74 A.3 HIV control plasmid (175d) ...................................................................................... 76 A.4 IL-7Rα PRF plasmid (835) ....................................................................................... 78 A.5 IL-2Rγ PRF plasmid (1800)...................................................................................... 80 Appendix B Sequenced PRF region of IL-7Rα PRF plasmid .................................................. 82 Appendix C In vitro synthesis with FluoroTect ........................................................................ 83 xi List of Tables Table 1.1 Examples of -1 PRF, adapted from Caliskan et al. [7]. .................................................. 5 Table 2.1 Primers used for DNA sequencing, PCR, and RT-qPCR. ............................................ 17 Table 2.2 Example delta-delta Ct calculation using HIV control plasmid qPCR samples. .......... 23 xii List of Figures Figure 1.1 The basic steps of protein translation are initiation, elongation, and termination, adapted from Jia et al. [1]. .............................................................................................................. 2 Figure 1.2 The putative IL-7Rα -1 PRF motif is located in the intracellular region of the wild type gene. ...................................................................................................................................... 12 Figure 3.1 Reporter plasmids used to assay for -1 PRF contain Rluc and Fluc. .......................... 26 Figure 3.2 Dual luciferase enzyme activity readout of control plasmids following nucleofection........................................................................................................................................................ 28 Figure 3.3 Fixation and permeabilization optimization for anti-Rluc and anti-Fluc antibodies. .. 31 Figure 3.4 Antibodies tested singly and together against cells transfected with plasmids singly and together. .................................................................................................................................. 33 Figure 3.5 Anti-Fluc antibody titrations. ...................................................................................... 35 Figure 3.6 Anti-Rluc antibody titrations. ...................................................................................... 37 Figure 3.7 Assessing dual luciferase protein expression in 293T cells. ....................................... 39 Figure 3.8 Frameshift signals from reporter plasmids can be detected by flow cytometry. ......... 41 Figure 3.9 IL-7Rα PRF plasmids in HeLa cells also had Rluc expression issues. ....................... 43 Figure 3.10 Subcloning the IL-7Rα PRF plasmid did not improve Rluc expression. .................. 45 Figure 3.11 The IL-7Rα PRF motif did not destabilize Rluc mRNA transcripts. ........................ 47 Figure 3.12 Rluc expression by western blot revealed impaired and/or abnormal Rluc production by the IL-7Rα PRF plasmid. ......................................................................................................... 50 Figure 3.13 Abnormal and/or impaired Rluc expression by the IL-7Rα PRF plasmid also observed in 293T cells 24 hours post-transfection. ...................................................................... 53 xiii Figure 3.14 IL-7Rα PRF plasmid Rluc expression issues persist in a cell-free system. .............. 57 xiv List of Abbreviations 175d HIV PRF control plasmid 175e Out-of-frame control plasmid 175f In-frame control plasmid 1800 IL-2Rγ PRF plasmid 4E-BP 4E-binding protein 835/J835 IL-7Rα PRF plasmid/Remade IL-7Rα PRF plasmid ACK Ammonium-Chloride-Potassium AGO1 Argonaute RISC component 1 ARE AU-rich element ATCC American Type Culture Collection BCA Bicinchoninic acid CCR5 C-C motif chemokine receptor 5 CD Cluster of differentiation cDNA Complementary DNA CHILP Common helper innate lymphoid precursor DEPC Diethyl pyrocarbonate DMEM Dulbecco's Modified Eagle Medium DNA Deoxyribonucleic acid eIF2 Eukaryotic translation initiation factor 2 eIF2α Eukaryotic translation initiation factor 2α eIF3 Eukaryotic translation initiation factor 3 xv eIF4E Eukaryotic translation initiation factor 4E eIF4G Eukaryotic translation initiation factor 4G EJC Exon junction complex FACS Fluorescence-activated cell sorting FBS Fetal bovine serum FL/Fluc Firefly luciferase Foxo1 Forkhead box protein O1 Foxp3 Forkhead box protein P3 GABPα GA-binding protein α GCN2 General control nonderepressible 2 GFI1B Growth factor independent 1B GFP Green fluorescent protein HIV Human immunodeficiency virus HRI Heme-regulated inhibitor IFN-γ Interferon γ IFN-γRα Interferon γ receptor α IL Interleukin IL-2Rγ Interleukin-2 receptor γ IL-7Rα Interleukin-7 receptor α ILC/ILCP Innate lymphoid cell/Innate lymphoid cell precursor JAK-STAT Janus kinase-signal transducer and activator of transcription LncRNA Long non-coding RNA LPS Lipopolysaccharide xvi miRNA Micro RNA mRNA Messenger RNA NGD No-go decay NMD Nonsense-mediated mRNA decay PBMC Peripheral blood mononuclear cell PBS Phosphate buffered saline PERK PKR-like endoplasmic reticulum kinase PFA Paraformaldehyde PKR Protein kinase R PLB Passive lysis buffer PRF Programmed ribosomal frameshift RL/Rluc Renilla luciferase RLU Relative light units RNA Ribonucleic acid RPL10-R98S Ribosomal protein L10-R98S mutation RT-qPCR Reverse transcription-quantitative polymerase chain reaction SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 SATB1 Special AT-rich sequence binding protein 1 SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis siRNA Small interfering RNA SOCS Suppressor of cytokine signaling SV40 Simian virus 40 T-ALL T cell acute lymphoblastic leukemia xvii TCR T cell receptor Th T helper TNF-α Tumor necrosis factor α tRNA Transfer RNA TSLP Thymic stromal lymphopoietin TSLPR Thymic stromal lymphopoietin receptor UTR Untranslated region xviii Acknowledgements Thank you to my supervisor, Dr. Ninan Abraham, for your support and mentorship. Your passion for research is inspiring and I appreciate your advocacy for diversity, equity, and inclusion in science. You truly care about your students, and it shows. Thank you to my committee members, Dr. Eric Jan and Dr. François Jean, for your guidance and support. Thank you to the Abraham lab – Dr. Jung Hee Seo, Dr. Etienne Melese, Dr. Abdalla Sheikh, and Julia Lu – your training and feedback were invaluable. Plus, work is much more enjoyable when you have fun with your coworkers. Special shout-out to Dr. Jung Hee Seo for keeping the lab functional and running smoothly. Thank you to Dr. Samia Khan, for your weekly writing workshops. Thank you to my friends, including my DSS homies, my tennis buddies, and Parse in Shambles, for keeping me sane. Thank you to my family, for your unconditional love, support, and encouragement. Thank you to my partner Deniz and our kitty Amber, for always being there for me, through the highs and the lows. I’m excited for what the future holds for us. xix Dedication “Well, this is it. The end of the path, and the beginning of whatever awaits. There will be greater hardships. We may be made to feel powerless. Yet come what may, let us have no regrets. No, let us have pride in what we have achieved. In what we achieved, and how it changed us for the better…” - Alphinaud Leveilleur (Final Fantasy 14) 1 Chapter 1: Introduction 1.1 Protein synthesis and its regulation 1.1.1 Fundamentals of translation The flow of genetic information, from DNA to RNA to protein, is essential for an organism’s survival. Ribosomes play an important role in this system at the stage of translating mRNA into protein. To initiate cap-dependent translation in eukaryotes, initiation factor proteins interact with the 5’ cap, the 5’ untranslated region (UTR), and the poly-A tail of a mRNA transcript (Figure 1.1). Initiation factors also bind to the small ribosomal subunit which is complexed with an initiator methionine tRNA. The initiation factors then hold the mRNA and the small ribosomal subunit together, and this complex will scan the mRNA to find the AUG start codon. Once in place, the large ribosomal subunit is recruited, and initiation factors are released. The complete ribosome can now commence translation elongation (Figure 1.1). With the help of elongation factor proteins, the ribosome moves along the mRNA from 5’ to 3’, catalyzing peptide bonds between the amino acids carried by tRNAs corresponding to the mRNA codons. Elongation continues until the ribosome encounters a stop codon, whereby release factor proteins facilitate separating the ribosome from the mRNA, the disassembly of the ribosome, and the release of the completed polypeptide (Figure 1.1). 2 Figure 1.1 The basic steps of protein translation are initiation, elongation, and termination, adapted from Jia et al. [1]. Translation is an integral process in the flow of genetic information. Ribosomes, mRNA, and tRNA, along with initiation factor proteins, elongation factor proteins, and release factor proteins, are all involved in the synthesis of new polypeptides. 3 1.1.2 Control of translation Translational control is important for regulating gene expression in cells. Generally, methods of translational control can either globally affect translation occurring in the cell, or specifically affect one or more defined mRNAs without impacting translation of other mRNAs. Global regulation is mainly mediated by modification of the translation machinery, while mRNA-specific regulation can be due to trans-acting factors such as regulatory protein complexes or microRNAs (miRNAs) binding to specific elements in the mRNA [2]. Global regulation of translation often modulates the initiation phase of protein synthesis. For example, eIF2 is a translation initiation factor that forms a complex with initiator methionine tRNA, which then binds to the small ribosomal subunit [2]. Phosphorylation of the α subunit of eIF2 inhibits its function, thereby globally decreasing mRNA translation [2]. There are four kinases in humans – GCN2, PKR, HRI, and PERK – that can phosphorylate eIF2α which are activated by various conditions of cellular stress. GCN2 is activated by amino-acid starvation, PKR is stimulated by the presence of double-stranded RNA during viral infection, HRI is triggered by heme deficiency, and PERK is activated by the unfolded protein response in the endoplasmic reticulum [2]. Another translation initiation factor linked to global protein regulation is the cap binding protein eIF4E. eIF4E binds to the 5’ cap on mRNA and helps recruit the small ribosomal subunit to the mRNA through a complex of proteins including eIF4G and eIF3 [2]. eIF4E is present in cells in limiting quantities, and therefore can be considered a rate-limiting component of cap-dependent translation [3]. 4E-BPs are proteins that modulate eIF4E function. When unphosphorylated, 4E-BP binds to eIF4E, blocking eIF4E from interacting with eIF4G and thus preventing the small ribosomal subunit from associating with the mRNA [4]. Interestingly, overexpression of eIF4E in fibroblast cell lines led to their 4 tumourigenic transformation, demonstrating the proto-oncogenic potential of translation factors [3]. This highlights the important role that translational control plays to ensure proper cell growth. 1.1.3 Programmed ribosomal frameshifts are a type of recoding event Maintenance of the open reading frame by ribosomes ensures that the correct sequence of amino acids is added to polypeptides during protein synthesis. However, unconventional mechanisms exist that allow alternative reading frames to be translated, which leads to the production of different protein products from a single region of mRNA. Programmed ribosomal frameshift (PRF) is one of these mechanisms. If a translating ribosome slips backwards by one base on the mRNA, this is termed -1 PRF, while a move forwards by one base is called +1 PRF. -2 and +2 PRF have been observed as well, but there are fewer reports of them compared to -1 and +1 PRF. An example of this phenomenon occurs in human immunodeficiency virus (HIV), which relies on -1 PRF to produce the correct ratio of Gag to Gag-pol proteins necessary for viral replication and viability [5, 6]. Much of what we know about PRF has come from studies on viruses, yeast, and bacteria (Table 1.1). Studies on PRF of mammalian genes are much less prevalent compared to the literature available for microorganisms. 5 Gene (protein function) System Insertion sequences of IS1 IS3 family Bacteria dnaX (polymerase III subunits) Bacteria (Escherichia coli and relatives) cdd (cytidine deaminase) Bacteria ermCL Bacteria (Erm resistant strains) orf 1a/1b Coronavirus (IBV, MHV, SARS Co-V, HCV) gag/pol (polymerase) Lentivirus (VMV, FIV) gag/pol (polymerase) Lentivirus (HIV, SIV) orf2/3 (p2/p3) Polerovirus (BWYV, ScYLV, PEMV) orf1/2 Totivirus (ScV-L-A) orf 1a/1b Astrovirus (Hast-V1) orf 1a/1b Arterivirus (BEV, EAV) orf 1a/1b (replicase) Arterivirus (PRRSV) Peg10/Edr (embryonic regulator) Higher eukaryotes Ma3 (paraneoplastic antigen) Higher eukaryotes Table 1.1 Examples of -1 PRF, adapted from Caliskan et al. [7]. Genes with various functions undergo -1 PRF. While -1 PRF is more common in microorganisms such as bacteria and viruses, a few examples in higher eukaryotes have also been identified. 