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Investigating the Translational Control Mechanisms Regulating Alternative Open Reading Frames

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Abstract

Translation initiation at alternative start sites has the potential to yield functionally and structurally distinct protein isoforms that can play decisive roles during various cellular processes including cell fate commitment. It further serves as a dynamic point of control in gene expression, enabling a rapid response to changes in the cellular environment through the translation of regulatory elements called upstream open reading frames (uORFs). Re-initiation after the translation of uORFs has been shown to regulate start site selection for downstream protein-coding genes, yet the molecular factors and mechanism underlying this choice has been largely uncharacterized. Here we identify the trans-acting factors that regulate start site usage in the key developmental transcription factor CEBPA using a sensitive and quantitative two-color fluorescence reporter coupled with CRISPRi screening. Our screen uncovered factors that have not been previously linked to re-initiation as well as recovered known regulators of re-initiation and CEBPA expression. In particular, we uncover a role for the ribosome recycling factor, Pelota (PELO), in the control of CEBPA translation. Using ribosome profiling and fluorescent based reporters, we provide evidence suggesting that PELO promotes long isoform expression by disfavoring CEBPA uORF translation in a manner that is partially dependent on mTOR activation. Our work provides further mechanistic insights into how ribosome recycling and translation re-initiation are coupled to regulate a major transcription factor with important implications for cell fate control.

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This item is under embargo until September 27, 2025.