| Summary: | Many genome-wide studies pointed out translation as a major regulator of gene expression, being a key post-transcriptional mechanism by which cells rapidly change their gene expression pattern in response to diverse stimuli. There are several cis-acting elements that can be involved in the regulation of translation initiation, including upstream open reading frames (uORFs). A uORF is defined as a coding sequence that is located within the 5’ untranslated region (5’UTR) of a transcript, and it is typically considered an inhibitor of downstream translation initiation at the main ORF (mORF). This can be due to the recognition of the uORF start codon by the preinitiation complex. In this case, when the translating ribosome encounters the stop codon of the uORF, the translation machinery disassembles, a fact that can avoid translation of the mORF if the ribosome cannot reinitiate at the main start codon. ATF4, CHOP and GADD34 are stress-response proteins encoded by uORF-harboring transcripts with translation repression activity, which is responsible for maintaining a low expression of these proteins in normal conditions. However, when ER stress occurs, the unfolded protein response (UPR) is activated and eIF2α is phosphorylated by PERK. In these cases, the availability of the preinitiation complex is reduced, favoring translation of the mORFs. The stress-response proteins are therefore up regulated, triggering a cascade of events aiming stress resolution and cell survival. Given that many factors of the PERK-pathway of the UPR are regulated by uORFs, in this work we intended to determine if PERK is also regulated at the translational level. We have started by validating the annotated sequence of PERK 5’UTR using 5’RACE. Then, we have selected uORFs based on ribosome profiling data already available. To study the role of these uORFs in translational regulation of PERK, we have cloned its 5’UTR into a reporter plasmid, in frame with firefly luciferase ORF. Using site-directed mutagenesis, we have made constructs with mutated uORFs to evaluate their impact in translation efficiency. Our data, suggests that the uORFs have a repressive effect in mORF translation, and we are now dissecting the mechanisms that drive this regulation, in normal and ER stress conditions.
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