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Multiple forms of poly(A) polymerases purified from HeLa cells function in specific mRNA 3'-end formation
L C Ryner1, Y Takagaki, J L Manley
1Department of Biological Sciences, Columbia University, New York, New York 10027.
Molecular and Cellular Biology
|October 1, 1989
Summary
Poly(A) polymerases (PAPs) are key enzymes in mRNA processing. This study purified and characterized multiple PAP forms, revealing distinct properties and confirming their role in polyadenylation, but not sequence recognition.
Area of Science:
- Molecular Biology
- Biochemistry
- Gene Expression
Background:
- Polyadenylation is a crucial post-transcriptional modification of eukaryotic messenger RNA (mRNA).
- Poly(A) polymerases (PAPs) are the enzymes responsible for catalyzing the addition of a poly(A) tail.
- Understanding the different forms and functions of PAPs is essential for comprehending gene regulation.
Purpose of the Study:
- To extensively purify and characterize Poly(A) polymerases (PAPs) from HeLa cell cytoplasmic and nuclear fractions.
- To investigate the enzymatic properties and cofactor preferences of different PAP forms.
- To elucidate the role of PAPs in pre-mRNA cleavage and polyadenylation, particularly in relation to sequence recognition.
Main Methods:
- Purification of PAPs using fast protein liquid chromatography (FPLC) and standard chromatographic techniques.
- Enzyme activity assays with varying divalent cations (Mn2+ vs. Mg2+).
- Reconstitution assays with nuclear fractions to assess cleavage and polyadenylation efficiency.
- Glycerol density gradient sedimentation to determine native enzyme size.
- Immunological inhibition assays using antibodies against rat hepatoma PAP.
Main Results:
- Three distinct PAP forms were identified: two nuclear (NE PAPs I and II) and one cytoplasmic (S100 PAP).
- NE PAP I and S100 PAP showed similar properties, preferring Mn2+ over Mg2+, while NE PAP II exhibited distinct chromatographic behavior and equal cofactor preference.
- All purified PAPs reconstituted efficient polyadenylation in the presence of cleavage and specificity factors, but lacked intrinsic AAUAAA sequence recognition.
- Native size of all three enzymes was estimated to be 50-60 kDa.
- Antibodies against a related PAP inhibited the activity of all three HeLa PAP forms.
Conclusions:
- HeLa cells contain multiple forms of Poly(A) polymerases with distinct biochemical properties.
- PAPs are essential for polyadenylation but rely on other nuclear factors for specific recognition of polyadenylation sites.
- The identified PAPs are likely related, as suggested by their similar native size and cross-reactivity with antibodies.