Polyadenylation of SV40 late pre-mRNA is dependent on phosphorylation of an essential component associated with the 3' end processing machinery.
We investigated whether phosphorylation of the essential components involved in the 3' end processing of mRNAs was required for mRNA polyadenylation. The proteins in HeLa nuclear extract were dephosphorylated with alkaline phosphatase, which is known to remove the phosphate moieties from serine and tyrosine. The dephosphorylated extract was used for analyzing cleavage-dependent polyadenylation of SV40 late pre-mRNA. The phosphatase treatment of the extract completely blocked the polyadenylation reaction, whereas dephosphorylation of the extract did not inhibit the cleavage reaction. Since the cleavage depends upon functional integrity of the specificity factor, it is unlikely that the phosphorylated state of the latter factor is required for the 3' end processing. Sodium vanadate, a potent inhibitor of alkaline phosphatase, markedly reduced the inhibitory effect of the phosphatase on the polyadenylation reaction. Dephosphorylation of the extract also prevented formation of the polyadenylation-specific complex with pre-mRNA, whereas the cleavage-specific complexes were formed under this condition. The Mn-dependent polyadenylation, which is largely poly(A) extension reaction, was relatively resistant to the phosphatase treatment. These data indicate that phosphorylation of a key factor is essential for the 3' end processing of pre-mRNA, and suggest that the factor may be poly(A) polymerase.
Chrislip
K M
KM
Department of Pharmacology and Molecular Biology, Chicago Medical School, North Chicago, Illinois 60064.
Hengst-Zhang
J A
JA
Jacob
S T
ST
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors; D002467:Cell Nucleus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D008345:Manganese; D010766:Phosphorylation; D011061:Poly A / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology*; D012367:RNA, Viral / Q000378:metabolism*; D013539:Simian virus 40 / Q000235:genetics*; D014638:Vanadates
D046911:Macromolecular Substances; D012322:RNA Precursors; D012367:RNA, Viral; D011061:Poly A; D014638:Vanadates; D008345:Manganese; D000469:Alkaline Phosphatase