3' cleavage and polyadenylation of mRNA precursors in vitro requires a poly(A) polymerase, a cleavage factor, and a snRNP.
We have separated and purified three factors from HeLa cell nuclear extracts that together can accurately cleave and polyadenylate pre-mRNAs containing the adenovirus L3 polyadenylation site. One of the factors is a poly(A) polymerase with a molecular weight of approximately 50-60 kd. The second activity is a cleavage factor with a native molecular weight in the range of 70-120 kd. The third component is a factor (cleavage and polyadenylation factor, CPF) that is needed for the cleavage reaction and, in addition, confers specificity to the poly(A) polymerase activity; the native molecular weight of CPF is approximately 200 kd. Poly(A) polymerase together with CPF is sufficient to specifically polyadenylate pre-mRNA substrates that have been precleaved at the poly(A) addition site. In contrast, all three components are required for accurate cleavage and polyadenylation of pre-mRNA substrates. Further purification of CPF by buoyant density centrifugation, ion exchange, and affinity column chromatography or by gel filtration demonstrates that CPF activity resides in a ribonucleoprotein and copurifies with U11 snRNP.
Christofori
G
G
Department of Cell Biology, Biocenter of the University of Basel, Switzerland.
Keller
W
W
D004722:Endoribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D009694:Nucleic Acid Precursors / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism*; D011062:Polynucleotide Adenylyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear
D009694:Nucleic Acid Precursors; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D011061:Poly A; D009713:Nucleotidyltransferases; D012319:RNA Polymerase II; D011062:Polynucleotide Adenylyltransferase; D004722:Endoribonucleases