Co-transcriptional Loading of RNA Export Factors Shapes the Human Transcriptome.

Nicolas Viphakone,Ian Sudbery,Stuart A Wilson,David Sims,Llywelyn Griffith,Catherine G Heath

doi:10.1016/j.molcel.2019.04.034

Nicolas Viphakone, Ian Sudbery + Show 4 more

Open Access

https://doi.org/10.1016/j.molcel.2019.04.034

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Abstract

SummaryDuring gene expression, RNA export factors are mainly known for driving nucleo-cytoplasmic transport. While early studies suggested that the exon junction complex (EJC) provides a binding platform for them, subsequent work proposed that they are only recruited by the cap binding complex to the 5′ end of RNAs, as part of TREX. Using iCLIP, we show that the export receptor Nxf1 and two TREX subunits, Alyref and Chtop, are recruited to the whole mRNA co-transcriptionally via splicing but before 3′ end processing. Consequently, Alyref alters splicing decisions and Chtop regulates alternative polyadenylation. Alyref is recruited to the 5′ end of RNAs by CBC, and our data reveal subsequent binding to RNAs near EJCs. We demonstrate that eIF4A3 stimulates Alyref deposition not only on spliced RNAs close to EJC sites but also on single-exon transcripts. Our study reveals mechanistic insights into the co-transcriptional recruitment of mRNA export factors and how this shapes the human transcriptome.

Highlights

RNA polymerase II (Pol II) transcribes most human genes as RNA precursors that mature via 50 capping, splicing, and 30 end processing, to reach their functional state
MRNA Export Factors Bind Protein Coding Transcripts with Specific Deposition Patterns To gain insight into the RNA species bound by mRNA export factors and their distribution along RNAs in vivo, we used individual nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP)
We generated stable cell lines expressing near endogenous levels of FLAG-tagged Alyref, Chtop, and Nxf1, which recapitulated known interactions and functionalities (Figure S1) (Fanis et al, 2012; Hautbergue et al, 2008; Chang et al, 2013), allowing us to use the same antibody and identical conditions to permit direct comparisons between iCLIP datasets

Summary

Introduction

RNA polymerase II (Pol II) transcribes most human genes as RNA precursors that mature via 50 capping, splicing, and 30 end processing (i.e., cleavage and polyadenylation [CPA]), to reach their functional state. This maturation deposits RNA binding proteins (RBPs) as completion marks for downstream events like RNA nuclear export, localization, translation, and stability (Singh et al, 2015). Up to 80% of pre-mRNAs can be affected by at least one inefficient splicing event (Middleton et al, 2017), causing the retention of 5%–15% of expressed introns within mRNAs (Boutz et al, 2015). While intron features influence retention events, the mechanisms involved remain unclear (Boutz et al, 2015)

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