Translational regulation is heavily employed during developmental processes to control the timely accumulation of proteins independently of gene transcription. In particular, mRNA poly(A) tail metabolism in the cytoplasm is a key determinant for balancing an mRNA's translational output and its decay rate. Noncanonical poly(A) polymerases (PAPs), such as germline development defective-2 (GLD-2), can mediate poly(A) tail extension. Little is known about the regulation and functional complexity of cytoplasmic PAPs. This study report the discovery of C. elegans GLD-4, a cytoplasmic PAP present in P granules that is orthologous to Trf4/5p from budding yeast. GLD-4 enzymatic activity is enhanced by its interaction with GLS-1, a protein associated with the RNA-binding protein GLD-3. GLD-4 is predominantly expressed in germ cells, and its activity is essential for early meiotic progression of male and female gametes in the absence of GLD-2. For commitment into female meiosis, both PAPs converge on at least one common target mRNA—i.e., gld-1 mRNA—and, as a consequence, counteract the repressive action of two PUF proteins and the putative deadenylase CCR-4. Together these findings suggest that two different cytoplasmic PAPs stabilize and translationally activate several meiotic mRNAs to provide a strong fail-safe mechanism for early meiotic progression (Schmid, 2009).
Poly(A) can be added to mRNAs both in the nucleus and in the cytoplasm. During oocyte maturation and early embryonic development, cytoplasmic polyadenylation of preexisting mRNAs provides a common mechanism of translational control. To begin to understand the regulation of polyadenylation activities during early development, poly (A) polymerases (PAPs) were analyzed in oocytes and early embryos of the frog, Xenopus laevis. A PAP cDNA that corresponds to a maternal mRNA present in frog oocytes was cloned and sequenced. This PAP is similar in size and sequence to mammalian nuclear PAPs. By immunoblotting using monoclonal antibodies raised against human PAP, it has been demonstrated that oocytes contain multiple forms of PAP that display different electrophoretic mobilities. The oocyte nucleus contains primarily the slower migrating forms of PAP, whereas the cytoplasm contains primarily the faster migrating species. The nuclear forms of PAP are phosphorylated, accounting for their retarded mobility. During oocyte maturation and early postfertilization development, preexisting PAPs undergo regulated phosphorylation and dephosphorylation events. Using the cloned PAP cDNA, it has been demonstrated that the complex changes in PAP forms seen during oocyte maturation may be due to modifications of a single polypeptide. These results demonstrate that the oocyte contains a cytoplasmic polymerase closely related to the nuclear enzyme and suggest models for how its activity may be regulated during early development (Ballantyne, 1995).
p34(cdc2)/cyclin B (MPF) hyperphosphorylates poly(A) polymerase (PAP) during M-phase of the cell cycle, causing repression of its enzymatic activity. Mutation of three cyclin-dependent kinase (cdk) consensus sites in the PAP C-terminal regulatory domain prevents complete phosphorylation and MPF-mediated repression. PAP also contains four nearby non-consensus cdk sites that are phosphorylated by MPF. Remarkably, full phosphorylation of all these cdk sites is required for repression of PAP activity, and partial phosphorylation has no detectable effect. The consensus sites are phosphorylated in vitro at a 10-fold lower concentration of MPF than the non-consensus sites. Consistent with this, during meiotic maturation of Xenopus oocytes, consensus sites are phosphorylated prior to the non-consensus sites at metaphase of meiosis I, and remain so throughout maturation, while the non-consensus sites do not become fully phosphorylated until after 12 h of metaphase II arrest. It is proposed that PAP's multiple cdk sites, and their differential sensitivity to MPF, provide a mechanism to link repression specifically to late M-phase. The possibility that this reflects a general means to control the timing of cdk-dependent regulatory events during the cell cycle is discussed (Colgan, 1998).
