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Multiple forms of poly(A) polymerases in human cells
A C Thuresson1, J Aström, A Aström
1Department of Medical Genetics, Uppsala University, Sweden.
Summary
Researchers cloned human poly(A) polymerase (PAP) mRNA, finding high similarity to bovine PAP. They identified three PAP forms in HeLa cells, with phosphorylation potentially regulating interactions with cleavage and polyadenylylation specificity factor (CPSF).
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Polyadenylation is a crucial post-transcriptional modification regulating mRNA stability and translation.
- Human poly(A) polymerase (PAP) is the key enzyme responsible for adding the poly(A) tail.
- Understanding PAP structure and function is vital for comprehending gene expression regulation.
Purpose of the Study:
- To clone and characterize human poly(A) polymerase (PAP) mRNA.
- To investigate the different forms of PAP in human cells and their localization.
- To explore the functional domains of PAP and its interaction with other polyadenylation factors.
Main Methods:
- Cloning of human PAP mRNA as cDNA in Escherichia coli.
- Nucleotide and amino acid sequence comparison with bovine PAP.
- Expression and purification of recombinant human PAP.
- Generation of monoclonal antibodies against recombinant PAP.
- Western blot analysis and cell fractionation to identify PAP forms and localization.
- In vitro activity assays and mutational analysis of PAP.
- Identification of potential phosphorylation sites.
Main Results:
- Human PAP mRNA sequence shows 97% nucleotide and 99% amino acid similarity to bovine PAP.
- Three PAP forms (90, 100, 106 kDa) were detected in HeLa cells.
- The 90-kDa PAP is nuclear, while 100- and 106-kDa forms are in both nuclear and cytoplasmic fractions.
- The 106-kDa PAP is likely a phosphorylated form of the 100-kDa PAP.
- Both N- and C-terminal regions of PAP are essential for enzymatic activity.
- The C-terminal region of PAP interacts with cleavage and polyadenylylation specificity factor (CPSF).
- Potential phosphorylation sites in the C-terminal region suggest regulation of PAP-CPSF interaction.
Conclusions:
- Human PAP shares high structural similarity with its bovine counterpart.
- Multiple PAP isoforms exist in human cells, with distinct subcellular localizations.
- Phosphorylation likely plays a regulatory role in PAP function and its interaction with CPSF.
- Both N- and C-terminal domains are critical for PAP activity and interaction with CPSF, highlighting a complex regulatory mechanism in polyadenylation.