Biochemical characterization of testis-specific poly(A) polymerase

Abstract

In mammals, poly(A) polymerase (PAP) plays a key role in the formation of the poly(A) tail at the 3``-end of precursor mRNA in the nucleus. Testis-specific poly(A) polymerase (TPAP), an isoform of PAP, shares 86% amino acid sequence identity with PAP. TPAP is involved in additional polyadenylation of pre-extended mRNA in the cytoplasm of spermatogenic cells. For further understanding of TPAP, in this study, histidine-tagged TPAP and PAP were expressed in insect cells and purified using $Ni^{2+}$ -NTA agarose. The purified recombinant TPAP and PAP were used for non-specific polyadenylation assays with RNA substrates in vitro. The activity of TPAP on those substrates was compared with that of PAP. TPAP had a higher activity in the reaction buffer containing $Mn^{2+}$ than $Mg^{2+}$. The TPAP activity for RNA substrates containing poly(A) at the 3’ end was better than those without poly(A) or containing poly(U). However, TPAP displayed the similar binding affinity to both the RNA substrates containing poly(A) and poly(U) tails, as determined by RNA gel-retardation experiments. In addition, overexpression of TPAP did not significantly exert influence on the length distribution of poly(A) tails in HeLa cells. Therefore, these results suggest that the polyadenylation activity of TPAP in poly(A) may require other trans-acting factors in vivo.