MULTIPLE FORMS OF POLY(A) POLYMERASES IN HUMAN-CELLS

We have cloned human poly(A) polymerase (PAP) mRNA as cDNA in Escheric hia coli. The primary structure of the mRNA was determined and compare d to the bovine PAP mRNA sequence. The two sequences were 97% identica l at the nucleotide level, which translated into 99% similarity at the amino acid level. Polypeptides representing recombinant PAP were expr essed in E. coli, purified, and used as antigens to generate monoclona l antibodies. Western blot analysis using these monoclonal antibodies as probes revealed three PAPs, having estimated molecular masses of 90 , 100, and 106 kDa in HeLa cell extracts. Fractionation of HeLa cells showed that the 90-kDa polypeptide was nuclear while the 100- and 106- kDa species were present in both nuclear and cytoplasmic fractions. Th e 106-kDa PAP was most likely a phosphorylated derivative of the 100-k Da species. PAP activity was recovered in vitro by using purified reco mbinant human PAP. Subsequent mutational analysis revealed that both t he N- and C-terminal regions of PAP were important for activity and su ggested that cleavage and polyadenylylation specificity factor (CPSF) interacted with the C-terminal region of PAP. Interestingly, tentative phosphorylation sites have been identified in this region, suggesting that phosphorylation/dephosphorylation may regulate the interaction b etween the two polyadenylylation factors PAP and CPSF.