@article {Byeon2020.06.29.165878, author = {Gun Woo Byeon and Elif Sarinay Cenik and Lihua Jiang and Hua Tang and Rhiju Das and Maria Barna}, title = {Functional and structural basis of extreme non-coding conservation in vertebrate mRNA 5{\textquoteright} untranslated regions}, elocation-id = {2020.06.29.165878}, year = {2020}, doi = {10.1101/2020.06.29.165878}, publisher = {Cold Spring Harbor Laboratory}, abstract = {The lack of RNA-level knowledge about extreme conservation in the genome remains a major gap in our understanding of the evolution of gene regulation. While previous findings have mainly focused on the role of extreme conservation in transcriptional regulation, its implications for RNA biology remains largely unexplored. Our results reveal an unexpected role of extremely conserved 5{\textquoteright}UTRs in translational regulation that is linked to the emergence of essential developmental features in vertebrate species. In particular, we identify widespread occurrence of non-canonical translation initiation sites in extremely conserved 5{\textquoteright}UTRs that may enable spatio-temporal regulation of protein expression during embryonic development. Understanding how these RNA sequences function requires an understanding of their structures. We develop in-cell mutate-and-map (icM2), a novel methodology that maps the RNA structural ensemble using high-throughput mutational analysis previously impossible to perform inside cells. Using icM2, we determine that the extremely conserved 5{\textquoteright}UTR of Csde1 can encode multiple alternative structures whose relative proportions are actively maintained by ATP-dependent RNA helicases. We further show that the dynamic range of protein expression can be precisely tuned according to the translational efficiency of the alternative conformations. These results explain how RNA-level biological functions can contribute to extreme sequence conservation in mRNA untranslated regions of vertebrate genomes.Competing Interest StatementThe authors have declared no competing interest.}, URL = {https://www.biorxiv.org/content/early/2020/06/29/2020.06.29.165878}, eprint = {https://www.biorxiv.org/content/early/2020/06/29/2020.06.29.165878.full.pdf}, journal = {bioRxiv} }