Pre-mRNA splicing is a critical and regulated processing event where introns are precisely excised from nascent RNA transcripts. As many as one third of all heritable disease mutations result in splicing defects. This research studies the role of branchpoints in determining splice site selection (3'ss) is utilized in vivo and also the effect branchpoints have on the life cycle o the intron. Each pre-mRNA splicing event creates a lariat and spliced exon junction. While a great deal is known about splice exon junctions almost nothing is known about lariats. By mapping all branchpoints in the human genome, we are opening up a whole new area of analysis. The identification of branchpoints by transcript data will facilitate the interpretation f clinical sequencing data. In addition to the intrinsic value of this data, the successful completio of this proposal will test some hypothesis about the fundamental catalysis and recognition that occurs in vivo in the processing of eukaryotic genes. Studying these intermediates at a system wide level will bring a biochemical-level understanding to hundreds of thousands of processing events. Furthermore, each intron lariat has a lifecycle - created by splicing of a transcribed product, recycled by debranching and degradation. The recycling of introns is vital to replenishing the intracellular levels of free nucleotides and to return splicing factors into activ spliceosomes. Some introns have a second life after splicing as non-coding RNAs (ncRNAs). As we are sampling steady state levels of introns we gain insight into both these processes. Our preliminary data indicates a novel degradation that is devoted to recycling large introns. This proposal seeks to follow this lead by defining the complete degradation pathways and exploring some of the reasons why certain introns appear stabilized.
A great deal of research effort has been invested in understanding how pre-mRNA are spliced into mRNAs. However each splicing event produces an excised intron as well as the spliced mRNA. Very little is known about lariat introns but their characterization is very informative about splicing and the mechanisms by which cells control RNA. We have recently discovered a new method for identifying these lariats in deep sequencing data. This proposal seeks to scale this up this method to map all branchpoints in the genome and to better understand the life cycle of introns in a cell.