Abstract
Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends.
Highlights
Export of mRNA from the nucleus to the cytoplasm is a highly regulated step in gene expression
Our previous work in P19 cells showed that individual SR proteins interact with distinct sets of mRNAs (Änkö et al 2010), suggesting that specific mRNAs may be controlled by SR protein family members independently (Björk et al 2009; Pandit et al 2013; Bradley et al 2015)
To test whether decreased cytoplasmic transcript levels are a good proxy for export defects in P19 cells, nuclear export factor 1 (NXF1) was depleted by RNAi, and changes in mRNA levels in cytoplasmic and nuclear fractions were quantified (Supplemental Fig. S1A,B)
Summary
Export of mRNA from the nucleus to the cytoplasm is a highly regulated step in gene expression. SR proteins regulate such diverse processes as 3′ end processing (Lou et al 1998; Bradley et al 2015), mRNA export (Masuyama et al 2004; Huang and Steitz 2005), mRNP packaging (Singh et al 2012), mRNA stability (Lemaire et al 2002), and translation (Michlewski et al 2008; Maslon et al 2014) They are recruited to pre-mRNA during transcription, consistent with cotranscriptional assembly of the spliceosome and splicing (Sapra et al 2009) and suggesting that SR proteins may couple sequential events and mark mRNAs as they transit from the nucleus to the cytoplasm. Extensive serine phosphorylation within the RS domain is crucial for SR protein recruitment to transcription sites and for spliceosome assembly; RS domain dephosphorylation occurs during splicing and is important for catalysis, release of the splicing machinery, and subsequent mRNP maturation (Huang and Steitz 2005; Shepard and Hertel 2009; Ghosh and Adams 2011)
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