Role of poly(A) polymerase in the cleavage and polyadenylation of mRNA precursor.
To determine the role of poly(A) polymerase in 3'-end processing of mRNA, the effect of purified poly(A) polymerase antibodies on endonucleolytic cleavage and polyadenylation was studied in HeLa nuclear extracts, using adenovirus L3 pre-mRNA as the substrate. Both Mg2+- and Mn2+-dependent reactions catalyzing addition of 200 to 250 and 400 to 800 adenylic acid residues, respectively, were inhibited by the antibodies, which suggested that the two reactions were catalyzed by the same enzyme. Anti-poly(A) polymerase antibodies also inhibited the cleavage reaction when the reaction was coupled or chemically uncoupled with polyadenylation. These antibodies also prevented formation of specific complexes between the RNA substrate and components of nuclear extracts during cleavage or polyadenylation, with the concurrent appearance of another, antibody-specific complex. These studies demonstrate that (i) previously characterized poly(A) polymerase is the enzyme responsible for addition of the poly(A) tract at the correct cleavage site and probably for the elongation of poly(A) chains and (ii) the coupling of these two 3'-end processing reactions appears to result from the potential requirement of poly(A) polymerase for the cleavage reaction. The results suggest that the specific endonuclease is associated with poly(A) polymerase in a functional complex.
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Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey 17033.
Jacob
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D000256:Adenoviridae / Q000378:metabolism; D000906:Antibodies; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism; D011062:Polynucleotide Adenylyltransferase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism
D000906:Antibodies; D012322:RNA Precursors; D012333:RNA, Messenger; D012367:RNA, Viral; D011061:Poly A; D008345:Manganese; D009713:Nucleotidyltransferases; D011062:Polynucleotide Adenylyltransferase