6 1.1.4 Elongation fidelity and the -1 PRF motif Translation elongation is a cycle where tRNA occupies in turn the A (aminoacyl), P (peptidyl), and E (exit) sites of a ribosome complexed with mRNA (Figure 1.1). At the end of the initiation phase, initiator methionine tRNA is in the P site [8]. During elongation, the A site receives the correct aminoacyl-tRNA for the mRNA codon at that position [9]. Next, the peptide bond is catalyzed between the amino acid on the aminoacyl-tRNA and the nascent peptide chain [9]. Finally, translocation brings the ribosome forward on the mRNA by one codon, or three bases, resulting in the deacetylated tRNA at the E site, the peptidyl-tRNA in the P site, and an empty A site [9]. The elongation cycle continues until the ribosome reaches a stop codon. The mechanisms of PRF are intrinsically linked to elongation fidelity. Most research on -1 PRF mainly focused on viruses. The viral -1 PRF motif on a mRNA is primarily composed of three parts: the heptameric slippery site, a short spacer typically less than 12 nucleotides long, and then a downstream stimulatory structure such as a mRNA pseudoknot [8]. The heptameric slippery site consists of the sequence N NNW WWH, where N is any three identical bases, W is any three identical weak bases (adenine or uracil), and H is anything but guanine [8]. During translation elongation, the ribosome moves along the mRNA. If the ribosome encounters a pseudoknot, it pauses at the slippery site. In brief, due to weak base pairing here between the mRNA codons and the tRNA anticodons, the ribosome can slip backwards on the mRNA, leading to a shift in the reading frame [8]. In the elongation cycle, ribosomal frameshifting can occur at three separate times: during translocation of the ribosome into or out of the slippery site, and when receiving an aminoacyl-tRNA into the A site while paused at the slippery site [10]. 7 1.1.5 -1 PRF is a form of translational control Programmed frameshift events have a number of documented outcomes. For example, they can produce carboxy-terminally extended fusion proteins like HIV Gag-pol [5, 6]. Conversely, they can also result in mRNA degradation through either the nonsense-mediated mRNA decay (NMD) pathway or the no-go decay (NGD) pathway [8]. The NMD pathway can be activated if the frameshift introduced a premature stop codon to the reading frame [11]. A multi-protein exon junction complex (EJC), normally displaced by translating ribosomes, remains on the mRNA instead [11]. The EJC then recruits NMD proteins that repress translation and initiate mRNA degradation [11]. The NGD pathway may be triggered when the mRNA secondary structure of the PRF motif causes the ribosome to be paused for too long at the slippery site [12]. Proteins recognize the stall occurring and direct the endonucleolytic cleavage of the mRNA close to the stall site [12]. This creates an opportunity for enzymes to come in and degrade the mRNA [12]. -1 PRF, therefore, is a method of translational control. Although about 10% of human genes are computationally predicted to contain -1 PRF signals, there is a distinct lack of functional studies [13, 14]. With potentially hundreds to thousands of human genes undergoing -1 PRF, its importance relative to other forms of translational control in homeostasis and disease progression is largely unknown. 1.2 Interleukin-7 and its receptor 1.2.1 Role in immune system The cytokine IL-7 and its receptor have major roles in proper immune system development, including T cell, B cell, and innate lymphoid cell (ILC) function. In the thymus, IL-7 is an important signal for thymocyte survival and maturation [15]. Once in the periphery, 8 naïve and memory T cells continue to rely on IL-7 as a pro-survival factor [16]. In the bone marrow, IL-7 is involved in determining B cell lineage commitment and the regulation of immunoglobulin gene rearrangement [15]. While mature B cells do not directly respond to IL-7 due to lack of IL-7R expression, IL-7 can still promote B cell survival indirectly through its effects on T cells [17]. ILCs are also dependent on IL-7R signaling during development, although its exact role at certain stages, such as the transition from common helper innate lymphoid precursors (CHILPs) to ILC precursors (ILCPs), requires further study [18]. IL-7 is thought to enhance the function of differentiated ILCs as well, like stimulating murine ILC2s to produce Th2 cytokines [18]. Due to the structure of the IL-7 receptor, it is involved in signaling pathways for more cytokines than just IL-7. IL-7R is composed of two subunits, IL-7Rα and IL-2Rγ, and both of these subunits also form alternate receptors when multimerized with other receptor subunits to transduce signals. For example, thymic stromal lymphopoietin (TSLP) is another cytokine that acts as an alarmin in response to injury or infection [19]. TSLP mediates type 2 inflammatory responses and can promote Th2 cell-mediated diseases by inducing the production of IL-4, IL-5, IL-9, and IL-13 [19]. The receptor for TSLP, comprised of the subunits IL-7Rα and TSLPR, can be found on dendritic cells, innate immune cells, T cells, epithelial cells, and neurons [19]. Additionally, IL-7 belongs to the gamma common chain family of cytokines which also includes IL-2, IL-4, IL-9, IL-15, and IL-21. All of the receptors for these cytokines have the subunit IL-2Rγ, meaning that IL-2Rγ is involved with multiple aspects of immune cell development and function [20]. Indeed, mutation of the gene for IL-2Rγ leads to severe combined immunodeficiency due to broad, defective cytokine signaling [20]. 9 1.2.2 Translational regulation of cytokines and their receptors Post-transcriptional control of cytokines and cytokine receptors remains an ongoing area of study. An advantage of translational regulation is that cells can rapidly respond to stimuli because the slower nuclear control mechanisms involving transcription, splicing, and RNA transport are avoided [21]. One method is that AU-rich elements (AREs) present in the 3’ UTR of certain cytokine mRNA, such as TNF-α, recruit proteins that influence mRNA stability, thereby affecting mRNA degradation and translation rates [22]. miRNA can also interact with cytokine and cytokine receptor mRNA to regulate expression. For example, miR-155 binds to the 3’ UTR of IFN-γRα mRNA in activated CD4+ T cells, inhibiting its expression, which helps promote Th1 differentiation [23]. Additionally, PKR activity can affect cytokine translation. IFN-γ mRNA contains a pseudoknot in its 5’ UTR that can activate PKR, which in turn phosphorylates eIF2α to inhibit translation [24]. 1.2.3 IL-7Rα expression IL-7Rα is known to be dynamically expressed throughout T cell development [25], so factors that affect its regulation are of interest to researchers. For example, IL-7Rα is expressed on naïve CD8+ T cells, but following T cell receptor activation during infection, IL-7Rα is downregulated on effector CD8+ T cells [26]. A small subset of effector CD8+ T cells do retain high expression of IL-7Rα, however, and these cells can become long-lived memory CD8+ T cells [26]. This characteristic of IL-7Rα expression, in combination with other markers, is used to help differentiate between CD8+ T cell subpopulations. Importantly, it signifies the need for tight regulation of this receptor subunit. 10 At present, most of the known methods by which IL-7Rα expression is regulated happen at the transcriptional level. There are multiple signals that can inhibit IL-7Rα transcription in T cells, such as IL-7 [27], IL-2 [28], and TCR signaling [29], while glucocorticoids can induce IL-7Rα transcription [30]. SATB1 [31] and GFI1B [32] are repressors of IL-7Rα expression that ensure genes are expressed at the correct time during T cell development. Conversely, the transcription factors PU.1 and GABPα can bind to the IL-7Rα promoter to activate transcription [33]. Foxo1 also increased IL-7Rα expression through direct binding to the IL-7Rα locus, albeit at an evolutionarily conserved noncoding region upstream of the transcription initiation site [34]. While many of the changes to IL-7Rα expression are due to transcriptional regulation, post-transcriptional regulation may also occur, although further study is required to determine the exact mechanisms. For instance, miR-17∼92, a cluster of 6 miRNAs, has been linked to IL-7Rα expression. Mice hematopoietic cells deficient in miR-17∼92 had negatively impacted T cell development, due to decreased IL-7Rα expression and reduced responsiveness to IL-7 [35]. In the course of that study, though, the authors were not able to identify a direct target of miR-17∼92 [35]. Additionally, the long noncoding RNA lnc-IL7R as well as IL-7Rα were both upregulated following LPS treatment of THP-1 cells, measured by real-time PCR [36]. Even though the gene for lnc-IL7R overlaps with the 3’ UTR of the IL-7Rα gene, lnc-IL7R was found to have a distinct role, different from IL-7Rα, as a suppressor of the LPS-induced inflammatory response [36]. However, because of the overlapping position of their genes, siRNAs targeting lnc-IL7R also knocked down IL-7Rα [36]. Post-translational regulation of IL-7Rα expression has been observed as well, when suppressor of cytokine signaling (SOCS) proteins induced by IL-7Rα signaling direct the receptor to the proteasome for degradation in a negative feedback loop [37]. 11 1.3 -1 PRF and IL-7R In 2014, a number of -1 PRF motifs in human cytokine receptors were identified by searching for putative -1 PRF motifs in silico based on a heptameric slippery site preceding a mRNA pseudoknot [13, 38]. Promising candidates were then tested in vitro using reporter constructs expressed in HeLa cells [38]. This study by the Dinman lab suggested that efficient -1 PRF occurred in mammalian cells [38]. Furthermore, knockdown of argonaute 1 (AGO1), a component of the RNA silencing complex, resulted in changes in the rate of -1 PRF in the reporter system, suggesting that miRNAs may regulate -1 PRF itself [38]. Two of the cytokine receptors showing -1 PRF activity in HeLa cells were IL-7Rα and IL-2Rγ [38], which together comprise the subunits of the heterodimeric IL-7 receptor. When aligned with the wildtype IL-7Rα gene, the putative IL-7Rα -1 PRF motif is found overlapping the junction between exon 7 and exon 8 within the intracellular domain (Figure 1.2). 12 The wild type IL-7Rα gene contains 8 exons that encode the extracellular, transmembrane, and intracellular domains. A) The wild type IL-7Rα gene product is a polypeptide 459 amino acids in length. B) The putative IL-7Rα -1 PRF motif overlaps the junction between exon 7 and exon 8. The predicted -1 frameshift product is a polypeptide 301 amino acids long, with a truncated intracellular domain compared to the wild type gene product. The exact amino acid sequence of the -1 frameshift product is dependent on where exactly the frameshift takes place over the slippery site. Figure 1.2 The putative IL-7Rα -1 PRF motif is located in the intracellular region of the wild type gene. 13 1.4 Hypothesis and research aims I hypothesized that translational regulation of the cytokine receptor IL-7Rα via -1 PRF modulated its expression in human T cells. Given the prior experimental evidence was derived from an epithelial cervical carcinoma cell line (HeLa cells), I sought to test the putative IL-7Rα -1 PRF in human T cells, which are a better model for intrinsic IL-7Rα signaling pathways. My main objective was to develop a single cell level assay to assess the role of -1 PRF in regulating IL-7Rα in primary CD8+ T cells. Our approach was to use plasmids containing our -1 PRF motif of interest flanked by Renilla (upstream) and firefly (downstream) luciferase reporter genes. Measurement of -1 PRF via firefly luciferase synthesis or activity at the single cell level would allow for the comparison of rates between different CD8+ T cell subpopulations, like naïve, effector, or memory, within the same sample. Establishment of such an assay would be used to compare the rate of -1 PRF in different primary CD8+ T cell subsets, enabling the examination of whether different conditions change the rate of -1 PRF and regulate the amount of IL-7Rα protein produced. Overall, definitive evidence of -1 PRF of IL-7Rα in a physiological context would represent an addition to our understanding of the translational control of cytokine receptors, and may help expand the developing field of -1 PRF in humans. 14 Chapter 2: Materials and Methods 2.1 Tissue culture and cell isolation 2.1.1 HeLa HeLa (CCL-2) human cervical carcinoma cells were obtained from the American Type Culture Collection (ATCC; Manassas, Virginia). Cells were passaged and expanded in DMEM (D6429, Sigma-Aldrich; St. Louis, Missouri) media supplemented with 10% fetal bovine serum (FBS), and maintained in a 5% CO2 incubator at 37° C. 2.1.2 293T 293T (CRL-3216) human embryonic kidney cells were obtained from the ATCC. Cells were passaged and expanded in DMEM media supplemented with 10% FBS and 2 mM L-glutamine (G7513, Sigma-Aldrich), and maintained in a 5% CO2 incubator at 37° C. 2.1.3 Primary CD8+ T cells 2.1.3.1 Human blood donation criteria All experiments involving human blood donation (protocol H17-02607) were conducted in accordance with UBC Research Ethics Board guidelines. Healthy adult volunteers were recruited by word of mouth or recruitment posters. To be eligible to participate, donors had no symptoms of blood-borne or inflammatory diseases, were not taking any medications, had no known blood abnormalities, and had not donated blood in the previous 3 weeks. Potential donors were excluded from participation if they had cancer, past or current intravenous drug abuse, a 15 history of hepatitis or jaundice, a history of anemia or abnormal blood clotting, or were taking anticoagulant or anti-inflammatory medication. 2.1.3.2 Primary CD8+ T cell isolation Peripheral blood mononuclear cells (PBMCs) were isolated from donated human blood by density centrifugation using Lymphoprep (07851) and SepMate tubes (85450) (Stemcell Technologies; Vancouver, British Columbia). PBMCs were then incubated with ACK lysis buffer (A1049201, Thermo Fisher; Waltham, Massachusetts). CD8+ T cells were separated out via a negative selection kit (17953, Stemcell Technologies) according to the manufacturer’s instructions. 