These experiments continue the studies of snRNP particle assembly focussing on the snRNP common core particle (suggested stoichiometry of (B2) (D'2) (D2EFG)) and the dimer U4/U6 snRNP particle. Chromatographic strategies are proposed for purifying the three previously identified snRNP core precursors of 1: 6S - (D2EFG), 2: 20S - (D', B, 70 kD) and 3: 4S - (B).Gel filtration of cytoplasmic extracts will be used as a first step followed by ion exchange chromatography to enrich for the individual precursors. The stoichiometry of the 20S complex and the identify of the non-snRNP 70 -kD """"""""chaperon like"""""""" protein in the 20S complex will then be investigated. The core particles will be assembled in vitro using the three purified precursors and in vitro synthesized snRNAs. Assembled complexes will be isolated using complementary methylated ribonucleotide probes and several criteria will be used to asses the integrity of the assembled particle. The exact assembly order of the precursors, the proteins in direct contact with the snRNA and requirements for assembly will be investigated. Synthesis and assembly of the core particles during Xenopus oogenesis and development will be studied to test the hypothesis that the 20S complex is rate limiting for snRNP particle assembly. The synthesis and assembly of the U4/U6 dimer snRNP particle will also be investigated. Experiments will investigate the pools of monomer and dimer particles and will ask where and when the two particles associate. Because the U6 snRNA is hypothesized to be the catalytic RNA for pre-mRNA splicing, this can give insight into the regulation of splicing. These studies provide a perspective on the structure and function of the snRNP particles and provide a model for RNA- protein recognition.
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| Fury, M G; Andersen, J (1997) In vitro interaction of U2 snRNA with cytoplasmic 6S protein complexes. FEBS Lett 404:70-4 |
| Fury, M G; Zhang, W; Christodoulopoulos, I et al. (1997) Multiple protein: protein interactions between the snRNP common core proteins. Exp Cell Res 237:63-9 |
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