Ribosome rescue factor PELOTA modulates translation start site choice and protein isoform levels of transcription factor C/EBPa [riboprofiling]

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 C/EBPa 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/EBPa 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/EBPa 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. Overall design: To determine the impact of reduced PELO levels on ribosome occupancy transcriptome-wide and on a CEBPA, we established a CEBPA fluorescent reporter in K562 cells. We then depleted PELO by sgRNA (CRISPRi). We determined changes in translation on our reporter (and transcriptome-wide) by ribosome profiling using the nucleases, RNAse1 and P1. A nontargeting sgRNA was used as a control and both sgRNA conditions (nontargeting and PELO KD) were performed in triplicate. Samples from each condition and replicate were also taken for matched RNA-seq gene expression analysis.