Publication Date
2013
Document Type
Thesis
Committee Members
Paula Bubulya (Advisor), Katherine Excoffon (Committee Member), Mill Miller (Committee Member)
Degree Name
Master of Science (MS)
Abstract
Pre-mRNA splicing requires proper splice site selection mediated by many factors including snRNPs and serine-arginine rich (SR) splicing factors. Son is the largest known SR splicing factor, and it has several putative functional domains including an RS domain, a glycine rich patch (G-patch) and double stranded RNA binding domain (DSRBD). One-third of Son's amino acid sequence consists of novel repetitive sequence motifs of unknown function (Sharma et al., 2010). Son is essential for organization of pre-mRNA processing factors in nuclear speckles and for cell cycle progression (Sharma et al., 2010; Sharma et al., 2011; Ahn et al 2011). Exon array analysis of Son-depleted HeLa cells revealed changes in 1061 transcripts showing exon inclusion or exclusion, and a total of 2067 splicing events that are potentially regulated by Son. We validated that Son is required for appropriate splice site choice in transcripts for several chromatin-modifying enzymes, including HDAC6, ADA and SETD8 (Sharma et al., 2011). However, the mechanism by which Son maintains accurate splicing is unknown. We are systematically generating model minigene cassettes for molecular and in situ analysis of Son-dependent splicing regulation. We have constructed a HDAC6 minigene reporter that contains the genomic sequence spanning exons 26 through 29. Following Son depletion in HeLa cells transiently transfected with the HDAC6 minigene reporter construct, we observed skipping of exons 27 and 28 on both the reporter and endogenous HDAC6 transcripts. Stable cell lines constructed using HDAC6 minigene reporter construct showed exclusion of exons 27 and 28 upon Son depletion. In order to study Son-dependent splicing on HDAC6 in situ, we aimed to localize splicing factor recruitment to the HDAC6 reporter minigene transcription site; however, RNA-FISH performed using probes designed to bind to HDAC6 minigene transcripts did not show reproducible labeling of the transcription locus. Finally, HEK293 cells stably expressing four different siRNA-refractory Son deletion mutants were used to show rescue splicing of HDAC6 minigene transcripts.
Page Count
98
Department or Program
Department of Biological Sciences
Year Degree Awarded
2013
Copyright
Copyright 2013, all rights reserved. This open access ETD is published by Wright State University and OhioLINK.