2.1.3.3 Primary CD8+ T cell culture Primary CD8+ T cells isolated from human donor PBMCs were plated in 12-well plates at a density of 1 x 106 cells per mL of media and maintained in a 5% CO2 incubator at 37° C. The media used on the day of isolation (day 0) was RPMI (30-2001, ATCC) supplemented with 10% FBS, penicillin-streptomycin (15140122, Thermo Fisher), IL-2 (78036, Stemcell Technologies), and CD3/CD28 (10971, Stemcell Technologies). On days 3, 5, and 7 of the culture, cells were replated in additional RPMI media containing 10% FBS, penicillin-streptomycin, and IL-2 to keep the density at 1 x 106 cells per mL. 16 2.2 Plasmid preparation 2.2.1 Plasmid stocks In-frame control (pJD175f, abbreviated as 175f), out-of-frame control (pJD175e, abbreviated as 175e), HIV control (pJD175d, abbreviated as 175d), IL-7Rα PRF (pJD835, abbreviated as 835), and IL-2Rγ PRF (pJD1800, abbreviated as 1800) dual luciferases plasmids were received from the Dinman lab (University of Maryland, College Park). The HIV control plasmid contains the -1 frameshift signal for Gag/Gag-pol in its PRF region. The full sequence of the dual luciferase plasmids can be found in Appendix A. The firefly control plasmid (FL-SV40) (115352) was from Addgene (Watertown, Massachusetts). The Renilla control plasmid (RL-SV40) (E2231) was from Promega (Madison, Wisconsin). The transfection control plasmid pUC-19 (54357) was from Thermo Fisher. All plasmids were transformed into DH5α competent cells and stored as glycerol stocks at -80° C. As needed, the glycerol stocks were used to inoculate liquid LB cultures where the plasmid DNA was extracted by a Midiprep kit (K0481, Thermo Fisher). Plasmid sequences were confirmed by diagnostic restriction enzyme digestion and/or DNA sequencing (Table 2.1). A pMaxGFP plasmid (VPA1002, Lonza; Basel, Switzerland) was also used to measure transfection efficiency by flow cytometry. 17 Target Primer sequences (5’ to 3’) In-frame control plasmid (175f) FWD: GCCTCGTGAAATCCCGTTAG REV: GTAGATGAGATGTGACGAAC Out-of-frame control plasmid (175e) FWD: GCCTCGTGAAATCCCGTTAG REV: GTAGATGAGATGTGACGAAC HIV control plasmid (175d) FWD: CGTCCAACATTATCATGGCC REV: CCAACCGAACGGACATTTCG IL-7Rα PRF plasmid (835) or remade IL-7Rα PRF plasmid (J835) FWD: CGTCCAACATTATCATGGCC REV: CCAACCGAACGGACATTTCG IL-2Rγ PRF plasmid (1800) FWD: CGTCCAACATTATCATGGCC REV: CCAACCGAACGGACATTTCG Renilla luciferase on plasmids FWD: GCAGAAGTTGGTCGTGAGG REV: TCATCCGTTTCCTTTGTTCTG β-actin FWD: GACGACATGGAGAAAATCTG REV: ATGATCTGGGTCATCTTCTC Table 2.1 Primers used for DNA sequencing, PCR, and RT-qPCR. 18 2.2.2 Subcloning the IL-7Rα PRF plasmid In order to recreate the IL-7Rα PRF plasmid to test its putative programmed ribosome frameshift functionality, we introduced the -1 PRF motif from the IL-7Rα PRF plasmid (insert) into the out-of-frame control plasmid (vector). Both the IL-7Rα PRF and out-of-frame control plasmids were digested with BamHI and SalI restriction enzymes. The digested plasmids were run on a gel, then the bands corresponding to the insert and vector were excised and purified from the gel slices using a kit (K0691, Thermo Fisher). Vector and insert were then ligated with T4 DNA ligase (M0202S, New England Biolabs; Ipswich, Massachusetts). The newly created plasmid J835, a remake of the original IL-7Rα PRF plasmid, was transformed into DH5α competent cells by heat shock and then spread onto LB-agar ampicillin plates. Single colonies were tested by colony PCR, using primers flanking the insert region (Table 2.1), and gel electrophoresis for the presence of correctly inserted and ligated plasmid. A colony with the correct plasmid was preserved as glycerol stock. The plasmid sequence was further verified by diagnostic restriction enzyme digestion and DNA sequencing. 2.3 Plasmid transfection 2.3.1 Nucleofection HeLa and primary CD8+ T cells were nucleofected with plasmid DNA using the Nucleofector 2b Device from Lonza. HeLa cells were nucleofected with a kit (MIR50112, Mirus Bio; Madison, Wisconsin) according to the manufacturer’s protocol for HeLa cells. Primary CD8+ T cells were nucleofected with a kit (VPA1002, Lonza) following the manufacturer’s protocol for unstimulated human T cells. 3 x 106 HeLa cells per sample were nucleofected with 2 µg plasmid DNA or GFP control using the I-013 program, while 5 x 106 primary CD8+ T cells 19 per sample were nucleofected with 2.5 µg plasmid DNA or 2 µg GFP control using the V-024 program. In a modification to both protocols, immediately after nucleofection, pre-warmed media was added to the cuvettes, then samples were incubated at room temperature for 10 minutes. HeLa and primary CD8+ T cells were collected for downstream analysis 16 hours after nucleofection. 2.3.2 Transfection During the optimization of our single cell level PRF assay, HeLa and 293T cells were transfected with plasmid DNA using Lipofectamine 3000 (L3000001, Thermo Fisher) as per the manufacturer’s instructions. DMEM with no supplements was used for the steps requiring serum-free media. Cells were collected for downstream analysis 48 hours after transfection unless noted otherwise. 2.4 Analysis of ribosome frameshifts 2.4.1 Protein collection, quantification, and analysis After the post-transfection incubation, cells were collected, counted, and then lysed using Passive Lysis Buffer (PLB) (E1941, Promega). If the cells were to be used for western blotting, a protease inhibitor tablet (11836153001, Sigma-Aldrich) was added to the PLB. The protein concentration of samples was determined using a bicinchoninic acid (BCA) assay kit (23227, Thermo Fisher). 20 2.4.1.1 Dual luciferase enzyme activity Dual luciferase enzyme activity assays were performed using a kit (E1910, Promega). 4 µg to 50 µg of protein in a maximum volume of 20 µL was loaded per assay. The same amount of protein was used for all samples within an experiment. In a modification from Promega’s protocol, 50 µL LARII and 50 µL Stop & Glo were added at the appropriate time. The rate that frameshifting occurred was calculated using the method described by Jacobs and Dinman [39]. Firefly and Renilla luciferase values of all technical replicates were averaged. To control for background luminescence, all averaged firefly and Renilla luciferase values were subtracted by the corresponding averaged readout from a control sample such as cells transfected with pUC-19. The firefly luciferase value was then divided by the Renilla luciferase value. The firefly:Renilla luciferase ratio of each sample was further divided by the firefly:Renilla luciferase ratio of the in-frame control sample. This number inferred the proportion of translating ribosomes in each sample that frameshifted, relative to the in-frame control sample. 2.4.1.2 Western blot Protein samples used for western blotting were mixed with 2X SDS loading buffer containing β-mercaptoethanol and boiled for 5 minutes. SDS-PAGE was performed using an 8% tris-glycine resolving gel. Proteins in the gel were transferred onto a nitrocellulose membrane, and successful transfer was verified with Ponceau S staining. The blocking buffer was prepared by mixing Intercept Blocking Buffer (927-70001, Li-cor; Lincoln, Nebraska) at a 1:1 ratio with PBS. The primary anti-Renilla luciferase antibody (ab185926, Abcam; Cambridge, United Kingdom) was diluted 1:10,000 in blocking buffer plus 0.1% Tween 20. Washes were done with PBS with 0.1% Tween 20. The secondary goat anti-rabbit antibody (A-21109, Thermo Fisher) 21 was diluted 1:10,000 in blocking buffer plus 0.1% Tween 20. The final wash was in PBS without Tween 20 added. Membranes were imaged on the Odyssey Classic from Li-cor and analyzed using their Image Studio Lite software. 2.4.2 Flow cytometry analysis After the post-transfection incubation, 293T and HeLa cells were rendered into single cell suspensions, collected, and counted. 1 x 106 cells per sample were used for staining and flow cytometry. All staining was completed at 4° C in the dark. Fixable viability dye (either 65-0865 or 65-0866, Thermo Fisher) was used. An Fc receptor blocker (422302, BioLegend; San Diego, California) was also used if cells were to be fixed and permeabilized. Cells were prepared for intracellular staining by fixing with 4% paraformaldehyde (PFA), permeabilizing with 90% methanol, and extensive washes with FACS buffer. Anti-Renilla luciferase (ab225339, Abcam) and anti-firefly luciferase (ab237253, Abcam) antibodies were used at concentrations of 1:150 and 1:200, respectively. Single stain compensation controls for the anti-Renilla luciferase and anti-firefly luciferase antibodies were performed using beads (01-3333, Thermo Fisher). Data was acquired on the Attune NxT Flow Cytometer from Thermo Fisher, and analyzed with FlowJo software from FlowJo LLC (Ashland, Oregon). 2.4.3 RT-qPCR analysis After the post-transfection incubation, cells were collected and counted. 5 x 105 cells were set aside for gene expression analysis and RNA extraction was performed using the RNeasy Micro Kit (74004, Qiagen; Hilden, Germany). RNA was reverse transcribed into cDNA with All-In-One 5X RT MasterMix (G592, Applied Biological Materials; Richmond, British 22 Columbia). qPCR reactions with primers targeting the 5’ end of the Renilla luciferase sequence and β-actin (Table 2.1) were completed using PerfeCTa SYBR Green SuperMix, Low ROX (95056, Quantabio; Beverly, Massachusetts). Data was collected on the QuantStudio 3 machine from Thermo Fisher. The presence of qPCR products was further verified by gel electrophoresis. Renilla luciferase gene expression was calculated using the delta-delta Ct method [40], with β-actin as the housekeeping gene, and results displayed relative to the in-frame control plasmid. As an example, the delta-delta Ct calculation for cells transfected with HIV control plasmid is shown here (Table 2.2). qPCR reactions were performed in triplicate so the Ct values for each group of 3 samples and their target gene were averaged. Delta Ct was calculated by subtracting the average Ct value of the housekeeping gene β-actin from the average Ct value of our gene of interest Renilla luciferase; for the HIV control sample, 15.453 – 23.098 = -7.645. Delta-delta Ct was calculated by subtracting the delta Ct of our reference sample, the in-frame control, from the delta Ct of the sample we are analyzing; for the HIV control sample, -7.645 – -7.808 = 0.163. Fold gene expression was calculated by 2^-(delta-delta Ct); for the HIV control sample, 2^-(0.163) = 0.893. 23 Sample Name Target Name Ct Value Average Ct Value ΔCt ΔΔCt Fold Gene Expression In-frame Control β-actin 22.998 23.079 -7.808 0 1.000 In-frame Control β-actin 23.186 In-frame Control β-actin 23.054 In-frame Control Renilla 15.268 15.271 In-frame Control Renilla 15.286 In-frame Control Renilla 15.259 HIV Control β-actin 23.159 23.098 -7.645 0.163 0.893 HIV Control β-actin 22.985 HIV Control β-actin 23.150 HIV Control Renilla 15.412 15.453 HIV Control Renilla 15.429 HIV Control Renilla 15.517 Table 2.2 Example delta-delta Ct calculation using HIV control plasmid qPCR samples. 24 2.4.4 In vitro synthesis Control and PRF plasmids were linearized by digestion with the restriction endonuclease PmlI. Linearized plasmids were then cleaned using a kit (A2891, Promega) and, if needed, concentrated to at least 400 ng/µL. Linearization was confirmed by gel electrophoresis. In vitro synthesis reactions were performed with a kit (L1101, Promega) that coupled transcription and translation reactions within the same tube. This kit contained an extract derived from the Spodoptera frugiperda (Sf21) cell line that performed the in vitro synthesis reactions. In a modification from Promega’s protocol, each reaction tube contained: 1000 ng of the linearized and cleaned plasmid DNA or a no DNA negative control, 7 µL of TNT T7 ICE Master Mix, 0.25 µL of 40X F-buffer (0.5 mM MgCl2, 40 mM KOAc pH 7.5), and DEPC H2O to a total volume of 10 µL. When all components were added, tubes were gently mixed, spun down, and incubated at 30° C for 2 hours. Tubes were then transferred to ice. In preparation for dual luciferase enzyme activity analysis, 1 µL from each reaction tube was added to 99 µL of PLB. 10 µL of this lysate was mixed with 50 µL LARII and 50 µL Stop & Glo during dual luciferase analysis as described in section 2.4.1.1. Following the dual luciferase assay, protein concentrations were determined via BCA assay. These in vitro synthesized proteins were then analyzed by SDS-PAGE and western blot as described in section 2.4.1.2. 25 Chapter 3: Results 3.1 Plasmid design The plasmids used to assay for -1 PRF were designed by and sourced from the Dinman lab [38]. They assess -1 PRF by means of the dual luciferase enzyme activity assay. The dual luciferase protocol measures within the same sample the luminescent signal of two reporters separated sequentially by a putative PRF motif. The reporters are firefly luciferase (“Fluc”) from Photinus pyralis and Renilla luciferase (“Rluc”) from Renilla reniformis. On the plasmid, the coding sequence for Rluc is in the 0 frame, and downstream of Rluc is the coding sequence for Fluc which is in the -1 frame (Figure 3.1). The PRF motif of interest, such as for IL-7Rα, has been inserted in between Rluc and Fluc. Therefore, Rluc is always produced, but Fluc is only produced (as a Rluc-Fluc fusion protein) when the PRF motif induces a -1 frameshift. The relative level of Fluc compared to Rluc shows the rate of -1 PRF occurring in the sample. During experimental optimization, in-frame or out-of-frame control plasmids were used. The in-frame control has both Rluc and Fluc in the 0 frame, so both are always produced. The out-of-frame control always has Fluc in the -1 frame, so only Rluc should be made. A HIV control plasmid was also utilized that contains the -1 frameshift signal for Gag/Gag-pol in its PRF region, which should frameshift approximately 5% of the time. 26 Figure 3.1 Reporter plasmids used to assay for -1 PRF contain Rluc and Fluc. In addition to the in-frame, out-of-frame, and HIV control plasmids, plasmids with putative IL-7Rα or IL-2Rγ PRF motifs inserted in between Rluc and Fluc were also used in experiments. 