Functional and structural basis of extreme conservation in vertebrate 5′ untranslated regions

The lack of knowledge about extreme conservation in genomes remains a major gap in our understanding of the evolution of gene regulation. Here, we reveal an unexpected role of extremely conserved 5′ untranslated regions (UTRs) in noncanonical translational regulation that is linked to the emergence of essential developmental features in vertebrate species. Endogenous deletion of conserved elements within these 5′ UTRs decreased gene expression, and extremely conserved 5′ UTRs possess cis-regulatory elements that promote cell-type-specific regulation of translation. We further developed in-cell mutate-and-map (icM2), a new methodology that maps RNA structure inside cells. Using icM2, we determined that an extremely conserved 5′ UTR encodes multiple alternative structures and that each single nucleotide within the conserved element maintains the balance of alternative structures important to control the dynamic range of protein expression. These results explain how extreme sequence conservation can lead to RNA-level biological functions encoded in the untranslated regions of vertebrate genomes. Extremely conserved 5′ UTRs act as cis-regulatory elements, playing an unsuspected role in translation regulation. A new in-cell mutational method, icM2, shows that these 5′ UTRs adopt alternative structures that depend on RNA helicase activity.