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Association of newly synthesized poly(A) polymerase with four distinct polypeptides
J A Hengst1, I Georgoff, H C Isom
1Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey 17033.
Abstract:
Nuclear poly(A) polymerase was isolated from [35S]methionine-labeled hepatoma McA-RH 7777 cells and subjected to DEAE-Sephadex chromatography. Flow-through and low salt wash fractions containing poly(A) polymerase activity were pooled and subjected to immunoblot analysis using anti-tumor type poly(A) polymerase antibodies and a biotinylated second antibody. The immune complex contained a single 48-kDa polypeptide band corresponding to the tumor-type enzyme. When immunoprecipitations were carried out using the same fraction and antibodies, at least five 35S-methionine-labeled proteins with approximate molecular masses of 74, 48, 35, 30, and 22 kDa were observed. Pulse-chase studies did not indicate a precursor-product relationship between the immunoprecipitated proteins. Preimmune sera did not react with poly(A) polymerase or other components in the protein complex. These data show that poly(A) polymerase exists as part of a complex with at least four other polypeptides and suggest that these polypeptides may be involved in the cleavage and/or polyadenylation reactions.
Insights
Nuclear poly(A) polymerase in hepatoma cells exists in a complex with other proteins. This protein complex may play a role in RNA cleavage and polyadenylation reactions.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Nuclear poly(A) polymerase is crucial for mRNA maturation.
- Understanding its cellular interactions is key to elucidating gene expression regulation.
Purpose of the Study:
- To investigate the protein complex associated with nuclear poly(A) polymerase in hepatoma cells.
- To identify potential interacting partners involved in polyadenylation.
Main Methods:
- Isolation and purification of nuclear poly(A) polymerase from [35S]methionine-labeled hepatoma cells.
- DEAE-Sephadex chromatography followed by immunoblot analysis.
- Immunoprecipitation using anti-tumor type poly(A) polymerase antibodies.
Main Results:
- A single 48-kDa polypeptide band corresponding to tumor-type poly(A) polymerase was identified.
- Immunoprecipitation revealed a complex containing at least five proteins (74, 48, 35, 30, and 22 kDa).
- Pulse-chase studies ruled out precursor-product relationships among these proteins.
Conclusions:
- Nuclear poly(A) polymerase forms a complex with at least four other polypeptides.
- These associated proteins may be involved in RNA cleavage and/or polyadenylation processes.
- The findings suggest a multi-protein machinery for mRNA polyadenylation.