27 3.2 Plasmid delivery via nucleofection In their study [38], the Dinman lab transfected the reporter plasmids into HeLa cells. However, we were interested in examining IL-7Rα signaling within a more physiologically relevant cell type, such as primary human T cells. Primary T cells are notoriously hard to transfect with lipid-based transfection reagents. Our first question was whether we could achieve high efficiency delivery of these plasmids using the nucleofection process. With 31.5% nucleofection efficiency as measured by GFP expression, nucleofection of HeLa cells with the in-frame and out-of-frame control plasmids showed expected results in the dual luciferase enzyme activity assay (Figure 3.2A). The in-frame control showed high activity of both luciferase enzymes while the out-of-frame control only expressed Rluc. Our next step was to confirm that we could observe dual luciferase enzyme activity readout from primary CD8+ T cells following nucleofection. We recruited healthy donors and collected whole blood from them at the UBC Centre for Blood Research facilities. In our lab, we processed the blood to isolate PBMCs, and then further purified for CD8+ T cells via magnetic bead sorting by negative selection. The CD8+ T cell cultures were grown for approximately 8 days to reach adequate cell numbers for experimentation. We attained 49.6% nucleofection efficiency assessed by GFP expression, and tested the in-frame and out-of-frame control plasmids (Figure 3.2B). The in-frame control showed similar levels of Fluc and Rluc, but the Rluc expressed by the out-of-frame control was much higher compared to the in-frame control. This may be because we did not have enough lysate to run both the BCA assay used to standardize protein amounts and the dual luciferase assay, which resulted in more variability. 28 Figure 3.2 Dual luciferase enzyme activity readout of control plasmids following nucleofection. Bars represent the average RLU (relative light units) values of 4 technical replicates (left hand histograms). Nucleofection efficiency was assessed via GFP plasmid delivery and expression (right hand plots). A) In HeLa cells. B) In primary CD8+ T cells. 29 3.3 Optimization of anti-Rluc and anti-Fluc flow cytometry antibodies CD8+ T cells are not a homogenous population, but rather can be divided into different subtypes, such as naïve, effector, or memory. Luciferase enzymatic assays are useful for bulk analysis of cells, but to examine potential -1 PRF differences between CD8+ T cell subpopulations, we required a technique that would let us study our samples at the single cell level, such as flow cytometry. Accordingly, to develop a single cell level PRF assay, we purchased anti-Rluc and anti-Fluc flow cytometry antibodies. Anti-Rluc recognizes amino acids 150 to 250 of Renilla luciferase while anti-Fluc recognizes firefly luciferase at the N terminus between amino acids 1 to 100. The plasmids used during testing include RL-SV40 and FL-SV40, which only express Rluc or Fluc constitutively, but are otherwise structurally very similar to the dual luciferase plasmids we received from the Dinman lab. We decided to use the 293T cell line for many optimization experiments because 293T cells are easier to culture and transfect than T cells, and SV40 large T antigen in 293T cells can interact with the SV40 origin of replication on our plasmids to enhance expression. 3.3.1 Testing different fixation and permeabilization methods 293T were transfected with FL-SV40 or RL-SV40 using Lipofectamine 3000. Three fixation/permeabilization methods were tested to determine which one allows the most antibodies into cells to bind to intracellular targets. We tested a Foxp3/transcription factor fixation/permeabilization kit, 1% paraformaldehyde (PFA) with 0.05% Tween-20, and 4% PFA with 90% methanol. Anti-Fluc was able to bind to the most targets when PFA/methanol was used, followed by PFA/Tween-20, and the Foxp3 protocol showing the least binding (Figure 3.3A). Likewise, Anti-Rluc was also able to bind the most targets after PFA/methanol fixation 30 and permeabilization, followed by the Foxp3 protocol, with PFA/Tween-20 as the least effective method (Figure 3.3B). Therefore, 4% PFA plus 90% methanol was used for all future experiments with these antibodies. Additionally, we did not see any off-target binding of anti-Rluc to Fluc (Figure 3.3A) or anti-Fluc to Rluc (Figure 3.3B). 31 Events shown are within the Live gate. Fluc and Rluc gates were set using the –DNA transfection control. A) 293T were transfected with firefly luciferase (FL-SV40) using Lipofectamine 3000. Transfection efficiency via GFP data was not available for this experiment. B) 293T were transfected with Renilla luciferase (RL-SV40) using Lipofectamine 3000. Transfection efficiency was 74.7% via GFP plasmid delivery and expression. Figure 3.3 Fixation and permeabilization optimization for anti-Rluc and anti-Fluc antibodies. 32 3.3.2 Testing flow cytometry antibodies together Next, we looked at both antibodies used together in a single sample, as well as FL-SV40 and RL-SV40 transfected together. It was important to make sure that the antibodies would not interact with each other, and were able to capture each Rluc and Fluc signal independently within one sample. Indeed, there does not seem to be any obvious antibody binding to antibody interactions, since samples with one antibody against a target had a similar percentage of positive events to samples with both antibodies used together against that target (Figure 3.4). When both antibodies were used together with both plasmids co-transfected within one sample, there was a population of double positive cells (Figure 3.4). 33 Events shown are within the Live gate. Fluc and Rluc gates were set using the –DNA transfection control. 293T were transfected using Lipofectamine 3000. Transfection efficiency was assessed via GFP plasmid delivery and expression. Figure 3.4 Antibodies tested singly and together against cells transfected with plasmids singly and together. 34 3.3.3 Anti-Fluc titrations To get the strongest signal from our antibodies while minimizing background noise, we titrated the flow cytometry antibodies. From previous titrations (data not shown), we decided to focus on testing anti-Fluc at 1:200 or 1:500. The 1:200 samples had more defined populations and less upward shifting than the 1:500 samples (Figure 3.5). Therefore, we decided to use anti-Fluc at 1:200 for all future experiments. 35 Figure 3.5 Anti-Fluc antibody titrations. Events shown are within the Live gate. Fluc and Rluc gates were set using the –DNA transfection control. 293T were transfected using Lipofectamine 3000. Transfection efficiency was assessed via GFP plasmid delivery and expression. 36 3.3.4 Anti-Rluc titrations Previous titrations including 1:500 indicated that 1:500 was too dilute and that higher concentrations had better staining results (data not shown). When comparing anti-Rluc at 1:300 and 1:150, more signal was captured when the antibody titre was higher (Figure 3.6). Therefore, anti-Rluc was used at 1:150 for future experiments. 37 Figure 3.6 Anti-Rluc antibody titrations. Events shown are within the Live gate. Fluc and Rluc gates were set using the –DNA transfection control. 293T were transfected using Lipofectamine 3000. Transfection efficiency was assessed via GFP plasmid delivery and expression. 38 3.4 Detecting control plasmids using flow cytometry After optimizing the fixation and permeabilization method and the titre of our antibodies, we needed to verify that we could detect signals from the dual luciferase plasmids that would be used to assay -1 PRF in CD8+ T cells. We began with the in-frame and out-of-frame control plasmids. The in-frame control, where both Rluc and Fluc are expressed together, displayed a double positive population (Figure 3.7). The out-of-frame control, which only expresses Rluc because Fluc is in a different reading frame, showed a Rluc positive population with no signal from Fluc (Figure 3.7). 39 Figure 3.7 Assessing dual luciferase protein expression in 293T cells. Events shown are within the Live gate. Fluc and Rluc gates were set using the –DNA transfection control. 293T were transfected using Lipofectamine 3000. Transfection efficiency was assessed via GFP plasmid delivery and expression. 40 3.5 Flow cytometry detection of PRF Next, we included plasmids with -1 PRF motifs in between Rluc and Fluc, such as an HIV PRF control which contains the Gag-pol frameshifting motif, as well as plasmids with the putative IL-7Rα or IL-2Rγ PRF motifs. The sample with the HIV control showed a population double positive for Rluc and Fluc, indicating that -1 PRF occurred and most notably, was detectable by flow cytometry (Figure 3.8A). In contrast, the IL-2Rγ PRF sample was only positive for Rluc, so PRF may not have occurred here (Figure 3.8A). The IL-7Rα PRF sample had essentially undetectable Rluc or Fluc (Figure 3.8A). The dual luciferase enzyme activity data obtained in parallel with this experiment followed similar patterns as the flow cytometry data, except that the IL-7Rα PRF sample did have a Rluc signal, albeit lower than all the other conditions (Figure 3.8B). Because of the Rluc expression discrepancy in the IL-7Rα PRF sample, the amount of plasmid transfected into each sample was double checked by running the plasmid DNA on a gel. All plasmids had bands of similar intensity, demonstrating that the transfections likely contained similar amounts of plasmid DNA (Figure 3.8C). 41 Figure 3.8 Frameshift signals from reporter plasmids can be detected by flow cytometry. A) Events shown are within the Live gate. Fluc and Rluc gates were set using the –DNA transfection control. 293T were transfected using Lipofectamine 3000. Transfection efficiency is assessed via GFP plasmid delivery and expression. B) Dual luciferase enzyme activity assay results from the same samples as part A. Bars represent the average RLU values of 4 technical replicates. C) 0.5 µg of each plasmid was ran on a gel. Expected band sizes: In-frame control = 5427 bp, Out-of-frame control = 5405 bp, HIV control = 5447 bp, IL-7Rα = 5531 bp, and IL-2Rγ = 5504 bp. 42 3.6 Examining potential cell line differences in PRF Since the results from the Dinman lab [38] were obtained in HeLa cells, it was possible that we did not see any IL-7Rα frameshifting in 293T due to cell line differences. Control plasmids and two replicates of IL-7Rα PRF plasmids were transfected into HeLa cells to test this. Flow cytometry results were inconclusive due to weak Rluc and Fluc signals, likely because of the lower transfection efficiency compared to past experiments (Figure 3.9A). However, dual luciferase enzymatic activity results did show the same pattern as in 293T (Figure 3.9B). The in-frame, out-of-frame, and HIV control plasmids expressed comparable amounts of Rluc. The in-frame control also produced a similar level of Fluc, while the out-of-frame control produced no Fluc, and the HIV control made a reduced amount of Fluc. Both IL-7Rα PRF plasmids expressed Rluc but not Fluc, and at a level lower than the control plasmids. 43 Figure 3.9 IL-7Rα PRF plasmids in HeLa cells also had Rluc expression issues. A) Events shown are within the Live gate. Fluc and Rluc gates were set using the –DNA transfection control. HeLa were transfected using Lipofectamine 3000. Transfection efficiency was assessed via GFP plasmid delivery and expression. B) Dual luciferase enzyme activity assay results from the same samples as part A. Bars represent the average RLU values of 4 technical replicates. 44 3.7 Investigating potential plasmid mutations We wondered if the IL-7Rα PRF plasmid had drifted since the frameshift signal previously detected by the Dinman lab and in past experiments from our lab failed to be observed in recent experiments. Perhaps the plasmid had acquired a mutation that interfered with its expression of Rluc and Fluc. The PRF region of the IL-7Rα PRF plasmid was sequenced to rule out a mutation here, and the results did not indicate that any base pair changes had occurred (Appendix B). Rather than trying to pinpoint the exact location of a possible mutation, we opted to recreate the IL-7Rα PRF plasmid by subcloning the PRF sequence in between Rluc and Fluc of the out-of-frame control plasmid. A dual luciferase enzyme activity experiment in HeLa cells using our original and remade IL-7Rα PRF plasmids showed a similar Rluc and Fluc readout between the two (Figure 3.10A). This suggests it is unlikely a discrepancy outside of the PRF region in our original IL-7Rα PRF plasmid accounts for the expression observed. Consistent with past results, we also observed that IL-7Rα PRF Rluc and Fluc expression was lower than control plasmids (Figure 3.10B). 45 Figure 3.10 Subcloning the IL-7Rα PRF plasmid did not improve Rluc expression. A) Dual luciferase enzyme activity assay results from HeLa transfected using Lipofectamine 3000. J835 denotes the remade IL-7Rα PRF plasmid. Bars represent the average RLU values of 4 technical replicates. B) Dual luciferase raw (i.e. not normalized to the no DNA control, or to the µg of lysate run) RLU values from part A. 46 3.8 Rluc transcript levels of reporter plasmids With an acquired mutation to the IL-7Rα PRF plasmid ruled out, we turned our attention towards its main unique feature: the PRF motif located in between Rluc and Fluc. An alternative hypothesis is that the IL-7Rα PRF sequence could be acting as a novel destabilizing element, leading to degradation of the mRNA and therefore less Rluc produced. To test this, we inspected Rluc transcript levels using RT-qPCR. 293T cells were transfected with the control and cytokine receptor PRF plasmids, and during cell collection, each sample was divided so that corresponding measurements from RT-qPCR, the dual luciferase enzyme activity assay, and western blot could be made. The Rluc primer for qPCR targets a 277 base region that begins upstream of Rluc and ends partway into the Rluc N-terminus. The ΔΔCt method was used to calculate Rluc fold gene expression relative to the in-frame control plasmid, with the housekeeping transcript β-actin as an internal standard for normalization. From the RT-qPCR results, we observed that the IL-7Rα and IL-2Rγ PRF plasmids expressed similar levels of Rluc mRNA as the HIV control plasmid (Figure 3.11). Furthermore, those three plasmids only have slightly lower Rluc expression than the in-frame control plasmid. These findings showed that there is no evidence of destabilization of mRNA by the IL-7Rα PRF motif. Therefore, the cause of reduced Rluc amounts does not appear to occur during transcription. 