The removal of introns from the nuclear precursors of mature cytoplasmic messenger RNA (pre-mRNA) is achieved by the spliceosome, an enormously complicated macromolecular machine. The spliceosome consists of hundreds of proteins and five snRNAs in addition to the pre-mRNA itself. In order to ensure faithful gene expression, it is critically important that each intron be removed from the pre-mRNA with single nucleotide precision. After splicing, the liberated intron is degraded within the nucleus. However, before intron turnover can proceed, the splicing apparatus that remains bound to the intron in a complex termed the lariat RNP, consisting of the U2, U5 and U6 snRNPs as well as numerous other factors, must be dissociated. Precious little is known about the disassembly of the lariat RNP, the recycling of the spliceosomal snRNPs for reuse in subsequent splicing cycles, and the degradation of the intron lariat RNA. In the work proposed here, functional assays will be performed on several newly discovered intron lariat RNP disassembly intermediates to gain insight into the mechanism underlying the recycling of spliceosome components. A key step in spliceosome disassembly is release of the intron lariat RNA followed by debranching, which is catalyzed by a phosphodiesterase designated Dbr1p. Crystals of this enzyme have been generated from two different organisms and their structures will be solved in the presence and absence of the substrate RNA. After debranching, the intron RNA is rapidly degraded in wild-type cells but accumulates in the prp27-1 mutant. We show that this mutation is an extreme loss- of-function allele of the 5'->3'exoribonuclease encoded by the RAT1 gene. In the final series of experiments, functional studies, both genetic and biochemical, will be used to determine precisely how Rat1p functions in the turnover of introns and its many other substrates. The long-term goal of this work is to understand the structure, function and dynamics of the RNPs implicated in all steps of gene expression. The specific objective of this application is to determine the mechanism of spliceosome disassembly and to understand the process of intron lariat RNA debranching and intron turnover. Our rationale is that the three factors under study in this application are critical and highly conserved players in spliceosome disassembly and intron turnover.
The human pre-mRNA processing machinery is co-opted or subverted by viral pathogens, such as HIV or influenza virus, which require the host for viral gene expression. Additionally, upwards of 50% of all cancers arise from aberrant gene expression, and several autoimmune disorders result from inappropriate immune responses to RNP components of the gene expression machinery. Understanding the details of the pre-mRNA splicing program will allow us to subvert a pathogen, specifically interrupt the replication of cancer cells and provide critical insight into autoimmunity. We are using Saccharomyces cerevisiae as a model system to allow comprehensive biochemical and genetic analyses as the core pre-mRNA splicing machinery and the mechanism are well conserved from yeast to humans;thus this work directly relates to human health.
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