Translation initiation at alternative start sites can dynamically control the synthesis of two or more functionally distinct protein isoforms from a single mRNA. Alternate isoforms of the hematopoietic transcription factor CCAAT-enhancer binding protein α (C/EBP α ) produced from different start sites exert opposing effects during myeloid cell development. This alternative initiation depends on sequence features of the CEBPA transcript, including a regulatory upstream open reading frame (uORF), but the molecular basis is not fully understood. Here we identify trans -acting factors that affect C/EBP α isoform choice using a sensitive and quantitative two-color fluorescence reporter coupled with CRISPRi screening. Our screen uncovered a role for the ribosome rescue factor PELOTA (PELO) in promoting expression of the longer C/EBP α isoform, by directly removing inhibitory unrecycled ribosomes and through indirect effects mediated by the mechanistic target of rapamycin (mTOR) kinase. Our work provides further mechanistic insights into coupling between ribosome recycling and translation reinitiation in regulation of a key transcription factor, with implications for normal hematopoiesis and leukemiagenesis.

Protein synthesis is a crucial but metabolically costly biological process that must be tightly coordinated with cellular needs and nutrient availability. In response to environmental stress, translation initiation is modulated to control protein output while meeting new demands. The cap-binding protein eIF4E-the earliest contact between mRNAs and the translation machinery-serves as one point of control, but its contributions to mRNA-specific translation regulation remain poorly understood. To survey eIF4E-dependent translational control, we acutely depleted eIF4E and determined how this impacts protein synthesis. Despite its essentiality, eIF4E depletion had surprisingly modest effects on cell growth and protein synthesis. Analysis of transcript-level changes revealed that long-lived transcripts were downregulated, likely reflecting accelerated turnover. Paradoxically, eIF4E depletion led to simultaneous upregulation of genes involved in catabolism of aromatic amino acids, which arose as secondary effects of reduced protein biosynthesis on amino acid pools, and genes involved in the biosynthesis of amino acids. These futile cycles of amino acid synthesis and degradation were driven, in part, by translational activation of GCN4, a transcription factor typically induced by amino acid starvation. Furthermore, we identified a novel regulatory mechanism governing translation of PCL5, a negative regulator of Gcn4, that provides a consistent protein-to-mRNA ratio under varied translation environments. This translational control was partial dependent on a uniquely long poly-(A) tract in the PCL5 5' UTR and on poly-(A) binding protein. Collectively, these results highlight how eIF4E connects translation to amino acid homeostasis and stress responses and uncovers new mechanisms underlying how cells tightly control protein synthesis during environmental challenges.

Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the roles of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks to many neurologically relevant mRNAs in NPCs. Our data reveal eIF3 predominantly interacts with 3' untranslated region (3'-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. High eIF3 crosslinking at 3'-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling. We identify the transcriptional regulator inhibitor of DNA binding 2 (ID2) mRNA as a case in which active translation levels and eIF3 crosslinking are dramatically increased upon early NPC differentiation. Furthermore, we find that eIF3 engagement at 3'-UTR ends is dependent on polyadenylation. The results presented here show that eIF3 engages with 3'-UTR termini of highly translated mRNAs, supporting a role of mRNA circularization in the mechanisms governing mRNA translation in NPCs.

The impact of synonymous codon choice on protein output has important implications for understanding endogenous gene expression and design of synthetic mRNAs. Previously, we used a neural network model to design a series of synonymous fluorescent reporters whose protein output in yeast spanned a seven-fold range corresponding to their predicted translation speed. Here, we show that this effect is not due primarily to the established impact of slow elongation on mRNA stability, but rather, that an active mechanism further decreases the number of proteins made per mRNA. We combine simulations and careful experiments on fluorescent reporters to argue that translation initiation is limited on non-optimally encoded transcripts. Using a genome-wide CRISPRi screen to discover factors modulating the output from non-optimal transcripts, we identify a set of translation initiation factors including multiple subunits of eIF3 whose depletion restored protein output of a non-optimal reporter. Our results show that codon usage can directly limit protein production, across the full range of endogenous variability in codon usage, by limiting translation initiation.

Disordered regions within RNA binding proteins are required to control mRNA decay and protein synthesis. To understand how these disordered regions modulate gene expression, we surveyed regulatory activity across the entire disordered proteome using a high-throughput functional assay. We identified hundreds of regulatory sequences within intrinsically disordered regions and demonstrate how these elements cooperate with core mRNA decay machinery to promote transcript turnover. Coupling high-throughput functional profiling with mutational scanning revealed diverse molecular features, ranging from defined motifs to overall sequence composition, underlying the regulatory effects of disordered peptides. Machine learning analysis implicated aromatic residues in particular contexts as critical determinants of repressor activity, consistent with their roles in forming protein-protein interactions with downstream effectors. Our results define the molecular principles and biochemical mechanisms that govern post-transcriptional gene regulation by disordered regions and exemplify the encoding of diverse yet specific functions in the absence of well-defined structure.