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UBC Theses and Dissertations

Characterization of the role of grainy head's 5'UTR uORFs in regulating translation during Drosophila melanogaster development Kirby, Mathew Pattee

Abstract

Precise control of the levels and spatiotemporal domains of protein synthesis is fundamental to cellular processes. Regulation of protein synthesis largely occurs at the rate-limiting step of translation initiation in which the translation start site is selected by the scanning ribosomal pre-initiation complex (PIC) and its associated initiation factors. Upstream open reading frames (uORFs) are prevalent regulatory elements located in the 5’ untranslated regions (5’UTR) of approximately 50% of mammalian transcripts. Generally, uORFs are viewed as constitutive repressors of translation of the downstream coding sequence (CDS) by sequestering ribosomes. Recent genome-wide studies have revealed that uORFs have widespread regulatory functions in different biological contexts, however our understanding of the roles played by uORFs is still in its infancy. In Drosophila melanogaster, the spatial and temporal expression of the transcription factor grainy head (grh) must be tightly controlled to ensure proper epithelial and central nervous system development. Intriguingly, grh’s eight mRNA isoforms display uORF-containing 5’UTRs ranging from 1 to 24 uORFs. To test for a role of these uORFs in Grh function, this thesis attempts to characterize the role of grh-RJ’s eleven uORFs in modulating the downstream CDS translation in order to fine-tune Grh’s spatiotemporal expression throughout Drosophila development. In this study, both in vitro translation assays and in vivo genetic analyses were used to analyze the regulatory role of grh-RJ’s uORFs on the downstream CDS translation. Our in vitro results showed that grh-RJ’s eleven uORFs severely repressed translation of the downstream CDS in translation extracts. Meanwhile, our transgenic in vivo results showed that that grh-RJ’s uORFs spatially restricted and repressed reporter expression in the Drosophila embryo. In general, we found that the role of grh-RJ’s uORFs is to repress translation of the downstream CDS, including restricting the spatial expression of Grh during Drosophila development. Together with the widespread prevalence of uORFs among species, this research suggests an extensive role of uORFs in regulating the level and spatiotemporal expression of proteins, which will likely contribute greatly to a fundamentally novel understanding of biological systems.

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