Alternative Splicing Coupled to Nonsense-Mediated Decay In Splicing Factor Expression Regulation

Alternative splicing of pre-mRNA can produce alternative isoforms that contain premature termination codons (PTCs). These PTC-containing messages, if translated, would produce truncated proteins. However, these mRNAs are rapidly degraded by nonsense-mediated decay (NMD), which preferentially degrades PTC-containing transcripts. NMD is thought to act upon PTC-containing isoforms as a type of quality control mechanism to degrade mRNAs with PTCs that arise from mutation, thus preventing their translation. New evidence suggests that not all NMD substrates are efficiently degraded, and it is unknown as to whether these are ever translated. Using the C. elegans model system, our lab has discovered examples of splicing factor transcripts with PTC-containing alternative isoforms that are inefficiently degraded by NMD. We also identified many alternative splicing events that change in NMD mutant strains, even though the alternative products do not contain PTCs. We propose that translation of PTC-containing splicing factor transcripts may produce truncated splicing factor proteins, and these may affect splicing patterns throughout the animal. We are currently testing whether PTC-containing transcripts are in fact translated. To do this, we are developing alternatively spliced, PTC-containing transgenes in which a FLAG epitope tag is inserted at the N-terminus of the protein. We are putting these tagged constructs into both wild type and NMD mutant animals and testing whether the truncated proteins are translated. If expression of the truncated proteins can be detected, it would suggest that the production of truncated splicing factors may be responsible for the global changes in alternative splicing previously observed in NMD mutants.