Identifying regulators of alternative CD33 exon-2 splicing Petra van Bergeijk1,2, Robert Stanton2, Samuel Hasson1 1Internal Medicine Research Unit, Pfizer, Cambridge, MA; 2Computational Sciences, Pfizer, Cambridge, MA
Genome wide association studies have linked a single nucleotide polymorphisms (SNP) in the CD33 gene to late-onset Alzheimer's disease susceptibility. Follow up studies showed that the identified SNP increases exon-2 skipping in the CD33 pre-mRNA. CD33 is a transmembrane receptor expressed on the surface of microglia that mediate neuroinflammatory responses such as cytokine secretion and phagocytosis. Interestingly, full length CD33, but not the exon-2 skipped isoform of CD33, suppressed microglia responses in vitro. Although the correction of CD33 exon-2 splicing could be a potential drug target to ameliorate AD, it is unknown how the alternative splicing of exon-2 is regulated. Here, we investigate splicing factors and their binding site in the CD33 pre-mRNA that play a role in CD33 exon-2 skipping. We used a CD33 splicing reporter cell line in which skipping, but not the retention of CD33 exon-2, results in the translation of the luminescent luciferase protein. In these cells, an siRNA sceen was performed to silence known splicing factors and luminescence levels were determined. We identified a specific splicing factor as a modifier of CD33 exon-2 splicing. In addition, by tiling the CD33 mRNA with Antisense Oligo's (ASO's) we identified an Exonic Splicing Enhancer (ESE) at the end of exon-2 to be involved in exon-2 splicing. To rule out effects of the splicing factor and the ASO on the luciferase reporter or total CD33 expression, results were validated in unmodified monocytic and phagocytic cell lines using a qPCR assay. This assay could detect both exon-2 skipped and exon-2 included CD33 mRNA and confirmed the effects on CD33 exon-2 splicing. Thus, we identified a splicing factor and an exonic splicing enhancer in the CD33 mRNA to be involved in CD33 exon-2 splicing and these new insights might help to target CD33 exon-2 skipping to mitigate AD.
Credits: None available.
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