Translational activation in oocytes and embryos is often regulated via increases in poly(A) length. Cleavage and polyadenylation specificity factor (CPSF), cytoplasmic polyadenylation element binding protein (CPEB), and poly(A) polymerase (PAP) have each been implicated in cytoplasmic polyadenylation in Xenopus laevis oocytes. Cytoplasmic polyadenylation activity first appears in vertebrate oocytes during meiotic maturation. Complexes containing both CPSF and CPEB are present in extracts of X. laevis oocytes prepared before or after meiotic maturation. Assessment of a variety of RNA sequences as polyadenylation substrates indicates that the sequence specificity of polyadenylation in egg extracts is comparable to that observed with highly purified mammalian CPSF and recombinant PAP. The two in vitro systems exhibit a sequence specificity that is similar, but not identical, to that observed in vivo, as assessed by injection of the same RNAs into the oocyte. These findings imply that CPSFs intrinsic RNA sequence preferences are sufficient to account for the specificity of cytoplasmic polyadenylation of some mRNAs. The hypothesis that CPSF is required for all polyadenylation reactions is discussed, but the polyadenylation of some mRNAs may require additional factors such as CPEB. To test the consequences of PAP binding to mRNAs in vivo, PAP was tethered to a reporter mRNA in resting oocytes using MS2 coat protein. Tethered PAP catalyzes polyadenylation and stimulates translation approximately 40-fold; stimulation is exclusively cis-acting, but is independent of a CPE and AAUAAA. Both polyadenylation and translational stimulation require PAPs catalytic core, but does not require the putative CPSF interaction domain of PAP. These results demonstrate that premature recruitment of PAP can cause precocious polyadenylation and translational stimulation in the resting oocyte, and can be interpreted to suggest that the role of other factors is to deliver PAP to the mRNA (Dickson, 2001).
Multiple forms of poly(A) polymerase (PAPs I, II, and III) cDNA have been isolated from bovine, human, and/or frog cDNA libraries. PAPs I and II are long forms of the enzyme that contain four functional domains: an apparent ribonucleoprotein-type RNA-binding domain, a catalytic region that may be related to the polymerase module, two nuclear localization signals (NLSs I and 2), and a C-terminal Ser/Thr-rich region. PAP III would encode a truncated protein that lacks the NLSs and the S/T-rich region. To investigate further the structure and expression of these forms, the mouse PAP gene and an intronless pseudogene were isolated from a mouse liver genomic library. The structure of the gene indicates that different forms of PAP are produced by alternative splicing (PAPs I and II) or by competition between polyadenylation and splicing (PAP III). The pseudogene appears to reflect yet another form of long PAP, which here is termed PAP IV. Mouse PAP III and two additional truncated forms, PAPs V and VI, which would be produced by use of poly(A) sites in adjacent introns, were also isolated from a mouse brain cDNA library. RNase protection and reverse transcription-PCR analyses showed that PAP II, V, and VI are expressed in all tissues tested but that PAP I and/or IV and III are tissue specific. However, immunoblot analysis detected only the long forms, raising the possibility that the short-form RNAs are not translated. Purified recombinant baculovirus-expressed PAPs were tested in several in vitro assays, and the short forms were found to be inactive (Zhao, 1996).
Deletion and substitution mutants of bovine poly(A) polymerase have been tested, and a small region has been identified that overlaps with a nuclear localization signal and binds to the RNA primer. Systematic mutagenesis of carboxylic amino acids led to the identification of three aspartates that are essential for catalysis. Sequence and secondary structure comparisons of regions surrounding these aspartates with sequences of other polymerases revealed a significant homology to the palm structure of DNA polymerase beta, terminal deoxynucleotidyltransferase and DNA polymerase IV of Saccharomyces cerevisiae, all members of the family X of polymerases. This homology extends as far as cca: tRNA nucleotidyltransferase and streptomycin adenylyltransferase, an antibiotic resistance factor (Martin, 1996).