47 Figure 3.11 The IL-7Rα PRF motif did not destabilize Rluc mRNA transcripts. 293T cells were transfected with plasmids using Lipofectamine 3000. After 48h, cells were collected in separate aliquots for RNA and protein work. Following RNA extraction, RNA was reverse-transcribed into cDNA. cDNA was then used with Rluc and β-actin primers for qPCR. Technical repeats were performed by running qPCR reactions in triplicate. Rluc fold gene expression relative to the in-frame control plasmid was calculated with the ΔΔCt method. β-actin was used as the housekeeping gene. 48 3.9 Evaluating Rluc protein expression We next assessed both enzymatic and protein expression by luciferase enzyme activity assay and immunoblot analyses, respectively. The dual luciferase enzyme activity results indicated again that there was distinctly lower Rluc expression for the IL-7Rα PRF plasmid compared to the controls and the IL-2Rγ PRF plasmid (Figure 3.12A). Dual luciferase enzyme activity data, combined with western blot data, would tell us if the Rluc and Rluc-Fluc fusion proteins made by these plasmids are actually being produced as expected. Evaluating the nature and level of proteins present would indicate if there were issues with the dual luciferase readout instead. To perform the immunoblot analysis, protein lysates taken from the same transfections as in Figure 3.11 and Figure 3.12A were run on an 8% acrylamide gel. After transfer and Ponceau S visualization, the nitrocellulose membrane was stained with a primary monoclonal antibody (clone EPR17792) against Rluc, the same clone as the anti-Rluc flow cytometry antibody used in earlier experiments. The Rluc band size is expected to be approximately 36 kDa. With Fluc’s estimated size of 61 kDa, a Rluc-Fluc fusion protein produced from a frameshift would be roughly 97 kDa. Ponceau S staining showed even intensity across wells, indicating that equal amounts of total protein were loaded and transferred onto the membrane (Figure 3.12B). The in-frame control showed a distinct band co-migrating near the 80 kDa molecular weight marker which is likely the main Rluc-Fluc fusion band. While the in-frame control plasmid theoretically only produces Rluc-Fluc fusion proteins, we can see that there was a smaller amount of protein appearing near the 30 kDa ladder mark, potentially representing Rluc. Other bands can be seen between 30 and 80 kDa as well. In contrast, the out-of-frame control only has a main band consistent with Rluc and no fusion proteins. The HIV control corresponds 49 with a frameshift signal, with mainly Rluc made with a smaller amount of Rluc-Fluc. The results for IL-2Rγ echo the dual luciferase enzyme activity findings, with Rluc levels similar to controls, but an Rluc-Fluc amount comparable to the out-of-frame control, i.e. indistinguishable from background levels. IL-7Rα, interestingly, showed a fainter band at 30 kDa and a more prominent band slightly above 30 kDa that no other samples exhibit. This band is quantitatively at a lower level and qualitatively different in predicted size. 50 Figure 3.12 Rluc expression by western blot revealed impaired and/or abnormal Rluc production by the IL-7Rα PRF plasmid. 293T cells were transfected with plasmids using Lipofectamine 3000. After 48h, cells were collected in separate aliquots for RNA and protein work. Following protein extraction, protein was quantified by BCA and used in A) the dual luciferase enzyme activity assay or B) western 51 blot. A) Bars represent the average RLU values of 4 technical replicates. B) 30 µg of protein lysates were run on an 8% acrylamide gel. After transfer and Ponceau S visualization, the nitrocellulose membrane was blocked and stained with a rabbit anti-Rluc primary antibody. After incubation with a goat anti-rabbit secondary, the membrane was imaged on a Licor Odyssey Classic. The Rluc band size is expected to be approximately 36 kDa. With Fluc’s estimated size of 61 kDa, a Rluc-Fluc fusion protein produced from a frameshift would be roughly 97 kDa. 52 3.10 24 hours versus 48 hours post-transfection timepoint comparison To verify that our observations of low Rluc expression by the IL-7Rα PRF reporter plasmid weren’t caused by the 48 hours post-transfection timepoint we were collecting cells at, we looked at the earlier timepoint of 24 hours. 293T cells were transfected with the control and cytokine receptor PRF plasmids and collected at 24 hours post-transfection for dual luciferase enzyme activity assay and immunoblot as previously performed. The dual luciferase enzyme activity results still showed lower Rluc expression in the IL-7Rα PRF sample 24 hours post-transfection, compared to the in-frame, out-of-frame, and HIV controls, and the IL-2Rγ PRF and RL-SV40 samples (Figure 3.13A). The immunoblot analysis also included the 48 hours timepoint in-frame and out-of-frame control samples from Figure 3.12B run together with the 24 hours timepoint samples (Figure 3.13B). The in-frame controls and the HIV control contain a band slightly larger than 100 kDa, consistent with the predicted Rluc-Fluc fusion band. The out-of-frame controls, the HIV control, and IL-2Rγ all have a band around 36 kDa matching where we expect Rluc to migrate on the gel. Once again, the main IL-7Rα band was a little above where we predict Rluc to be. From both the dual luciferase enzyme activity and immunoblot data, the HIV control data indicated frameshifting, but there does not appear to be frameshifting in the IL-2Rγ and IL-7Rα PRF samples. Therefore, our observations of abnormal Rluc expression by the IL-7Rα PRF reporter plasmid are not exclusive to the 48 hours post-transfection timepoint because the issue was also documented at 24 hours post-transfection. . 53 Figure 3.13 Abnormal and/or impaired Rluc expression by the IL-7Rα PRF plasmid also observed in 293T cells 24 hours post-transfection. 293T cells were transfected with plasmids using Lipofectamine 3000. After 24h, cells were collected. Following protein extraction, protein was quantified by BCA and used in A) the dual 54 luciferase enzyme activity assay or B) western blot. A) Bars represent the average RLU values of 4 technical replicates. B) 4 µg of 24 hours timepoint lysates and 0.6 µg of 48 hours timepoint lysates (from Figure 3.12B) were run on an 8% acrylamide gel. After transfer and Ponceau S visualization, the nitrocellulose membrane was blocked and stained with a rabbit anti-Rluc primary antibody. After incubation with a goat anti-rabbit secondary, the membrane was imaged on a Licor Odyssey Classic. The Rluc band size is expected to be approximately 36 kDa. With Fluc’s estimated size of 61 kDa, a Rluc-Fluc fusion protein produced from a frameshift would be roughly 97 kDa. 55 3.11 In vitro transcription and translation of reporter plasmids Additionally, we wanted to investigate in vitro synthesis of the reporter plasmids to see if protein production in a cell-free system would have less variation than transfection into cells. In vitro synthesis reactions were performed using extracts from the Sf21 cell line which couple transcription and translation within the same tube. The in vitro synthesized proteins were analyzed by dual luciferase enzyme activity assay and immunoblot. From the dual luciferase enzyme activity results, we observed that Rluc expression from the IL-7Rα PRF plasmid continued to be lower than the IL-2Rγ PRF sample and the in-frame, out-of-frame, and HIV controls (Figure 3.14A). The immunoblot analysis also included the 293T transfection in-frame and out-of-frame control samples from Figure 3.12B run together with the in vitro synthesis samples (Figure 3.14B). The in-frame controls contain a band slightly above the 100 kDa ladder mark, predicted to be the main Rluc-Fluc fusion band. The out-of-frame controls have a band just above the 35 kDa ladder mark, which lines up with Rluc’s expected size. Both the HIV control and IL-2Rγ have bands indicating Rluc expression but little or no Rluc-Fluc expression, consistent with the dual luciferase enzyme activity findings. Echoing the observations from Figure 3.12B, the IL-7Rα PRF sample displayed a prominent band a little larger than the expected size for Rluc, that seems to be unique to the IL-7Rα PRF sample (Figure 3.14C). Overall, switching to a cell-free system did not resolve the Rluc expression issues we originally flagged in transfected cells. 56 57 Figure 3.14 IL-7Rα PRF plasmid Rluc expression issues persist in a cell-free system. Plasmids were linearized and cleaned, and then used in in vitro synthesis reactions catalyzed by extracts from the Sf21 cell line. The in vitro translation products were analyzed by A) the dual luciferase enzyme activity assay, and following protein quantification by BCA, B) western blot. A) Bars represent the average RLU values of 4 technical replicates. B) 6 µg of in vitro synthesized lysates and 3 µg of transfected 293T lysates (from Figure 3.12B) were run on an 8% acrylamide gel. After transfer and Ponceau S visualization, the nitrocellulose membrane was blocked and stained with a rabbit anti-Rluc primary antibody. After incubation with a goat anti-rabbit secondary, the membrane was imaged on a Licor Odyssey Classic. The Rluc band size is expected to be approximately 36 kDa. With Fluc’s estimated size of 61 kDa, a Rluc-Fluc fusion protein produced from a frameshift would be roughly 97 kDa. C) Band intensity quantification. 58 Chapter 4: Conclusion 4.1 Summary of results The goal of this thesis was to investigate if translational regulation of cytokine receptors via -1 PRF modulates their expression. Previous studies examined a putative IL-7Rα -1 PRF motif in HeLa cells using the dual luciferase enzyme activity bulk assay. My objective was to develop a single cell level assay to assess the role of -1 PRF in regulating IL-7Rα in primary CD8+ T cells. Not only are CD8+ T cells a physiologically relevant model unlike the cervical carcinoma HeLa cells, but a single cell level assay would allow for comparison of -1 PRF between different CD8+ T cell subpopulations. To that end, I successfully established a single cell level assay to detect -1 PRF using flow cytometry. This assay used plasmids originally designed for dual luciferase enzyme activity, in combination with detection of the reporters using anti-Renilla and anti-firefly antibodies. -1 PRF was validated in the HIV control plasmids by discernment of frameshift products at the single cell level. However, when I tested the putative IL-7Rα PRF plasmid, I observed consistently lower Renilla expression. On the reporter plasmids, Renilla is always in frame and is upstream of any PRF motifs, so theoretically all the plasmids should express similar amounts of it. The IL-7Rα PRF plasmid, alongside our other PRF plasmids, was studied at the stages of transcription and translation using various assays to assess possible causes of Renilla expression variation. I demonstrated that the IL-7Rα PRF plasmid had impaired and/or abnormal Renilla protein production, which impeded the original intention of using this plasmid in human primary CD8+ T cells. While the findings from the Dinman lab [38] about potential unprecedented -1 PRF in higher eukaryotes were initially exciting and led to this thesis project, 59 my findings do not permit a definitive determination of whether IL-7Rα -1 PRF occurs in human CD8+ T cells or not. 4.2 Overall discussion Since most validated examples of PRF occur in viruses, yeast, or bacteria, the possibility of cytokine receptor -1 PRF taking place in higher eukaryote mammalian cells proposed by the Dinman lab [38] was intriguing to us. Their research had the potential to expand knowledge of the role -1 PRF may play in human homeostasis or disease progression, as well as the types of translational regulation that could affect cytokine receptors. IL-7Rα protein expression specifically is tightly regulated in naïve, effector, and memory CD8+ T cells, so an additional layer of regulation could be important to understand. With the goal of assessing -1 PRF of IL-7Rα in primary CD8+ T cells, we received dual luciferase enzyme activity assay plasmids from the Dinman lab. These in-frame control, out-of-frame control, HIV control, IL-7Rα PRF, and IL-2Rγ PRF plasmids were the same ones used in their 2014 paper [38] (Figure 3.1). Even though the Dinman lab used HeLa cells for their -1 PRF reporter assays to suggest evidence of -1 PRF in IL-7Rα translation, a more physiologically relevant cell type like T cells would a better model for intrinsic IL-7Rα signaling pathways. We confirmed that these plasmids could be delivered into HeLa and primary human CD8+ T cells using nucleofection (Figure 3.2). Furthermore, we wanted to overcome a particular limitation of the dual luciferase enzyme activity assay that these plasmids were designed for. CD8+ T cells can be divided into different subpopulations based on marker expression, such as naïve, effector, or memory. Luciferase enzymatic assays are a bulk analysis of cells, so data cannot be associated with cell subpopulations unless cells were sorted before the assay was performed. Our solution was to 60 develop a single cell level PRF assay utilizing flow cytometry. We used anti-Rluc and anti-Fluc antibodies, which in conjunction with a CD8+ T cell marker panel, would be able to provide information on the state of IL-7Rα PRF within