Protein translation control of organism development

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Embryonic development requires precise spatiotemporal synthesis of thousands of proteins. Correspondingly, translational control of mRNAs is essential for proper embryogenesis. The long-term vision of our research program is to explore the roles played by a novel, potentially widespread contributor to translation regulation during embryogenesis, upstream open reading frames (uORFs). Most transcripts in eukaryotes have uORFs in their 5'untranslated regions (5'UTRs) that recruit translating ribosomes and sequester translation machinery away from the main coding sequence (mCDS). This suppresses protein synthesis. In spite of the potential that uORFs thereby represent as widespread translational regulators, there are very few descriptions of their role or impact in any biological context. Rationale and Preliminary data: Ribo-seq of Drosophila embryos identified >22,000 translating uORFs through development; many at levels higher than their downstream mCDS. Our analysis of these datasets identified 305 transcription factor transcripts with high uORF translation rates during Drosophila embryogenesis. To test a role for these uORFs, using in vitro translation assays and in vivo transgenic reporter analysis, we found that 5'UTR uORFs within the transcription factor grainy head (grh), suppress and spatially regulate translation of grh mCDS. Our short-term objective is to start systematic mutant analysis of transcription factor uORFs to test their role in regulating rates and spatiotemporal patterns of protein synthesis required for embryogenesis. We hypothesize that transcription factor translation is regulated by uORFs in the 5'UTR of their transcripts. We predict that mutation of uORF start codons - to prevent uORF translation - will induce ectopic, precocious and/or upregulated expression of transcription factor, causing severe defects in embryonic development. Aim 1. Genetic analysis of uORF function in transcription factor translation and developmental function. Transcripts for analysis will be prioritized, based on high uORF translation and the gene's role in embryogenesis. The impact of uORF mutagenesis will be assessed: (i) We will screen transcripts using transgenic reporters to identify uORFs that suppress main coding sequence translation. (ii) Suppressive uORFs will be disrupted by genome editing, followed by assessment of transcription factor expression and impact on embryogenesis. Aim 2. In vitro translation assays of uORF activity. We will systematically screen transcripts for uORFs that suppress main coding sequence translation. For transcripts with multiple uORFs, we will explore the relative contribution of each uORF to translation suppression. Significance. Our innovative objectives will provide ground-breaking evidence that uORFs play essential roles in translation control required for embryogenesis. This sets the stage for our long-term vision to examine the many roles and mechanisms underlying uORF function in development.