Co-transcriptional loading of RNA export factors shapes the human transcriptome

Abstract

During gene expression, RNA export factors are mainly known for driving nucleocytoplasmic transport. Whilst early studies suggested that the Exon Junction Complex 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 actually 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 eIF4A3 stimulates Alyref deposition not only on spliced RNAs close to EJC sites, but also single exon transcripts. Our study reveals mechanistic insights into the cotranscriptional recruitment of mRNA export factors and how this shapes the human transcriptome. Kaida et al., 2010;Millevoi et al., 2006;Rot et al., 2017), and Thoc5, which plays a role in mRNA export (Katahira et al., 2013).A major pathway for nuclear mRNA export uses the TREX complex (Strässer et al., 2002) which contains a core THO sub-complex, the RNA helicase Ddx39b, and RNA export adaptors and co-adaptors that Ddx39b loads onto mRNAs (Heath et al., 2016). A major adaptor is Alyref (Stutz et al., 2000), though some shuttling SR proteins can also work as adaptors (Huang and Steitz, 2003). Several co-adaptors have been characterized: Chtop, Thoc5, Cpsf6 and Rbm15 (Chang et al., 2013;Katahira et al., 2009;Ruepp et al., 2009;Zolotukhin et al., 2009). These proteins together recruit the export receptor Nxf1 and stimulate its RNA binding activity, which promotes mRNA export Hautbergue et al., 2008;Viphakone et al., 2015;. The topology of TREX components on RNAs and the molecular mechanisms involved in their deposition are still unclear. It has been suggested that the EJC may serve as a binding platform for RNA export factors (Le Hir et al., 2001;Singh et al., 2012).Yet, these interactions were not seen in other studies which instead revealed a key role for the cap binding complex (CBC, containing Ncbp1 and Ncbp2) in recruiting TREX, consistent with the idea that mRNPs may be exported 5' end first (Cheng et al., 2006;Chi et al., 2013). Thus, the relative contribution of the EJC and CBC to TREX deposition on the RNA in vivo remains unresolved.Here, we address some of these outstanding questions by performing individual nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) (Broughton and Pasquinelli, 2013;König et al., 2011;Lee and Ule, 2018) on the mRNA export factors Alyref, Chtop, and Nxf1. Our in vivo results suggest co-transcriptional recruitment of these proteins all along the RNA during splicing but before CPA, which grants them additional roles in gene expression. Alyref can bind inefficiently spliced introns and regulates their splicing, and Chtop binds last exons and participates in APA regulation. We est...

Nuclear sorting of RNA

“…ALYREF associates with CBCA‐bound pre‐mRNA through NCBP1 and recruits the transcription/export machinery (TREX) to promote nuclear export via the nuclear export factor 1 (NXF1/TAP) (Figure c,i) (Cheng et al, ; Masuda et al, ; Shi et al, ). ALYREF also binds to the exon junction complex (EJC) and recent data suggests that the initial recruitment of ALYREF by the CBC leads to its transfer throughout the RNA at sites adjacent to bound EJCs (Viphakone et al, ). This highlights that proper cap formation, together with efficient splicing, cements ALYREF binding and mark these transcripts towards nuclear export.…”

“…However, this is not yet a full commitment as exemplified by nuclear retained lncRNAs, such as XIST . These transcripts can be capped, spliced and bound by export factors, including ALYREF and CHTOP, but still show comparatively reduced binding of NXF1 compared to exported mRNAs (Viphakone et al, ). Other signals, bound by proteins that mediate XIST retention, such as HNRNPU, must therefore overrule the drive towards export (Sakaguchi et al, ).…”

“…Additionally, how ALYREF distinguishes stable RNAs destined for export from RNAs destined for decay is yet to be fully determined. One clue is that unstable RNAs, such as PROMPTs, eRNAs and some lncRNAs, are often not spliced, preventing the stable deposition of ALYREF (Schlackow et al, ; Viphakone et al, ) and thus prolonging nuclear residence time contributing to decay (Fan et al, ).…”

NCBP3 is a productive mRNP component

“…The spliced RNA will then be marked by the Exon Junction Complex (EJC), composed of its four core components EIF4A3, RBM8A/Y14, MAGOH and MLN51/CASC3/BTZ, as well as its multiple peripheral factors (19,20). In addition to the EJC, the Transcription and EXport (TREX) complex is recruited co-transcriptionally (21,22). A central TREX component ALYREF can be recruited by the CBC directly to the 5' end of RNA, and joined by other TREX proteins, e.g.…”

“…A central TREX component ALYREF can be recruited by the CBC directly to the 5' end of RNA, and joined by other TREX proteins, e.g. DDX39B and the THO complex, together with the EJC, leading to the formation of a nuclear export competent mRNP (10,(22)(23)(24).…”