Identification of a Novel Isoform of Poly(A) Polymerase, TPAP, Specifically Present in the Cytoplasm of Spermatogenic Cells

Abstract

We have identified cDNA clones encoding a testis-specific poly(A) polymerase, termed TPAP, a candidate molecule responsible for cytoplasmic polyadenylation of preexisting mRNAs in male haploid germ cells. The TPAP gene was most abundantly expressed coincident with the additional elongation of mRNA poly(A) tails in round spermatids. The amino acid sequence of TPAP contained 642 residues, and shared a high degree of identity (86%) with that of a nuclear poly(A) polymerase, PAP II. Despite the sequence conservation of functional elements, including three catalytic Asp residues, an ATP-binding site, and an RNA-binding domain, TPAP lacked an approximately 100-residue C-terminal sequence carrying one of two bipartite-type nuclear localization signals, and part of a Ser/Thr-rich domain found in PAP II. Recombinant TPAP produced by an in vitro transcription/translation system was capable of incorporating the AMP moiety from ATP into an oligo(A)(12) RNA primer in the presence of MnCl(2). Moreover, an affinity-purified antibody against the 12-residue C-terminal sequence of TPAP recognized a 70-kDa protein in the cytoplasm of spermatogenic cells. These results suggest that TPAP may participate in the additional extension of mRNA poly(A) tails in the cytoplasm of male germ cells, and may play an important role in spermiogenesis, probably through the stabilization of mRNAs.

Possible Function of the ADAM1a/ADAM2 Fertilin Complex in the Appearance of ADAM3 on the Sperm Surface

“…Total cellular RNA was prepared from testicular tissues using ISOGEN (Nippon Gene, Toyama, Japan) (24). The RNA samples were glyoxylated, separated by agarose gel electrophoresis, and transferred onto the nylon membranes.…”

“…The RNA samples were glyoxylated, separated by agarose gel electrophoresis, and transferred onto the nylon membranes. The blots were probed by 32 P-labeled DNA fragments and analyzed by a BAS2000 Bio-Image Analyzer (Fuji Photo Film, Tokyo, Japan) (24).…”

Synthesis, Processing, and Subcellular Localization of Mouse ADAM3 during Spermatogenesis and Epididymal Sperm Transport

“…Affinity-purified polyclonal antibodies against A D A M 1 b , A D A M 3 , s p 3 2 , a n d T P A P w e r e prepared as described previously [12,13,19,20]. Monoclonal anti-ADAM2 (9D2.2) and anti-ADAM3 (7C1.2) antibodies were purchased from Chemicon.…”

A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans

“…5). Canonical PAPs, which include nuclear and cytoplasmic enzymes, are all closely related [12][13][14][15] ; they are monomeric and possess three key domains (Fig. 5, left) 8,9 .…”