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Polyadenylation of SV40 late pre-mRNA is dependent on phosphorylation of an essential component associated with the
K M Chrislip1, J A Hengst-Zhang, S T Jacob
1Department of Pharmacology and Molecular Biology, Chicago Medical School, North Chicago, Illinois 60064.
Abstract:
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.
Insights
Phosphorylation is essential for messenger RNA (mRNA) polyadenylation, a key step in gene expression. Removing phosphate groups blocked this process, indicating a critical role for phosphorylated proteins in mRNA 3' end processing.
Area of Science:
- Molecular Biology
- Biochemistry
- Gene Expression
Background:
- Messenger RNA (mRNA) undergoes 3' end processing, including polyadenylation, for stability and translation.
- The role of protein phosphorylation in mRNA 3' end processing, specifically polyadenylation, remains incompletely understood.
Purpose of the Study:
- To investigate the requirement of phosphorylation for mRNA polyadenylation.
- To determine if specific phosphorylation events are essential for the 3' end processing of pre-mRNA.
Main Methods:
- Dephosphorylation of HeLa nuclear extracts using alkaline phosphatase.
- Analysis of cleavage-dependent polyadenylation of SV40 late pre-mRNA using treated extracts.
- Assessing the formation of pre-mRNA-protein complexes under dephosphorylated conditions.
Main Results:
- Alkaline phosphatase treatment completely inhibited mRNA polyadenylation but not pre-mRNA cleavage.
- Sodium vanadate partially reversed the inhibitory effect of phosphatase on polyadenylation.
- Dephosphorylation prevented polyadenylation-specific complex formation, while cleavage-specific complexes still formed.
- Mn-dependent poly(A) extension was relatively resistant to phosphatase treatment.
Conclusions:
- Phosphorylation of a key factor is essential for mRNA 3' end processing and polyadenylation.
- This critical factor is likely poly(A) polymerase, highlighting its post-translational modification requirements.