<p>Interleukin 7 receptor, IL7R, is expressed exclusively on cells of the lymphoid lineage and its expression is crucial for development and maintenance of T cells. While transcriptional regulation of IL7R expression has been widely studied, its posttranscriptional regulation has only recently been uncovered. Alternative splicing of IL7R exon 6, the only exon that encodes the transmembrane domain of the receptor, results in membrane-bound (exon 6 included) and soluble (exon 6 skipped) IL7R isoforms, respectively. Interestingly, the inclusion of exon 6 is affected by a single-nucleotide polymorphism associated with the risk of developing multiple sclerosis, a prototypic demyelinating disease of the central nervous system. 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.</p><p>We utilized mutagenesis of exon 6 and surrounding introns to identify multiple exonic and intronic cis-acting regulatory elements that impact inclusion of exon 6. At least two of these elements, one exonic splicing silencer and one exonic splicing enhancer, are located in the direct vicinity of the MS-associated SNP. We also uncovered a consensus polyadenylation signal, AAUAAA in intron 6 of IL7R, 16 nucleotides downstream from exon 6 5' splice site, and showed that mutations to this site resulted in an increase in exon 6 inclusion. Additionally, we determined that the 5' splice site of exon 6 is weak. We propose that this site may be responsible for exon 6 splicing regulation.</p><p>Using tobramycin RNA affinity chromatography followed by mass spectrometry, we identified 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. siRNA-mediated knockdown of CPSF1 resulted in an increase in exon 6 inclusion, consistent with the results of mutations to the CPSF1 binding site. Correspondingly, CPSF1 depletion had no effect on a minigene with a mutation in the intronic polyadenylation site. Finally, 3'RACE and RT-PCR experiments on RNA from Jurkat cells suggested that the intronic AAUAAA site is utilized at low frequency by the polyadenylation machinery to produce a novel polyadenylated mRNA isoform. Together, our results suggest that competing pre-mRNA splicing and polyadenylation may regulate exon 6 inclusion and resultant levels of functional IL7R produced. Since the intronic polyadenylated isoform of IL7R is predicted to be translated into a membrane-bound protein product with a shortened, signal transduction-incompetent cytoplasmic tail, this may be relevant for both T cell biology and development of multiple sclerosis.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/5599 |
| Date | January 2012 |
| Creators | Evsyukova, Irina |
| Contributors | Garcia-Blanco, Mariano A, Been, Michael D |
| Source Sets | Duke University |
| Detected Language | English |
| Type | Dissertation |
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