A single pre-mRNA could generate multiple forms of mammalian poly(A) polymerase mRNAs by alternative splicing or alternative polyadenylation. A cDNA encoding a testis-specific poly(A) polymerase has been isolated. The transcription level of Papt in testis of a 2 weeks old mouse is much lower than that of the general poly(A) polymerase gene, Pap. However, the transcription ratio of Papt to Pap is reversed in testis of a 4 weeks old mouse. Transient expression analysis showed that GFP-Papt fusion protein is present both in the nucleus and cytoplasm of HeLa cells. These results suggest that Papt is involved in polyadenylation of transcripts expressed during spermatogenesis (Lee, 2000).
cDNA clones have been identified 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 is most abundantly expressed coincident with the additional elongation of mRNA poly(A) tails in round spermatids. The amino acid sequence of TPAP contains 642 residues, and shares 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 lacks 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 is capable of incorporating the AMP moiety from ATP into an oligo(A)12 RNA primer in the presence of MnCl2. Moreover, an affinity-purified antibody against the 12-residue C-terminal sequence of TPAP recognizes 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 (Kashiwabara, 2000).
The 3'-terminal adenylic acid residue in several human small RNAs including signal recognition particle (SRP) RNA, nuclear 7SK RNA, U2 small nuclear RNA, and ribosomal 5S RNA is caused by a post-transcriptional adenylation event. Using the Alu portion of the SRP RNA as a substrate in an in vitro adenylation assay, an adenylating enzyme has been identified that adds adenylic acid residues to SRP/Alu RNA from the HeLa cell nuclear extract. All the peptide sequences obtained by microsequencing of the purified enzyme matched a unique human cDNA corresponding to a new adenylating enzyme having homologies to the well characterized mRNA poly(A) polymerase. The amino terminus region of the human SRP RNA adenylating enzyme showed approximately 75% homology to the amino terminus of the human mRNA poly(A) polymerase that includes the catalytic domain. The carboxyl terminus of the human SRP RNA adenylating enzyme showed less than 25% homology to the carboxyl terminus of poly(A) polymerase, which interacts with other factors and provides specificity. The SRP RNA adenylating enzyme is coded for by a gene located on chromosome 2 in contrast to the poly(A) polymerase gene, which is located on chromosome 14. A recombinant protein for the SRP RNA adenylating enzyme was prepared, and its activity was compared with the purified enzyme from HeLa cells. The data indicate that in addition to the SRP RNA adenylating enzyme, other factors may be required to carry out accurate 3'-end adenylation of SRP RNA (Perumal, 2001).
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. A human PAP, called neo-PAP, is encoded by a previously uncharacterized gene. cDNA was isolated from a tumor-derived cDNA library encoding an 82.8-kDa protein bearing 71% overall similarity to human PAP. Strikingly, the organization 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 assays of both specific and nonspecific polyadenylation and also endonucleolytic cleavage. Neo-PAP produced by transfection is exclusively nuclear, as demonstrated by immunofluorescence microscopy. However, notable sequence divergence between the C-terminal domains of neo-PAP and PAP suggests that the two enzymes might be differentially regulated. While PAP is phosphorylated throughout the cell cycle and hyperphosphorylated during M phase, neo-PAP does not show evidence of phosphorylation on Western blot analysis; this was unexpected in the context of a conserved cyclin recognition motif and multiple potential cyclin-dependent kinase (cdk) phosphorylation sites. Intriguingly, Northern blot analysis demonstrates that each PAP displays distinct mRNA splice variants, and both PAP mRNAs are significantly overexpressed in human cancer cells compared to expression in normal or virally transformed cells. Neo-PAP may therefore be an important RNA processing enzyme that is regulated by a mechanism distinct from that utilized by PAP (Topalian, 2001).
A detailed study was performed of the spatial distribution of a set of mRNA 3' processing factors in human T24 cells. A key enzyme in RNA 3' processing, poly(A) polymerase (PAP), was found in the cytoplasm and throughout the nucleus in a punctated pattern. A subset of the various isoforms of PAP is specifically concentrated at sites of RNA synthesis in the nucleoplasm. Additionally, the other factors necessary for RNA 3' processing, such as CstF, CPSF, and PABII, were also found at these transcription sites. These data show that the set of 3' processing factors that are presumed to be necessary for most RNA 3' cleavage and polyadenylation is indeed found at sites of RNA synthesis in the nucleoplasm. Furthermore, sites of RNA synthesis that are particularly enriched in both PAP and PABII are found at the periphery of irregularly shaped domains, called speckles, which are known to contain high concentrations of splicing factors and poly(A) RNA. Disruption of RNA 3' processing by the drug 9-beta-D-arabinofuranosyladenine causes the speckles to break up into smaller structures. These findings indicate that there is a spatial and structural relationship between 3' processing and the nuclear speckles. These studies reveal a complex and distinct organization of the RNA 3' processing machinery in the mammalian cell nucleus (Schul, 1998).
