title: 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 5' 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 5' 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.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Irina
authors.initials: I
authors.lastname: Evsyukova
authors.forename: Shelton S
authors.initials: SS
authors.lastname: Bradrick
authors.forename: Simon G
authors.initials: SG
authors.lastname: Gregory
authors.forename: Mariano A
authors.initials: MA
authors.lastname: Garcia-Blanco
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D039223:Cleavage And Polyadenylation Specificity Factor / Q000235:genetics / Q000378:metabolism*; D005091:Exons / Q000235:genetics*; D020868:Gene Silencing; D006801:Humans; D007438:Introns; D013058:Mass Spectrometry; D009103:Multiple Sclerosis / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms / Q000096:biosynthesis; D022821:RNA Splice Sites; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020395:Receptors, Interleukin-7 / Q000235:genetics*; D017381:Spliceosomes / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism
chemical_list: D039223:Cleavage And Polyadenylation Specificity Factor; D020033:Protein Isoforms; D022821:RNA Splice Sites; D012333:RNA, Messenger; D020395:Receptors, Interleukin-7; D015534:Trans-Activators