GENOME-WIDE STUDIES ON POST-TRANSCRIPTIONAL REGULATION

Regulation of gene expression is essential for cellular survival and complex responses or phenotypes. Improvements in sequencing technology enable investigation of RNA transcription as a means to understand complex biological systems. However, there is increasing evidence that the correlation between RNA abundance and protein abundance is weak, as the range of associated protein abundances observed for transcripts with similar abundance spans two orders of magnitude. This discrepancy in abundances can be partially explained by changes in transcript stability or translational efficiency, collectively known as post-transcriptional regulation. Single gene studies have demonstrated that post-transcriptional regulation by trans- or cis-acting factors is essential for cell survival and differentiation. Post-transcriptional regulation can be driven by trans-acting RNA binding proteins (RBP) or cis-acting RNA-encoded elements. Genome-wide RNA association studies have identified an extensive number of RBPs; however, the regulatory roles of these RBPs are largely unclear. We provide a method for generating and functionally characterizing post-transcriptional regulators in genome-wide expression libraries. Briefly, we generate genome-wide expression libraries utilizing large-volume turbidostats. We then characterize the post-transcriptional regulation activity of each expression fragment through tethering to a reporter RNA encoding a fluorescent protein. Our method recovers known post-transcriptional regulators as well as novel regulators. We also performed a genome-wide survey for cis-acting RNA elements known as upstream open reading frames (uORFs). uORFs are thought to negatively regulate transcript translation by titrating ribosomes away from downstream coding sequence (CDS). Under translation-limited conditions, uORFs are thought to be bypassed in favor of the CDS. We identified several previously uncharacterized uORFs as important regulators of the cellular stress response. In addition, we demonstrate that uORFs are occupied in stress, arguing against the current model for uORF regulation. Collectively, our data provides a genome-wide view of post-transcriptional mechanisms by both trans- and cis-acting factors