Poly(A) polymerase (PAP) is present in multiple forms in mammalian cells and tissues. The 90-kDa isoform is the product of the gene PAPOLG, which is distinct from the previously identified genes for poly(A) polymerases. The 90-kDa isoform is referred to as human PAP gamma (hsPAP gamma). hsPAP gamma shares 60% identity to human PAPII (hsPAPII) at the amino acid level. hsPAP gamma exhibits fundamental properties of a bona fide poly(A) polymerase, specificity for ATP, and cleavage and polyadenylation specificity factor/hexanucleotide-dependent polyadenylation activity. The catalytic parameters indicate similar catalytic efficiency to that of hsPAPII. Mutational analysis and sequence comparison revealed that hsPAP gamma and hsPAPII have similar organization of structural and functional domains. hsPAP gamma contains a U1A protein-interacting region in its C terminus, and PAP gamma activity can be inhibited, as can hsPAPII, by the U1A protein. hsPAPgamma is restricted to the nucleus as revealed by in situ staining and by transfection experiments. Based on these studies, it is obvious that multiple isoforms of PAP are generated by three distinct mechanisms: gene duplication, alternative RNA processing, and post-translational modification. The exclusive nuclear localization of hsPAP gamma establishes that multiple forms of PAP are unevenly distributed in the cell, implying specialized roles for the various isoforms (Kyriakopoulou, 2001).
The 3' ends of nearly all eukaryotic pre-mRNAs undergo cleavage and polyadenylation, thereby acquiring a poly(A) tail added by the enzyme poly(A) polymerase (PAP). Two well-characterized examples of regulated poly(A) tail addition in the nucleus consist of spliceosomal proteins, either the U1A or U170K proteins, binding to the pre-mRNA and inhibiting PAP via their PAP regulatory domains (PRDs). These two proteins are the only known examples of this type of gene regulation. On the basis of sequence comparisons, it was predicted that many other proteins, including some members of the SR family of splicing proteins, contain functional PRDs. The putative PRDs found in the SR domains of the SR proteins SRP75 and U2AF65, via fusion to a heterologous MS2 RNA binding protein, specifically and efficiently inhibit PAP in vitro and pre-mRNA polyadenylation in vitro and in vivo. A similar region from the SR domain of SRP40 does not exhibit these activities, indicating that this is not a general property of SR domains. The polyadenylation- and PAP-inhibitory activity of a given polypeptide can be accurately predicted based on sequence similarity to known PRDs and can be measured even if the polypeptides' RNA target is unknown. These results also indicate that PRDs function as part of a network of interactions within the pre-mRNA processing complex and suggest that this type of regulation will be more widespread than previously thought (Ko, 2002).
The steady-state levels of microRNAs (miRNAs) and their activities are regulated by the post-transcriptional processes. It is known that 3' ends of several miRNAs undergo post-dicing adenylation or uridylation. The liver-specific miR-122 from human hepatocytes and mouse livers. Direct analysis by mass spectrometry revealed that one variant of miR-122 has a 3'-terminal adenosine that is introduced after processing by Dicer. GLD-2, which is a regulatory cytoplasmic poly(A) polymerase, was demonstrated to be responsible for the 3'-terminal adenylation of miR-122 after unwinding of the miR-122/miR-122* duplex. In livers from GLD-2-null mice, the steady-state level of the mature form of miR-122 was specifically lower than in heterozygous mice, whereas no reduction of pre-miR-122 was observed, demonstrating that 3'-terminal adenylation by GLD-2 is required for the selective stabilization of miR-122 in the liver (Katoh, 2009).
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