Identification and functional characterization of neo-poly(A) polymerase, an RNA processing enzyme overexpressed in human tumors

Poly(A) polymerase (PAP) plays an essential role in polyadenylation of mRNA precursors, and it has long been thought that mammalian cells contain only a single PAP gene. We describe here the unexpected existence of a human PA P, which we call neo-PAP, encoded by a previously uncharacterized gene. cDN A was isolated from a tumor-derived cDNA library encoding an 82.8-kDa prote in bearing 71% overall similarity to human PAP. Strikingly, the organizatio n of the two PAP genes is nearly identical, indicating that they arose from a common ancestor. Neo-PAP and PAP were indistinguishable in in vitro assa ys of both specific and nonspecific polyadenylation and also endonucleolyti c cleavage. Neo-PAP produced by transfection was exclusively nuclear, as de monstrated by immunofluorescence microscopy. However, notable sequence dive rgence between the C-terminal domains of neo-PAP and PAP suggested that the two enzymes might be differentially regulated. While PAP is phosphorylated throughout the cell cycle and hyperphosphorylated during M phase, neo-PAP did not show evidence of phosphorylation on Western blot analysis, which wa s unexpected in the context of a conserved cyclin recognition motif and mul tiple potential cyclin-dependent kinase (cdk) phosphorylation sites. Intrig uingly, Northern blot analysis demonstrated that each PAP displayed distinc t mRNA splice variants, and both PAIR mRNAs were significantly overexpresse d in human cancer cells compared to expression in normal or virally transfo rmed cells. Neo-PAP may therefore be an important RNA processing enzyme tha t is regulated by a mechanism distinct from that utilized by PAP.