Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved alternative splicing nonsense-mediated decay pathways

摘要

a (Top) mRNA metabolism from transcription to degradation. EJCs (purple) deposited upstream of exon junctions and other RNA-binding proteins (RBPs; gray) are cleared by ribosomes (orange) during the pioneer round of translation. While protein-coding isoforms are subject to multiple rounds of translation prior to decay, NMD isoforms are rapidly eliminated. Steps affected by harringtonine treatment are indicated. (Bottom) Hypothetical abundance throughout the mRNA lifecycle in the libraries analyzed in this paper: EJC-bound RIPiT-Seq (purple) and RNA-Seq libraries treated with (+; dark green) or without (−; light green) harringtonine. b–d Genome browser tracks of library coverage across individual genes (gray: protein-coding isoform(s); blue: NMD isoform) containing poison cassette exons (b, TRA2B and c, U2AF2) or 3′ UTR introns (d, hnRNPA1). Shown are all three EJC RIPiT-Seq replicates and replicate 1 for both (+) and (−) harringtonine RNA-Seq libraries. Conservation tracks show phyloP basewise scores derived from Multiz alignment of 30 vertebrate species. Numbers below tracks indicate mean reads per million (RPM) spanning each exon junction. Numbers on the right in b and c are percent spliced in (PSI) values for poison exon inclusion events; PSI values for RNA-Seq libraries are replicate means. See “Methods” for PSI formula