each cell as well as its specific CD8+ T cell subtype. Another way a single cell level flow cytometry PRF assay could have been executed is by removing the Rluc and Fluc genes in our plasmids and replacing them with fluorescent proteins detectable by flow cytometry, such as GFP and mCherry. Potentially, our method with anti-Rluc and anti-Fluc antibodies provided signal amplification compared to detecting fluorescent proteins. When optimizing the flow cytometry PRF assay, we used 293T cells for ease of culturing and transfection, and for SV40-mediated enhanced expression. Of the fixation/permeabilization methods tested, 4% PFA plus 90% methanol was the most effective at allowing anti-Fluc and anti-Rluc antibodies into cells to bind to their targets (Figure 3.3). Next, I showed that the antibodies didn’t have off target effects, such as anti-Fluc and anti-Rluc binding together, and that both Fluc and Rluc signals could be measured from one sample (Figure 3.4). We then titrated the antibodies, settling on 1:200 for anti-Fluc (Figure 3.5) and 1:150 for anti-Rluc (Figure 3.6). It is conceivable that further titrations could determine an even better concentration. Subsequently, we verified that the flow cytometry PRF assay could detect signals from the in-frame control and out-of-frame control plasmids (Figure 3.7). This was followed up by testing all of our control and putative PRF plasmids. Frameshift signals from reporter plasmids were successfully detected by this flow cytometry PRF assay (Figure 3.8). However, the IL-7Rα PRF sample had essentially undetectable Rluc or Fluc by cytometry (Figure 3.8A), and dual luciferase enzyme activity data obtained in parallel showed the IL-7Rα PRF sample expressed no Fluc and lower Rluc than all other samples (Figure 3.8B). Based on the way the plasmids were 61 designed, we would expect variation in the Fluc readout, but Rluc is upstream of Fluc and as internal control, was predicted to be expressed similarly across all tested plasmids. This Rluc discrepancy was not due to transfecting uneven amounts of plasmid DNA into the 293T cells (Figure 3.8C). Neither was it specific to 293T cells because HeLa cells transfected with the IL-7Rα PRF plasmid also had lower Rluc compared to all other samples (Figure 3.9). Next we examined the possibility that the IL-7Rα PRF plasmid had acquired a mutation affecting Rluc expression. A number of different mutations may have that result, such as one in the PRF region, or one in a promoter region. The PRF region of the IL-7Rα PRF plasmid was sequenced but no mutation was found. To rule out the possibility of a mutation elsewhere, we recreated the IL-7Rα PRF plasmid by subcloning the IL-7Rα PRF sequence in between Rluc and Fluc of the out-of-frame control plasmid. We had no issues with signal strength from our out-of-frame control plasmid, and this is how the IL-7Rα PRF plasmid was originally generated by the Dinman lab. Despite this, Rluc expression from the original and remade IL-7Rα PRF plasmids were similar to each other, and lower than control plasmids, so it was unlikely a mutation caused the discrepancy (Figure 3.10). We then investigated if transcript stability was affecting Rluc expression at the mRNA level. The IL-7Rα PRF motif might be sensitive to miRNA stabilization or destabilization, and the miRNAs within our lab’s cell lines could differ from the Dinman lab’s cell lines. Alternatively, the IL-7Rα PRF insert sequence itself may have destabilizing properties such that the whole mRNA is affected. Using RT-qPCR, we observed similar levels of Rluc mRNA from the IL-7Rα PRF, IL-2Rγ PRF, and HIV control plasmids, which overall were only slightly lower than the in-frame control plasmid (Figure 3.11). Therefore, reduced Rluc expression from the IL-7Rα PRF plasmid does not seem linked to mRNA stability. 62 Finally, we explored whether post-transcriptional processes may be impacting Rluc expression by the IL-7Rα PRF plasmid. Rluc protein was analyzed via dual luciferase enzyme activity and immunoblot analyses, and the results from each assay are consistent with each other (Figure 3.12). On the western blot, the Rluc band seems to comigrate with the 30 kDa marker, while the Rluc-Fluc fusion protein band comigrates with the 80 kDa marker. Other bands seen between 30 and 80 kDa may arise if the translational machinery does not always make it all the way from the start of Rluc to the end of Fluc. The IL-7Rα PRF plasmid sample had both a fainter band at 30 kDa and a unique band slightly above 30 kDa, compared to the other samples and controls. This mirrors the observation of lower Rluc expression from the IL-7Rα PRF plasmid measured by dual luciferase enzyme activity. Our results suggest that the IL-7Rα PRF plasmid had impaired and/or abnormal Rluc production arising after transcription. One explanation for these findings might be that Rluc could be C-terminally extended in such a way that the dual luciferase enzymatic reactions were hindered, yet the longer Rluc band could still be seen on a western blot. Alternatively or concurrently, Rluc may also be targeted for degradation for some reason. It can be noted that the data from this experiment, acquired from cells 48 hours post-transfection, was quite comparable to dual luciferase enzyme activity and immunoblot data from cells collected 24 hours post-transfection (Figure 3.13). The anomalous Rluc expression by the IL-7Rα PRF plasmid was therefore not exclusive to just one timepoint. In order to rule out variation from cell transfection as the cause of Rluc expression disparity, we tested our control and reporter PRF plasmids using a cell-free in vitro synthesis reaction. In vitro synthesis allowed us to better control variables related to transcript input, including promoter efficiency and transcription efficiency, post-transcription, and translation efficiency. For example, the same amount of polymerases, nucleotides, tRNAs, ribosomes, and 63 translation initiation/elongation/termination factors can be added to each reaction. Cellular variables that affect expression are also eliminated, such as DNA uptake, miRNAs, or pathways for DNA, mRNA, or protein degradation. This means that in vitro synthesis is a useful system for studying proteins that are degraded quickly, or are toxic to cells. Our in vitro synthesis reactions used extracts from the Sf21 cell line and coupled transcription and translation within the same tube. Only the Sf21 extract system was tested, so it could be worth it to repeat the experiment in a different cell-free synthesis system, such as rabbit reticulocyte lysate. The same amount of plasmid DNA was added to each reaction and we assumed that transcription efficiency was equal between samples. The in vitro synthesized proteins were analyzed by dual luciferase enzyme activity assay and immunoblot. Both assays once again captured impaired and/or abnormal Rluc expression by the IL-7Rα PRF plasmid compared to control plasmids, eliminating cell transfection variation as the cause for poor expression of Rluc from the IL-7Rα PRF plasmid (Figure 3.14). The western blot results for the IL-7Rα PRF plasmid showed a band slightly larger in size than the predicted band for Rluc, which was unique to this sample, and corresponded with lower Rluc expression measured by dual luciferase enzyme activity. This corroborates similar bands seen from western blots with the IL-7Rα PRF plasmid when transfecting cells (Figure 3.12 and Figure 3.13), although the band appeared much more abundant when in vitro synthesized (Figure 3.14). Because the potentially C-terminally extended Rluc expressed by the IL-7Rα PRF plasmid was observed at higher quantities when produced by in vitro synthesis, it is possible that Rluc expressed by the IL-7Rα PRF plasmid when transfected into cells is impacted by miRNA interactions and/or protein degradation. An additional consideration for my project was the publication of an article [41] calling into question some of the findings that the Dinman lab published in 2014 [38]. Although the 64 focus of the critique argues that CCR5 mRNA actually does not frameshift as the Dinman group claimed, no mention was made of the data on cytokine receptor -1 PRF on which my project is based. The report disputed the Dinman lab’s ability to determine essential -1 PRF elements following their own observation that mutation of the CCR5 slippery site did not affect -1 PRF rates [41]. Some of the issues raised also involve how the dual luciferase plasmids were constructed. This has bearing on my project since the control and PRF dual luciferase plasmids I have been using are from the Dinman lab and are the same ones from their contested paper. The in-frame and out-of-frame control plasmids, for example, could have been designed with the IL-7Rα PRF motif present in between Rluc and Fluc while still having Fluc in-frame or out-of-frame to better match the experimental IL-7Rα PRF plasmid. The authors note that production of Rluc-Fluc fusion proteins is not ideal because there is the potential that fusion may impact individual reporter activity or stability [41]. To account for this possibility, we attempted one experiment directly measuring the expression of Rluc and Fluc, via labelled lysine tRNA incorporated into all proteins synthesized by in vitro synthesis, but the results were inconclusive (Appendix C). Instead, plasmids can be designed where the putative PRF motif is flanked by tandem StopGo sequences that express Rluc and Fluc as two separate proteins. It is unknown how relevant other questions raised about the CCR5 PRF plasmid, like cryptic splicing affecting the frameshift signal detected, relate to the cytokine receptor PRF plasmids [41]. Therefore, my findings need to be viewed within the context of the limitations of this particular plasmid reporter system amid concerns raised about its conception and design. 65 4.3 Clinical significance Any evidence of -1 PRF of IL-7Rα would represent a possible addition to our understanding of the translational control of cytokine receptors, and may help expand the developing field of -1 PRF in humans. Furthermore, if -1 PRF contributes to disease progression, it presents a new target for novel therapeutics. For example, merafloxacin was identified as a -1 PRF inhibitor for SARS-CoV-2 and other betacoronaviruses that prevented viral replication in vitro [42]. As well, mouse lymphoid cells with the ribosomal protein mutation RPL10-R98S had altered frameshifting in Jak-Stat genes, including IL-7Rα, which promoted JAK-STAT upregulation and T-cell acute lymphoblastic leukemia (T-ALL) pathogenesis [43]. 4.4 Future directions Even though we did not test the IL-7Rα PRF plasmid in human primary CD8+ T cells as originally planned because of Renilla expression issues, the single cell flow cytometry PRF assay we established can be adapted for further experimentation. The next apparent step would be to conduct the flow cytometry PRF assay in CD8+ T cells using a different IL-7Rα PRF plasmid that does not have Renilla expression issues. With a flow panel of markers for CD8+ T cell subsets of interest, such as naïve, effector, and memory, for example, it could finally be determined whether there are -1 PRF differences between CD8+ T cell subpopulations. After proper optimization, the flow cytometry PRF assay can conceivably be used with any putative PRF motif plasmids and cell types as long as the plasmids contain Rluc and Fluc in the correct configuration. Alongside human IL-7Rα and IL-2Rγ, the Dinman lab also identified a putative -1 PRF signal in mouse IL-7Rα [38], so promising results in human CD8+ T cells could warrant study of mouse IL-7Rα in murine immune cells. Since dual luciferase enzyme activity bulk 66 assays are a common way to test putative PRF motifs, many labs could use their existing plasmids with the single cell flow cytometry PRF assay to acquire data from a new angle. Another option for examining -1 PRF in CD8+ T cells could be to directly measure what is happening in the cells, which would avoid some of the variables that arise when using a proxy system like plasmids transfected into cells. For example, mass spectrometry could be conducted to try and capture the presence of the predicted wild type IL-7Rα -1 PRF product in CD8+ T cells. Furthermore, IL-7Rα expression could be tested after treating CD8+ T cells with an inhibitor or inducer of PRF, such as 5-fluorouracil [44]. Additionally, experiments could be performed to assess the role that miRNAs may have on IL-7Rα -1 PRF in CD8+ T cells if -1 PRF is confirmed to occur. In Chapter 1 it was noted that miRNA can interact with cytokine receptor mRNA, and the miRNA cluster miR-17∼92 has been linked to IL-7Rα expression. Evidence that miRNAs may affect -1 PRF was also presented by the Dinman lab’s paper [38] that initially led to us further pursuing the question of IL-7Rα -1 PRF. 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Genes 8: 295. 