This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The spliceosome is the cellular machine responsible for removing the introns that interrupt nearly all human gene transcripts. In a highly dynamic series of molecular interactions, the spliceosome is assembled on each intron from on the order of 150 proteins, as well as 5 structural RNAs. The Jurica group has developed a protocol isolate to spliceosomes assembled in vitro on a synthetic splicing substrate from HeLa cell nuclear extract. LC-MS/MS analysis of these complexes identified a potential 'parts list' of over 200 proteins, although the stoichiometric presence of many of these proteins remains to be verified. Control experiments indicate that a subset of the proteins bind the pre-mRNA even in the absence of splicing. In order to further define the composition of the core spliceosome complex Jurica will use mass spectrometry to identify proteins associated with the spliceosome when flanking pre-mRNA is released. They will also compare the composition of C complex assembled on other pre-mRNA splicing substrates and employ isotope tagging to allow more quantitative comparison of these different samples. Additionally, they will identify proteins that are exposed on the surface of the spliceosome by chemically modifying purified spliceosome complexes under both native and denaturing conditions. Using mass spectrometry to analyze peptides derived from these samples, they can conclude that peptides modified in both samples are surface exposed, while those modified only in the denatured complexes are buried within the complex. The limiting amount of material and high complexity of their samples will require mass spectrometric instrumentation with very high resolution and very high sensitivity.
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