Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro.
We have partially purified a poly(A) polymerase (PAP) from HeLa cell nuclear extract which is involved in the 3'-end formation of polyadenylated mRNA. PAP had a molecular weight of approximately 50 to 60 kilodaltons. In the presence of manganese ions, PAP was able to polyadenylate RNA nonspecifically. However, in the presence of magnesium ions PAP required the addition of a cleavage and polyadenylation factor to specifically polyadenylate pre-mRNAs that contain an intact AAUAAA sequence and end at the poly(A) addition site (precleaved RNA substrates). The purified fraction containing PAP was also required in combination with a cleavage and polyadenylation factor and a cleavage factor for the correct cleavage at the poly(A) site of pre-mRNAs. Since the two activities of the PAP fractions, PAP and cleavage activity, could not be separated by extensive purification, we concluded that the two activities are contained in a single component, a PAP that is also required for the specific cleavage preceding the polyadenylation of pre-mRNA.
Christofori
G
G
Department of Cell Biology, University of Basel, Switzerland.
Keller
W
W
D002457:Cell Extracts; D002478:Cells, Cultured; D005591:Chemical Fractionation; D002845:Chromatography; D006367:HeLa Cells; D006801:Humans; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010957:Plasmids; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D015138:RNA, Nuclear / Q000378:metabolism*
D002457:Cell Extracts; D012322:RNA Precursors; D012333:RNA, Messenger; D015138:RNA, Nuclear; D011061:Poly A; D009713:Nucleotidyltransferases; D011062:Polynucleotide Adenylyltransferase