RRM
2007 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Pre-messenger mRNAs are often alternatively spliced to give rise to multiple mRNA isoforms thus increasing proteome diversity. Tissue specific alternative mRNA splicing is essential for functionally different gene products from a single gene, and thus it is an important mechanism for cellular differentiation. Various human diseases are caused by the disruption of the splicing machinery. The molecular mechanisms by which alternative splicing is regulated appear varied and remain poorly understood.
A transgenic alternative splicing reporter gene in C.elegans has been developed by Kuroyanagi et al (Nature Methods, November 2006). This reporter derived from the egl-15 gene which encodes the sole homologue of fibroblast growth factor receptor (FGFR). Egl-15 has two mutually exclusive exons 5A and 5B correspond to a portion of the protein’s extracellular domain. To monitor the expression of mutually exclusive exons, GFP and RFP were introduced downstream of exon 5A and 5B respectively. Transgenic worms show differential expression of GFP and RFP proteins depending upon the tissue specific splice patterns.
Here this worm based system is used to investigate the function of phylogenetically conserved pre-mRNA splicing factors on a whole-animal basis. Specifically, we are interested in a group of proteins weakly associated with the U2 snRNP particle’s SF3b subunit that, in yeast, are necessary for efficient splicing of only a minor subset of yeast introns. The approach is to use selective RNAi-based degradation of selected splicing factors and then to monitor the impact of the knockdown on reporter gene expression and pre-mRNA splicing. Based on RNAi knockdowns in a number of tissue culture systems, I hypothesize that knockdowns of the yeast splicing factors in C. elegans will alter the pattern of splicing. This approach will provide the first experimental data for activity in splicing for some of these gene products and may provide insight into how diminished levels selective components of the splicing apparatus influence splice site choice.
Preliminary data are encouraging as clear changes in reporter gene activity occur with targeted knockdown of selective splicing factors. Furthermore, the precise differences in GFP and RFP expression that occur in response to RNAi addition appear to vary with the specific splicing factor targeted. Future studies will employ bioinformatic approaches to investigate genome-wide changes in splice site selection after knockdown of specific alternative splicing factors and core components of the spliceosome.
Keywords: splicing factor, Celegans