72 Appendices Appendix A DNA sequences of the dual luciferase reporter plasmids A.1 In-frame control plasmid (175f) agatcgatctgcgcagcaccatggcctgaaataacctctgaaagaggaacttggttaggtaccttctgaggcggaaagaa ccagctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatc tcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattag tcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatgg ctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggctttt ttggaggcctaggcttttgcaaaaagcttgattcttctgacacaacagtctcgaacttaagctgcagaagttggtcgtga ggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaag actcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccactcccagt tcaattacagctcttaaggctagagtacttaatacgactcactataggctagccaccatgacttcgaaagtttatgatcc agaacaaaggaaacggatgataactggtccgcagtggtgggccagatgtaaacaaatgaatgttcttgattcatttatta attattatgattcagaaaaacatgcagaaaatgctgttatttttttacatggtaacgcggcctcttcttatttatggcga catgttgtgccacatattgagccagtagcgcggtgtattataccagaccttattggtatgggcaaatcaggcaaatctgg taatggttcttataggttacttgatcattacaaatatcttactgcatggtttgaacttcttaatttaccaaagaagatca tttttgtcggccatgattggggtgcttgtttggcatttcattatagctatgagcatcaagataagatcaaagcaatagtt cacgctgaaagtgtagtagatgtgattgaatcatgggatgaatggcctgatattgaagaagatattgcgttgatcaaatc tgaagaaggagaaaaaatggttttggagaataacttcttcgtggaaaccatgttgccatcaaaaatcatgagaaagttag aaccagaagaatttgcagcatatcttgaaccattcaaagagaaAGGTGAAGTTCGTCGTCCaacattatcatggcctcgt gaaatcccgttagtaaaaggtggtaaacctgacgttgtacaaattgttaggaattataatgcttatctacgtgcaagtga tgatttaccaaaaatgtttattgaatcggacccaggattcttttccaatgctattgttgAAGGTGCCAAGAAGTTTCcta atactgaatttgtcaaagtaaaaggtcttcatttttcgcaagaagatgcacctgatgaaatgggaaaatatatcaaatcg ttcgttgagcgagttctcaaaaatgaacaaatgtcgacgggggcccctaggagatctagcgctggatcccccggggagct catggaagacgccaaaaacataaagaaaggcccggcgccattctatcctctagaggatggaaccgctggagagcaactgc ataaggctatgaagagatacgccctggttcctggaacaattgcttttacagatgcacatatcgaggtgaacatcacgtac gcggaatacttcgaaatgtccgttcggttggcagaagctatgaaacgatatgggctgaatacaaatcacagaatcgtcgt atgcagtgaaaactctcttcaattctttatgccggtgttgggcgcgttatttatcggagttgcagttgcgcccgcgaacg acatttataatgaacgtgaattgctcaacagtatgaacatttcgcagcctaccgtagtgtttgtttccaaaaaggggttg caaaaaattttgaacgtgcaaaaaaaattaccaataatccagaaaattattatcatggattctaaaacggattaccaggg atttcagtcgatgtacacgttcgtcacatctcatctacctcccggttttaatgaatacgattttgtaccagagtcctttg atcgtgacaaaacaattgcactgataatgaattcctctggatctactgggttacctaagggtgtggcccttccgcataga actgcctgcgtcagattctcgcatgccagagatcctatttttggcaatcaaatcattccggatactgcgattttaagtgt tgttccattccatcacggttttggaatgtttactacactcggatatttgatatgtggatttcgagtcgtcttaatgtata gatttgaagaagagctgtttttacgatcccttcaggattacaaaattcaaagtgcgttgctagtaccaaccctattttca ttcttcgccaaaagcactctgattgacaaatacgatttatctaatttacacgaaattgcttctgggggcgcacctctttc gaaagaagtcggggaagcggttgcaaaacgcttccatcttccagggatacgacaaggatatgggctcactgagactacat cagctattctgattacacccgagggggatgataaaccgggcgcggtcggtaaagttgttccattttttgaagcgaaggtt gtggatctggataccgggaaaacgctgggcgttaatcagagaggcgaattatgtgtcagaggacctatgattatgtccgg ttatgtaaacaatccggaagcgaccaacgccttgattgacaaggatggatggctacattctggagacatagcttactggg acgaagacgaacacttcttcatagttgaccgcttgaagtctttaattaaatacaaaggatatcaggtggcccccgctgaa ttggaatcgatattgttacaacaccccaacatcttcgacgcgggcgtggcaggtcttcccgacgatgacgccggtgaact tcccgccgccgttgttgttttggagcacggaaagacgatgacggaaaaagagatcgtggattacgtcgccagtcaagtaa caaccgcgaaaaagttgcgcggaggagttgtgtttgtggacgaagtaccgaaaggtcttaccggaaaactcgacgcaaga aaaatcagagagatcctcataaaggccaagaagggcggaaagtccaaattgtaacacgtgtaattctagagcggccgctt cgagcagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtga aatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcatttta tgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtaaaatcgataagttt aaacagatccaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatg tatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccg tgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatg ctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgcccc gaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaaga 73 gcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatg gcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatc ggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagct gaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactg gcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgc tcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcact ggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagac agatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgat ttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtga gttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatct gctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaag gtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactc tgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccg ggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttg gagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggc ggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatcttt atagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaa aacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatggctcgac Appendix A.1 Complete DNA sequence of the in-frame control plasmid (175f). Renilla luciferase is highlighted in blue, and firefly luciferase is highlighted in red. 74 A.2 Out-of-frame control plasmid (175e) agatcgatctgcgcagcaccatggcctgaaataacctctgaaagaggaacttggttaggtaccttctgaggcggaaagaa ccagctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatc tcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattag tcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatgg ctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggctttt ttggaggcctaggcttttgcaaaaagcttgattcttctgacacaacagtctcgaacttaagctgcagaagttggtcgtga ggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaag actcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccactcccagt tcaattacagctcttaaggctagagtacttaatacgactcactataggctagccaccatgacttcgaaagtttatgatcc agaacaaaggaaacggatgataactggtccgcagtggtgggccagatgtaaacaaatgaatgttcttgattcatttatta attattatgattcagaaaaacatgcagaaaatgctgttatttttttacatggtaacgcggcctcttcttatttatggcga catgttgtgccacatattgagccagtagcgcggtgtattataccagaccttattggtatgggcaaatcaggcaaatctgg taatggttcttataggttacttgatcattacaaatatcttactgcatggtttgaacttcttaatttaccaaagaagatca tttttgtcggccatgattggggtgcttgtttggcatttcattatagctatgagcatcaagataagatcaaagcaatagtt cacgctgaaagtgtagtagatgtgattgaatcatgggatgaatggcctgatattgaagaagatattgcgttgatcaaatc tgaagaaggagaaaaaatggttttggagaataacttcttcgtggaaaccatgttgccatcaaaaatcatgagaaagttag aaccagaagaatttgcagcatatcttgaaccattcaaagagaaaggtgaagttcgtcgtccaacattatcatggcctcgt gaaatcccgttagtaaaaggtggtaaacctgacgttgtacaaattgttaggaattataatgcttatctacgtgcaagtga tgatttaccaaaaatgtttattgaatcggacccaggattcttttccaatgctattgttgaaggtgccaagaagtttccta atactgaatttgtcaaagtaaaaggtcttcatttttcgcaagaagatgcacctgatgaaatgggaaaatatatcaaatcg ttcgttgagcgagttctcaaaaatgaacaaatgtcgacggatccttcaacttccctgagctcgaagacgccaaaaacata aagaaaggcccggcgccattctatcctctagaggatggaaccgctggagagcaactgcataaggctatgaagagatacgc cctggttcctggaacaattgcttttacagatgcacatatcgaggtgaacatcacgtacgcggaatacttcgaaatgtccg ttcggttggcagaagctatgaaacgatatgggctgaatacaaatcacagaatcgtcgtatgcagtgaaaactctcttcaa ttctttatgccggtgttgggcgcgttatttatcggagttgcagttgcgcccgcgaacgacatttataatgaacgtgaatt gctcaacagtatgaacatttcgcagcctaccgtagtgtttgtttccaaaaaggggttgcaaaaaattttgaacgtgcaaa aaaaattaccaataatccagaaaattattatcatggattctaaaacggattaccagggatttcagtcgatgtacacgttc gtcacatctcatctacctcccggttttaatgaatacgattttgtaccagagtcctttgatcgtgacaaaacaattgcact gataatgaattcctctggatctactgggttacctaagggtgtggcccttccgcatagaactgcctgcgtcagattctcgc atgccagagatcctatttttggcaatcaaatcattccggatactgcgattttaagtgttgttccattccatcacggtttt ggaatgtttactacactcggatatttgatatgtggatttcgagtcgtcttaatgtatagatttgaagaagagctgttttt acgatcccttcaggattacaaaattcaaagtgcgttgctagtaccaaccctattttcattcttcgccaaaagcactctga ttgacaaatacgatttatctaatttacacgaaattgcttctgggggcgcacctctttcgaaagaagtcggggaagcggtt gcaaaacgcttccatcttccagggatacgacaaggatatgggctcactgagactacatcagctattctgattacacccga gggggatgataaaccgggcgcggtcggtaaagttgttccattttttgaagcgaaggttgtggatctggataccgggaaaa cgctgggcgttaatcagagaggcgaattatgtgtcagaggacctatgattatgtccggttatgtaaacaatccggaagcg accaacgccttgattgacaaggatggatggctacattctggagacatagcttactgggacgaagacgaacacttcttcat agttgaccgcttgaagtctttaattaaatacaaaggatatcaggtggcccccgctgaattggaatcgatattgttacaac accccaacatcttcgacgcgggcgtggcaggtcttcccgacgatgacgccggtgaacttcccgccgccgttgttgttttg gagcacggaaagacgatgacggaaaaagagatcgtggattacgtcgccagtcaagtaacaaccgcgaaaaagttgcgcgg aggagttgtgtttgtggacgaagtaccgaaaggtcttaccggaaaactcgacgcaagaaaaatcagagagatcctcataa aggccaagaagggcggaaagtccaaattgtaacacgtgtaattctagagcggccgcttcgagcagacatgataagataca ttgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgcttta tttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggt gtgggaggttttttaaagcaagtaaaacctctacaaatgtggtaaaatcgataagtttaaacagatccaggtggcacttt tcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataac cctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattccctttttt gcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacg agtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatga gcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacac tattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatg cagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccg cttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgac gagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttc ccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggt ttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcc cgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctc actgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaattta aaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtca 75 gaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacc accgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgc agataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctc gctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagtt accggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaac tgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggc agggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgcca cctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttt tacggttcctggccttttgctggccttttgctcacatggctcgac Appendix A.2 Complete DNA sequence of the out-of-frame control plasmid (175e). Renilla luciferase is highlighted in blue, and firefly luciferase is highlighted in red. 76 A.3 HIV control plasmid (175d) agatcgatctgcgcagcaccatggcctgaaataacctctgaaagaggaacttggttaggtaccttctgaggcggaaagaa ccagctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatc tcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattag tcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatgg ctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggctttt ttggaggcctaggcttttgcaaaaagcttgattcttctgacacaacagtctcgaacttaagctgcagaagttggtcgtga ggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaag actcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccactcccagt tcaattacagctcttaaggctagagtacttaatacgactcactataggctagccaccatgacttcgaaagtttatgatcc agaacaaaggaaacggatgataactggtccgcagtggtgggccagatgtaaacaaatgaatgttcttgattcatttatta attattatgattcagaaaaacatgcagaaaatgctgttatttttttacatggtaacgcggcctcttcttatttatggcga catgttgtgccacatattgagccagtagcgcggtgtattataccagaccttattggtatgggcaaatcaggcaaatctgg taatggttcttataggttacttgatcattacaaatatcttactgcatggtttgaacttcttaatttaccaaagaagatca tttttgtcggccatgattggggtgcttgtttggcatttcattatagctatgagcatcaagataagatcaaagcaatagtt cacgctgaaagtgtagtagatgtgattgaatcatgggatgaatggcctgatattgaagaagatattgcgttgatcaaatc tgaagaaggagaaaaaatggttttggagaataacttcttcgtggaaaccatgttgccatcaaaaatcatgagaaagttag aaccagaagaatttgcagcatatcttgaaccattcaaagagaaaggtgaagttcgtcgtccaacattatcatggcctcgt gaaatcccgttagtaaaaggtggtaaacctgacgttgtacaaattgttaggaattataatgcttatctacgtgcaagtga tgatttaccaaaaatgtttattgaatcggacccaggattcttttccaatgctattgttgaaggtgccaagaagtttccta atactgaatttgtcaaagtaaaaggtcttcatttttcgcaagaagatgcacctgatgaaatgggaaaatatatcaaatcg ttcgttgagcgagttctcaaaaatgaacaaatgtcgacggatccttttttagggaagatctggccttcccacaaggggag gccagggaattttcttccgagctcgaagacgccaaaaacataaagaaaggcccggcgccattctatcctctagaggatgg aaccgctggagagcaactgcataaggctatgaagagatacgccctggttcctggaacaattgcttttacagatgcacata tcgaggtgaacatcacgtacgcggaatacttcgaaatgtccgttcggttggcagaagctatgaaacgatatgggctgaat acaaatcacagaatcgtcgtatgcagtgaaaactctcttcaattctttatgccggtgttgggcgcgttatttatcggagt tgcagttgcgcccgcgaacgacatttataatgaacgtgaattgctcaacagtatgaacatttcgcagcctaccgtagtgt ttgtttccaaaaaggggttgcaaaaaattttgaacgtgcaaaaaaaattaccaataatccagaaaattattatcatggat tctaaaacggattaccagggatttcagtcgatgtacacgttcgtcacatctcatctacctcccggttttaatgaatacga ttttgtaccagagtcctttgatcgtgacaaaacaattgcactgataatgaattcctctggatctactgggttacctaagg gtgtggcccttccgcatagaactgcctgcgtcagattctcgcatgccagagatcctatttttggcaatcaaatcattccg gatactgcgattttaagtgttgttccattccatcacggttttggaatgtttactacactcggatatttgatatgtggatt tcgagtcgtcttaatgtatagatttgaagaagagctgtttttacgatcccttcaggattacaaaattcaaagtgcgttgc tagtaccaaccctattttcattcttcgccaaaagcactctgattgacaaatacgatttatctaatttacacgaaattgct tctgggggcgcacctctttcgaaagaagtcggggaagcggttgcaaaacgcttccatcttccagggatacgacaaggata tgggctcactgagactacatcagctattctgattacacccgagggggatgataaaccgggcgcggtcggtaaagttgttc cattttttgaagcgaaggttgtggatctggataccgggaaaacgctgggcgttaatcagagaggcgaattatgtgtcaga ggacctatgattatgtccggttatgtaaacaatccggaagcgaccaacgccttgattgacaaggatggatggctacattc tggagacatagcttactgggacgaagacgaacacttcttcatagttgaccgcttgaagtctttaattaaatacaaaggat atcaggtggcccccgctgaattggaatcgatattgttacaacaccccaacatcttcgacgcgggcgtggcaggtcttccc gacgatgacgccggtgaacttcccgccgccgttgttgttttggagcacggaaagacgatgacggaaaaagagatcgtgga ttacgtcgccagtcaagtaacaaccgcgaaaaagttgcgcggaggagttgtgtttgtggacgaagtaccgaaaggtctta ccggaaaactcgacgcaagaaaaatcagagagatcctcataaaggccaagaagggcggaaagtccaaattgtaacacgtg taattctagagcggccgcttcgagcagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtga aaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaa caacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaat gtggtaaaatcgataagtttaaacagatccaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttt tctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagt atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaac gctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaaga tccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcc cgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcac agaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggcca acttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgcctt gatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaac gttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaag ttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtct cgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaac tatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttact catatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatg 77 accaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcc tttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagc taccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagtta ggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtgg cgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggtt cgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacg cttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagg gggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcag gggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatg gctcgac Appendix A.3 Complete DNA sequence of the HIV control plasmid (175d). Renilla luciferase is highlighted in blue, firefly luciferase is highlighted in red, and the PRF sequence is highlighted in yellow with black background. 