This renewal application represents the Principal Investigator's decision to combine two active NIH-grants GM21595 and GM28274 into one competing renewal of the former. Both have supported increasingly congruent studies of pre-messenger RNA: nuclear RNP particles (hnRNP) and small nuclear RNP's (snRNP's), once-separate research fields that have now attractively converged. The central focus now, as before, is the relationship between ribonucleoprotein structure and RNA processing. This is a fundamental aspect of gene expression in normal eukaryotic cells. The proposed research employs human small nuclear RNA's and premRNA's transcribed from SP6 promoters, in vitro systems for snRNA 3' processing and snRNP assembly, and a mRNA splicing and hnRNP assembly system, together with contemporary methods of RNP analysis. Many of the experimental systems and procedures to be used in this study of RNA processing and RNP assembly were developed with the previous support of this grant. The 3' processing of a human U2 RNA precursor will be investigated in an efficient and accurate in vitro system. The intracellular size of U2 RNA processing will be determined; further purification of the processing activity will be undertaken and the role of specific U2 RNA sequences and/or secondary structure in 3' processing will be investigated. The relationship between the assembly of pre-U1 and pre-U2 RNA's into RNP form and their 3' processing will be determined. Proteins binding to the pre-snRNA's will be identified by RNA- protein crosslinking. The role of cap hypermethylation and internal modifications of U1 and U2 RNA precursors on their RNP assembly will also be studied. The DNA sequences adjacent to and within a human U6 RNA gene important for transcriptional control will be investigated. The in vivo maturation of U6 RNA, a polymerase III transcript, will be studied with particular reference to its dimerization with U4 snRNA, a pol II transcript. Proteins specifically bound to newlytranscribed U6 RNA have been identified and will be investigated further. A fourth group of studies will characterize proteins present in active spliceosomes. The possibility of base-pairing among U1, U2, U5 and other snRNP's within the spliceosome will also be investigated.
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