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Jackson, Jennie Lynne

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.

Text

2025-04-25

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/90793

Microbiology and Immunology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/