RNA G-quadruplexes mark repressive upstream open reading frames in human mRNAs

Translational control is a key determinant of protein abundance, which in turns defines the physiology and pathology of human cells. Initiation of translation is highly regulated in eukaryotes and is considered as the rate-limiting step of protein synthesis. mRNA secondary structures in 5' untranslated region (UTR) and associated helicases have been characterised as key determinants of translation initiation. Nevertheless the transcriptome-wide contribution of non-canonical secondary structures, such as RNA G-quadruplexes (rG4s), to the translation of human mRNAs remains largely unappreciated. Here we use a ribosome profiling strategy to investigate the translational landscape associated to rG4s-containing mRNAs and the contribution of two rG4s-specialised DExH-box helicases, DHX9 and DHX36, to translation initiation in human cells. We show that rG4-forming sequences in 5'-UTR is associated with decreased translation efficiency which correlate with an increased ribosome density within the 5'-UTRs. We found that rG4s contribute to the translation of upstream open reading frames, and as a consequence, thwart the translation of the associated protein coding sequences (CDS). Depletion of the DHX36 and DHX9 helicases demonstrated that the formation of the rG4 structural motif rather than its nucleotide sequence mediate translation initiation. Our findings unveil a role for non-canonical structures in defining alternative 5' starts for human mRNAs translation initiation. Overall design: 11 Ribo-Seq and RNA-Seq matched samples; 1 iCLIP libraries containing two barcoded replicates