Alternative Splicing Coupled to Nonsense-Mediated Decay in Regulation of Splicing Factor Expression

In humans, 95% of all genes are alternatively spliced (AS).  Many human diseases have been attributed to mutations that cause mistakes in pre-mRNA splicing.  An SR protein is a splicing factor that has an RNA-binding domain and a splicing activation domain.  A truncated splicing factor that is missing the SR domain, translated from a transcript containing a premature termination codon (PTC), could interfere with splicing patterns throughout the cell by competing with full length splicing factors for RNA binding sites.  PTC-containing messages are degraded by nonsense-mediated decay (NMD).  However, using the C. elegans model system, our lab has discovered PTC-containing splicing factor transcripts that are not completely degraded.  We also found many AS events that change in an NMD mutant background, even though the alternative products do not contain PTCs. We hypothesize that translation of PTC-containing splicing factor transcripts into truncated splicing factor proteins that interfere with normal splicing events may be causing this phenomenon. In order to detect whether truncated splicing factors are expressed, we inserted a N-terminal FLAG tag into six splicing factor genes, and expressed them as extra-chromosomal arrays in wild-type and NMD (smg-2) mutant backgrounds.  Reverse transcription and PCR were performed to confirm the expected alternative splicing of the transgenes.  Immunoblots are being performed to detect whether the truncated proteins are expressed from the transgenes.   If truncated splicing factors can be detected, it would support our hypothesis that the production of truncated splicing factors may be responsible for the global changes in alternative splicing previously observed.