Byeon, G. W. | Search bioRxiv

bioRxiv2020

VELCRO-IP RNA-seq explores ribosome expansion segment function in translation genome-wide

SUMMARYRoles for ribosomal RNA (rRNA) in gene regulation remain largely unexplored. With hundreds of rDNA units scattered across multiple chromosomal loci, it is not possible to genetically modify rRNA in mammalian cells, hindering understanding of ribosome function. Emerging evidence suggests that expansion segments (ESs), tentacle-like rRNA extensions that vary in sequence and size across eukaryotic evolution, may provide platforms for the binding of proteins and mRNAs. Here, we develop VELCRO-IP RNA-seq: a versatile methodology to generate species-adapted ESs and map specific mRNA regions across the transcriptome that preferentially associate with ESs. By applying VELCRO-IP RNA-seq to a mammalian ES, ES9S, we identified a large array of mRNAs that are selectively recruited to ribosomes via an ES. We further characterize a set of specific 5’ UTRs that facilitate cap-independent translation through ES9S-mediated ribosome recruitment. These data provide a novel technology for studying the enigmatic ESs of the ribosome in gene-specific translation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Functional and structural basis of extreme non-coding conservation in vertebrate mRNA 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. While previous findings have mainly focused on the role of extreme conservation at the level of DNA in transcriptional regulation, its implications for RNA biology remains largely unexplored. Here, we reveal an unexpected role of extremely conserved 5UTRs in translational regulation that is linked to the emergence of essential developmental features in vertebrate species. Endogenous deletion of conserved elements within these 5UTRs decreased gene expression at the post-transcriptional level. A large-scale reporter library of extremely conserved 5UTRs revealed the widespread presence of cis-regulatory elements that promote cell-type specific regulation of translation. As these elements function as RNA molecules, further understanding of their potential structures was essential. We therefore developed in-cell mutate-and-map (icM2), a novel methodology that maps RNA structure using high-throughput mutational analysis, previously impossible to perform inside cells. Using icM2, we determined that an extremely conserved 5UTR encodes multiple alternative structures whose relative proportions are actively maintained by ATP-dependent RNA helicases. We further show that each single nucleotide within the extremely 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.

genetics↗