78 A.4 IL-7Rα PRF plasmid (835) agatcgatctgcgcagcaccatggcctgaaataacctctgaaagaggaacttggttaggtaccttctgaggcggaaagaa ccagctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatc tcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattag tcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatgg ctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggctttt ttggaggcctaggcttttgcaaaaagcttgattcttctgacacaacagtctcgaacttaagctgcagaagttggtcgtga ggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaag actcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccactcccagt tcaattacagctcttaaggctagagtacttaatacgactcactataggctagccaccatgacttcgaaagtttatgatcc agaacaaaggaaacggatgataactggtccgcagtggtgggccagatgtaaacaaatgaatgttcttgattcatttatta attattatgattcagaaaaacatgcagaaaatgctgttatttttttacatggtaacgcggcctcttcttatttatggcga catgttgtgccacatattgagccagtagcgcggtgtattataccagaccttattggtatgggcaaatcaggcaaatctgg taatggttcttataggttacttgatcattacaaatatcttactgcatggtttgaacttcttaatttaccaaagaagatca tttttgtcggccatgattggggtgcttgtttggcatttcattatagctatgagcatcaagataagatcaaagcaatagtt cacgctgaaagtgtagtagatgtgattgaatcatgggatgaatggcctgatattgaagaagatattgcgttgatcaaatc tgaagaaggagaaaaaatggttttggagaataacttcttcgtggaaaccatgttgccatcaaaaatcatgagaaagttag aaccagaagaatttgcagcatatcttgaaccattcaaagagaaaggtgaagttcgtcgtccaacattatcatggcctcgt gaaatcccgttagtaaaaggtggtaaacctgacgttgtacaaattgttaggaattataatgcttatctacgtgcaagtga tgatttaccaaaaatgtttattgaatcggacccaggattcttttccaatgctattgttgaaggtgccaagaagtttccta atactgaatttgtcaaagtaaaaggtcttcatttttcgcaagaagatgcacctgatgaaatgggaaaatatatcaaatcg ttcgttgagcgagttctcaaaaatgaacaaatgtcgacACCAAGAAAAAATTTAAATGTGAGttCAATCCTGAAAGTTTC CTGGACTGCCAGATTCATAGGGTGGATGACATTCAAGCTAGAGATGAAGTGGAAGGTTTTCTGCAAGATACGTTTCCTCA GCAAggatccttcaacttccctgagctcgaagacgccaaaaacataaagaaaggcccggcgccattctatcctctagagg atggaaccgctggagagcaactgcataaggctatgaagagatacgccctggttcctggaacaattgcttttacagatgca catatcgaggtgaacatcacgtacgcggaatacttcgaaatgtccgttcggttggcagaagctatgaaacgatatgggct gaatacaaatcacagaatcgtcgtatgcagtgaaaactctcttcaattctttatgccggtgttgggcgcgttatttatcg gagttgcagttgcgcccgcgaacgacatttataatgaacgtgaattgctcaacagtatgaacatttcgcagcctaccgta gtgtttgtttccaaaaaggggttgcaaaaaattttgaacgtgcaaaaaaaattaccaataatccagaaaattattatcat ggattctaaaacggattaccagggatttcagtcgatgtacacgttcgtcacatctcatctacctcccggttttaatgaat acgattttgtaccagagtcctttgatcgtgacaaaacaattgcactgataatgaattcctctggatctactgggttacct aagggtgtggcccttccgcatagaactgcctgcgtcagattctcgcatgccagagatcctatttttggcaatcaaatcat tccggatactgcgattttaagtgttgttccattccatcacggttttggaatgtttactacactcggatatttgatatgtg gatttcgagtcgtcttaatgtatagatttgaagaagagctgtttttacgatcccttcaggattacaaaattcaaagtgcg ttgctagtaccaaccctattttcattcttcgccaaaagcactctgattgacaaatacgatttatctaatttacacgaaat tgcttctgggggcgcacctctttcgaaagaagtcggggaagcggttgcaaaacgcttccatcttccagggatacgacaag gatatgggctcactgagactacatcagctattctgattacacccgagggggatgataaaccgggcgcggtcggtaaagtt gttccattttttgaagcgaaggttgtggatctggataccgggaaaacgctgggcgttaatcagagaggcgaattatgtgt cagaggacctatgattatgtccggttatgtaaacaatccggaagcgaccaacgccttgattgacaaggatggatggctac attctggagacatagcttactgggacgaagacgaacacttcttcatagttgaccgcttgaagtctttaattaaatacaaa ggatatcaggtggcccccgctgaattggaatcgatattgttacaacaccccaacatcttcgacgcgggcgtggcaggtct tcccgacgatgacgccggtgaacttcccgccgccgttgttgttttggagcacggaaagacgatgacggaaaaagagatcg tggattacgtcgccagtcaagtaacaaccgcgaaaaagttgcgcggaggagttgtgtttgtggacgaagtaccgaaaggt cttaccggaaaactcgacgcaagaaaaatcagagagatcctcataaaggccaagaagggcggaaagtccaaattgtaaca cgtgtaattctagagcggccgcttcgagcagacatgataagatacattgatgagtttggacaaaccacaactagaatgca gtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagtta acaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctac aaatgtggtaaaatcgataagtttaaacagatccaggtggcacttttcggggaaatgtgcgcggaacccctatttgttta tttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaa gagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccag aaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggt aagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtatt atcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccag tcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcg gccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcg ccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaa caacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggat aaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgg gtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcagg caactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtt 79 tactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatct catgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgag atcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaa gagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgta gttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgcca gtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacgggg ggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgc cacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttc cagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcg tcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctca catggctcgac Appendix A.4 Complete DNA sequence of the IL-7Rα PRF plasmid (835). Renilla luciferase is highlighted in blue, firefly luciferase is highlighted in red, and the PRF sequence is highlighted in yellow with black background. 80 A.5 IL-2Rγ PRF plasmid (1800) agatcgatctgcgcagcaccatggcctgaaataacctctgaaagaggaacttggttaggtaccttctgaggcggaaagaa ccagctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatc tcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattag tcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatgg ctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggctttt ttggaggcctaggcttttgcaaaaagcttgattcttctgacacaacagtctcgaacttaagctgcagaagttggtcgtga ggcactgggcaggtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaag actcttgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacaggtgtccactcccagt tcaattacagctcttaaggctagagtacttaatacgactcactataggctagccaccatgacttcgaaagtttatgatcc agaacaaaggaaacggatgataactggtccgcagtggtgggccagatgtaaacaaatgaatgttcttgattcatttatta attattatgattcagaaaaacatgcagaaaatgctgttatttttttacatggtaacgcggcctcttcttatttatggcga catgttgtgccacatattgagccagtagcgcggtgtattataccagaccttattggtatgggcaaatcaggcaaatctgg taatggttcttataggttacttgatcattacaaatatcttactgcatggtttgaacttcttaatttaccaaagaagatca tttttgtcggccatgattggggtgcttgtttggcatttcattatagctatgagcatcaagataagatcaaagcaatagtt cacgctgaaagtgtagtagatgtgattgaatcatgggatgaatggcctgatattgaagaagatattgcgttgatcaaatc tgaagaaggagaaaaaatggttttggagaataacttcttcgtggaaaccatgttgccatcaaaaatcatgagaaagttag aaccagaagaatttgcagcatatcttgaaccattcaaagagaaAGGTGAAGTTCGTCGTCCaacattatcatggcctcgt gaaatcccgttagtaaaaggtggtaaacctgacgttgtacaaattgttaggaattataatgcttatctacgtgcaagtga tgatttaccaaaaatgtttattgaatcggacccaggattcttttccaatgctattgttgAAGGTGCCAAGAAGTTTCcta atactgaatttgtcaaagtaaaaggtcttcatttttcgcaagaagatgcacctgatgaaatgggaaaatatatcaaatcg ttcgttgagcgagttctcaaaaatgaacaaatgtcgacCCCAAAAtcGGAGGGGCCCTTGGGGAGGGGCCTGGGGCCTCC CCATGCAACCAGCATAGCCCCTACTGGGCCCCCCCATGTTACACCCTAAAGCCTGAAACCggatcccccggggagctcat ggaagacgccaaaaacataaagaaaggcccggcgccattctatcctctagaggatggaaccgctggagagcaactgcata aggctatgaagagatacgccctggttcctggaacaattgcttttacagatgcacatatcgaggtgaacatcacgtacgcg gaatacttcgaaatgtccgttcggttggcagaagctatgaaacgatatgggctgaatacaaatcacagaatcgtcgtatg cagtgaaaactctcttcaattctttatgccggtgttgggcgcgttatttatcggagttgcagttgcgcccgcgaacgaca tttataatgaacgtgaattgctcaacagtatgaacatttcgcagcctaccgtagtgtttgtttccaaaaaggggttgcaa aaaattttgaacgtgcaaaaaaaattaccaataatccagaaaattattatcatggattctaaaacggattaccagggatt tcagtcgatgtacacgttcgtcacatctcatctacctcccggttttaatgaatacgattttgtaccagagtcctttgatc gtgacaaaacaattgcactgataatgaattcctctggatctactgggttacctaagggtgtggcccttccgcatagaact gcctgcgtcagattctcgcatgccagagatcctatttttggcaatcaaatcattccggatactgcgattttaagtgttgt tccattccatcacggttttggaatgtttactacactcggatatttgatatgtggatttcgagtcgtcttaatgtatagat ttgaagaagagctgtttttacgatcccttcaggattacaaaattcaaagtgcgttgctagtaccaaccctattttcattc ttcgccaaaagcactctgattgacaaatacgatttatctaatttacacgaaattgcttctgggggcgcacctctttcgaa agaagtcggggaagcggttgcaaaacgcttccatcttccagggatacgacaaggatatgggctcactgagactacatcag ctattctgattacacccgagggggatgataaaccgggcgcggtcggtaaagttgttccattttttgaagcgaaggttgtg gatctggataccgggaaaacgctgggcgttaatcagagaggcgaattatgtgtcagaggacctatgattatgtccggtta tgtaaacaatccggaagcgaccaacgccttgattgacaaggatggatggctacattctggagacatagcttactgggacg aagacgaacacttcttcatagttgaccgcttgaagtctttaattaaatacaaaggatatcaggtggcccccgctgaattg gaatcgatattgttacaacaccccaacatcttcgacgcgggcgtggcaggtcttcccgacgatgacgccggtgaacttcc cgccgccgttgttgttttggagcacggaaagacgatgacggaaaaagagatcgtggattacgtcgccagtcaagtaacaa ccgcgaaaaagttgcgcggaggagttgtgtttgtggacgaagtaccgaaaggtcttaccggaaaactcgacgcaagaaaa atcagagagatcctcataaaggccaagaagggcggaaagtccaaattgtaacacgtgtaattctagagcggccgcttcga gcagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaat ttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgt ttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtaaaatcgataagtttaaa cagatccaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtat ccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgt cgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctg aagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaa gaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagca actcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggca tgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcgga ggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaa tgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcg aactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcg gcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggg gccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacaga tcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgattta 81 aaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagtt ttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgct gcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggta actggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgt agcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggt tggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggag cgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcgga caggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttata gtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaac gccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatggctcgac Appendix A.5 Complete DNA sequence of the IL-2Rγ PRF plasmid (1800). Renilla luciferase is highlighted in blue, firefly luciferase is highlighted in red, and the PRF sequence is highlighted in yellow with black background. 82 Appendix B Sequenced PRF region of IL-7Rα PRF plasmid Appendix B.1 No mutations detected in PRF region of the IL-7Rα PRF plasmid. DNA sequencing was performed by the UBC Sequencing and Bioinformatics Consortium. Results from the sequencer (top) were aligned with the expected sequence of the IL-7Rα PRF plasmid (bottom). Blue is the 3’ end of Renilla luciferase, yellow is the PRF motif, and red is the 5’ end of firefly luciferase. There are no mismatches in the sequenced sample compared to the expected sequence of IL-7Rα PRF plasmid. 83 Appendix C In vitro synthesis with FluoroTect In vitro synthesis reactions were performed as described in section 2.4.4 except with the addition of 0.3 µL of FluoroTect (L5001, Promega) per reaction. Once the in vitro synthesis reactions were complete, dual luciferase enzyme activity was measured as per section 2.4.4. SDS-PAGE was also performed. In vitro synthesis reaction samples were mixed with 5X SDS loading buffer containing β-mercaptoethanol and heated for 3 minutes at 95° C. Samples and a ladder (PM2700, SMOBiO Technology) were loaded onto a 12% tris-glycine resolving gel, and after running, the gel was imaged on the Amersham Typhoon from Cytiva. FluoroTect contains fluorescently labelled lysine tRNA, which get incorporated into polypeptides produced during in vitro synthesis. Following SDS-PAGE, this allows for visualization of all proteins made from in vitro translation. Our dual luciferase reporter plasmids produce fused Rluc-Fluc as the product of frameshifting, which may affect individual reporter activity or stability. Therefore, the intention of this FluoroTect experiment was to directly confirm the expression of Rluc and Fluc, in case the fused Rluc-Fluc was interfering with dual luciferase enzyme activity or anti-Rluc antibody measurements, but the results were inconclusive (Appendix C.1). Further experimental optimization, such as trying a different cell-free synthesis system, may be required to interpret the FluoroTect gel readout correctly (Appendix C.1A). The fluorescently labelled lysine tRNA in FluoroTect obstructed dual luciferase enzyme activity RLU measurements (Appendix C.1B). 84 Appendix C.1 In vitro synthesis reactions containing FluoroTect were inconclusive in verifying Rluc and Fluc protein expression. Plasmids were linearized and cleaned, and then used in in vitro synthesis reactions catalyzed by extracts from the Sf21 cell line. The in vitro translation products were analyzed by A) SDS-PAGE, and B) the dual luciferase enzyme activity assay. A) 3 µL of in vitro synthesized lysates were run on a 12% acrylamide gel. The gel was imaged on an Amersham Typhoon with the Cy2 and Cy5 channels. The Rluc band size is expected to be approximately 36 kDa. With Fluc’s estimated size of 61 kDa, a Rluc-Fluc fusion protein produced from a frameshift would be roughly 97 kDa. B) Bars represent the average RLU values of 2 technical replicates.
Thesis/Dissertation
eng
Microbiology and Immunology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Abraham, Ninan
Assessment of potential :1 programmed ribosomal frameshifting by the human cytokine receptor IL-7Rα
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