Cleavage and polyadenylation specificity factor 1 (CPSF1) regulates alternative splicing of interleukin 7 receptor (IL7R) exon 6

Abstract

Interleukin 7 receptor, IL7R, is expressed exclusively on cells of the lymphoid lineage, and its expression is crucial for the development and maintenance of T cells. Alternative splicing of IL7R exon 6 results in membrane-bound (exon 6 included) and soluble (exon 6 skipped) IL7R isoforms. Interestingly, the inclusion of exon 6 is affected by a single-nucleotide polymorphism associated with the risk of developing multiple sclerosis. Given the potential association of exon 6 inclusion with multiple sclerosis, we investigated the cis-acting elements and trans-acting factors that regulate exon 6 splicing. We identified multiple exonic and intronic cis-acting elements that impact inclusion of exon 6. Moreover, we utilized RNA affinity chromatography followed by mass spectrometry to identify trans-acting protein factors that bind exon 6 and regulate its splicing. These experiments identified cleavage and polyadenylation specificity factor 1 (CPSF1) among protein-binding candidates. A consensus polyadenylation signal AAUAAA is present in intron 6 of IL7R directly downstream from the 59 splice site. Mutations to this site and CPSF1 knockdown both resulted in an increase in exon 6 inclusion. We found no evidence that this site is used to produce cleaved and polyadenylated mRNAs, suggesting that CPSF1 interaction with intronic IL7R pre-mRNA interferes with spliceosome binding to the exon 6 59 splice site. Our results suggest that competing mRNA splicing and polyadenylation regulate exon 6 inclusion and consequently determine the ratios of soluble to membrane-bound IL7R. This may be relevant for both T cell ontogeny and function and development of multiple sclerosis.

Human Epistatic Interaction Controls IL7R Splicing and Increases Multiple Sclerosis Risk

“…5). This result is consistent with exon 6 skipping being augmented by two insults: strengthening of a weak silencer by the risk allele of rs6897932 (Evsyukova et al, 2013; Gregory et al, 2007) and reduced expression of the activator DDX39B. Collectively, our genetic and functional studies indicate that carriers of both IL7R and DDX39B risk alleles are at highest risk of developing MS, very likely due to increased expression of sIL7R.…”

“…Using different sets of IL7R exon 6 RNAs and HeLa nuclear extracts we identified a total of 89 candidate trans -factors (Evsyukova et al, 2013). Importantly, we showed that both HeLa and Jurkat T cells recapitulate the SNP rs6897932- and ESE2-dependent changes in IL7R exon 6 splicing (Fig.…”

“…We previously showed that the rs6897932 C allele in exon 6 of IL7R , which is strongly associated with MS susceptibility, increases skipping of exon 6 (Evsyukova et al, 2013; Gregory et al, 2007), and this correlates with elevated levels of circulating sIL7R (Hoe et al, 2010; Lundstrom et al, 2013). This is predicted to have important repercussions on the pathogenesis of MS as sIL7R has been shown to exacerbate the severity of EAE, a mouse model of MS (Lundstrom et al, 2013).…”

“…This variant introduces a non-synonymous threonine to isoleucine change at amino acid position 244, but this change does not appear to alter IL7 signaling (unpublished results). Importantly, we showed that the risk allele enhances skipping of exon 6 (Evsyukova et al, 2013; Gregory et al, 2007), increasing the fraction of mRNAs that code for a secreted form of the receptor (sIL7R), and this correlates with increased plasma levels of sIL7R (Hoe et al, 2010; Lundstrom et al, 2013). Elevated levels of sIL7R have been shown to exacerbate the severity of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, presumably by enhancing the activity or bioavailability of IL7 (Lundstrom et al, 2013).…”

“…Previously we identified several important cis -acting elements, among them a critically important exonic splicing enhancer (ESE2), immediately downstream of rs6897932, whose mutation (ΔESE2) inhibited inclusion of exon 6 (Evsyukova et al, 2013). By combining RNA affinity chromatography with mass spectrometry, we discovered factors interacting with exon 6 and flanking sequences (Evsyukova et al, 2013) and here we identified those that required ESE2 to bind. We showed that knockdown of one of these factors, the DEAD Box Polypeptide 39B (DDX39B, also known as UAP56/BAT1), increased IL7R exon 6 skipping in cell lines and in primary CD4 + T cells, and up-regulated sIL7R secretion.…”