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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032467-05
Application #
3281328
Study Section
Molecular Biology Study Section (MBY)
Project Start
1983-07-01
Project End
1991-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
5
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
Organized Research Units
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Garcia-Blanco, M A; Jamison, S F; Sharp, P A (1989) Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns. Genes Dev 3:1874-86
Garcia-Blanco, M A; Clerc, R G; Sharp, P A (1989) The DNA-binding homeo domain of the Oct-2 protein. Genes Dev 3:739-45
Virtanen, A; Sharp, P A (1988) Processing at immunoglobulin polyadenylation sites in lymphoid cell extracts. EMBO J 7:1421-9
Ganguly, S; Sharp, P A; RajBhandary, U L (1988) Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo. Mol Cell Biol 8:361-70
Moore, C L; Skolnik-David, H; Sharp, P A (1988) Sedimentation analysis of polyadenylation-specific complexes. Mol Cell Biol 8:226-33
Sedivy, J M; Capone, J P; RajBhandary, U L et al. (1987) An inducible mammalian amber suppressor: propagation of a poliovirus mutant. Cell 50:379-89
Skolnik-David, H; Moore, C L; Sharp, P A (1987) Electrophoretic separation of polyadenylation-specific complexes. Genes Dev 1:672-82
Berkner, K L; Schaffhausen, B S; Roberts, T M et al. (1987) Abundant expression of polyomavirus middle T antigen and dihydrofolate reductase in an adenovirus recombinant. J Virol 61:1213-20
Moore, C L; Skolnik-David, H; Sharp, P A (1986) Analysis of RNA cleavage at the adenovirus-2 L3 polyadenylation site. EMBO J 5:1929-38
Capone, J P; Sedivy, J M; Sharp, P A et al. (1986) Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells. Mol Cell Biol 6:3059-67

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