Polyadenylation of messenger RNA precursors is a critical step in the synthesis of mRNAs in eucaryotic organisms and an event that is regulated for some transcription units. We have recently developed a soluble reaction from extracts of Hela cells that accurately polyadenylates exogenous substrate RNAs. Processing of the L3 site of Adenovirus 5 has been partially characterized. We propose to extend this study by identification of the precise phosphodiester bonds cleaved and newly synthesized during the polyadenylation reaction. Processing at the L3 site involves endonucleolytic cleavage and thus generation of both an upstream cleavage product and a downstream cleavage product. Under normal conditions the upstream cleavage product is polyadenylation. Under specific reaction conditions, synthesis of the poly A tract can be inhibited and the upstream cleavage product generated with little or no added adenosing residues. We will isolate and characterize the termini of both the downstream and upstream cleavage RNAs. This should reveal whether the precise site of poly A addition is produced by endonucleolytic cleavage at that position or by endonucleolytic cleavage downstream and exonucleolytic processing to the poly A addition site. These results will also demonstrate that the processing reaction at the L3 site can be mechanistically separated from the poly A synthesis reaction. Site specific mutagenesis will be used to identify the sequences and structure essential for processing at the L3 site. Polyadenylation in the soluble reaction is probably dependent upon the activity of small nuclear ribonucleoprotein (snRNP) particles since a monoclonal antibody (Y12) which reacts with sn-RNP inhibits the reaction. We will identify the specific snRNPs required and test whether processing at different poly A sites requires different snRNPs. Although not definitely proven it is likely that the regulated synthesis of the two forms of mRNAs for the heavy chain immunoglobulin genes is due to regulation of the efficiency of polyadenylation at the two sites. Polyadenylation at the upstream site in mature B cells generates mRNA encoding the secreted antibody. Polyadenylation at the downstream site in immature B cells generates mRNA for membrane bound antibody., We will develop RNA substrates for these two polyadenylation sites and characterize the in vitro processing in both Hela and B cell extracts.
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