A novel nuclear human poly(A) polymerase (PAP), PAP gamma.

Poly(A) polymerase (PAP) is present in multiple forms in mammalian cells and tissues. Here we show that 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 hsPAPII, by the U1A protein. hsPAPgamma is restricted to the nucleus as revealed by in situ staining and by transfection experiments. Based on this and previous 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.     

Entamoeba histolytica: cloning and expression of the poly(A) polymerase EhPAP

In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability, and translation. Here we identified and cloned a gene codifying for a putative nuclear poly(A) polymerase (EhPAP) in Entamoeba histolytica. Protein sequence alignments with eukaryotic PAPs showed that EhPAP has the RNA-binding region and the PAP central domain with the catalytic nucleotidyl transferase domain described for other nuclear PAPs. Recombinant EhPAP expressed in bacteria was used to generate specific antibodies, which recognized two EhPAP isoforms of 60 and 63kDa in nuclear and cytoplasmic extracts by Western blot assays. RT-PCR assays showed that EhPap mRNA expression varies in multidrug-resistant trophozoites growing in different emetine concentrations. Moreover, EhPap mRNA expression is about 10- and 7-fold increased in G1 and S phase, respectively, through cell cycle progression. These results suggest the existence of a link between EhPAP expression and MDR and cell cycle regulation, respectively.     

Deregulation of Poly(A) Polymerase Interferes with Cell Growth

Vertebrate poly(A) polymerase (PAP) contains a catalytic domain and a C-terminal Ser-Thr-rich regulatory region. Consensus and nonconsensus cyclin-dependent kinase (cdk) sites are conserved in the Ser-Thr-rich region in vertebrate PAPs. PAP is phosphorylated by cdc2-cyclin B on these sites in vitro and in vivo and is inactivated by hyperphosphorylation in M-phase cells, when cdc2-cyclin B is active. In the experiments described here, we undertook a genetic approach in chicken DT40 cells to study the function of PAP phosphorylation. We found that PAP is highly conserved in chicken and is essential in DT40 cells. While cells could tolerate reduced levels of PAP, even modest overexpression of either wild-type PAP or a mutant PAP with two consensus cdk sites mutated (cdk⁻ PAP) was highly deleterious and at a minimum resulted in reduced growth rates. Importantly, cells that expressed cdk⁻ PAP had a significantly lower growth rate than did cells that expressed similar levels of wild-type PAP, which was reflected in increased accumulation of cells in the G₀-G₁ phase of the cell cycle. We propose that the lower growth rate is due to the failure of hyperphosphorylation and thus M-phase inactivation of cdk⁻ PAP.     

Characterization of Genes Encoding Poly(A) Polymerases in Plants: Evidence for Duplication and Functional Specialization

Background

Poly(A) polymerase is a key enzyme in the machinery that mediates mRNA 3′ end formation in eukaryotes. In plants, poly(A) polymerases are encoded by modest gene families. To better understand this multiplicity of genes, poly(A) polymerase-encoding genes from several other plants, as well as from Selaginella, Physcomitrella, and Chlamydomonas, were studied.

Methodology/Principal Findings

Using bioinformatics tools, poly(A) polymerase-encoding genes were identified in the genomes of eight species in the plant lineage. Whereas Chlamydomonas reinhardtii was found to possess a single poly(A) polymerase gene, other species possessed between two and six possible poly(A) polymerase genes. With the exception of four intron-lacking genes, all of the plant poly(A) polymerase genes (but not the C. reinhardtii gene) possessed almost identical intron positions within the poly(A) polymerase coding sequences, suggesting that all plant poly(A) polymerase genes derive from a single ancestral gene. The four Arabidopsis poly(A) polymerase genes were found to be essential, based on genetic analysis of T-DNA insertion mutants. GFP fusion proteins containing three of the four Arabidopsis poly(A) polymerases localized to the nucleus, while one such fusion protein was localized in the cytoplasm. The fact that this latter protein is largely pollen-specific suggests that it has important roles in male gametogenesis.

Conclusions/Significance

Our results indicate that poly(A) polymerase genes have expanded from a single ancestral gene by a series of duplication events during the evolution of higher plants, and that individual members have undergone sorts of functional specialization so as to render them essential for plant growth and development. Perhaps the most interesting of the plant poly(A) polymerases is a novel cytoplasmic poly(A) polymerase that is expressed in pollen in Arabidopsis; this is reminiscent of spermatocyte-specific cytoplasmic poly(A) polymerases in mammals.     

Control of poly(A) polymerase level is essential to cytoplasmic polyadenylation and early development in Drosophila

Poly(A) polymerase (PAP) has a role in two processes, polyadenylation of mRNA precursors in the nucleus and translational control of certain mRNAs by cytoplasmic elongation of their poly(A) tails, particularly during early development. It was found recently that at least three different PAP genes exist in mammals, encoding several PAP isoforms. The in vivo specificity of function of each PAP isoform currently is unknown. Here, we analyse PAP function in Drosophila: We show that a single PAP isoform exists in Drosophila that is encoded by the hiiragi gene. This single Drosophila PAP is active in specific polyadenylation in vitro and is involved in both nuclear and cytoplasmic polyadenylation in vivo. Therefore, the same PAP can be responsible for both processes. In addition, in vivo overexpression of PAP does not affect poly(A) tail length during nuclear polyadenylation, but leads to a dramatic elongation of poly(A) tails and a loss of specificity during cytoplasmic polyadenylation, resulting in embryonic lethality. This demonstrates that regulation of the PAP level is essential for controlled cytoplasmic polyadenylation and early development.     

Characterization of the E.coli poly(A) polymerase: nucleotide specificity, RNA-binding affinities and RNA structure dependence

Polyadenylation of RNA molecules in bacteria and chloroplasts has been implicated as part of the RNA degradation pathway. The polyadenylation reaction is performed in Escherichia coli mainly by the enzyme poly(A) polymerase I (PAP I). In order to understand the molecular mechanism of RNA polyadenylation in bacteria, we characterized the biochemical properties of this reaction in vitro using the purified enzyme. Unlike the PAP from yeast nucleus, which is specific for ATP, E.coli PAP I can use all four nucleotide triphosphates as substrates for addition of long ribohomopolymers to RNA. PAP I displays a high binding activity to poly(U), poly(C) and poly(A) ribohomopolymers, but not to poly(G). The 3′-ends of most of the mRNA molecules in bacteria are characterized by a stem–loop structure. We show here that in vitro PAP I activity is inhibited by a stem–loop structure. A tail of two to six nucleotides located 3′ to the stem–loop structure is sufficient to overcome this inhibition. These results suggest that the stem–loop structure located in most of the mRNA 3′-ends may function as an inhibitor of polyadenylation and degradation of the corresponding RNA molecule. However, RNA 3′-ends produced by endonucleolytic cleavage by RNase E in single-strand regions of mRNA molecules may serve as efficient substrates for polyadenylation that direct these molecules for rapid exonucleolytic degradation.     

Crystal structure of the RRM domain of poly(A)-specific ribonuclease reveals a novel m(7)G-cap-binding mode

Poly(A)-specific ribonuclease (PARN) is a processive 3′-exoribonuclease involved in the decay of eukaryotic mRNAs. Interestingly, PARN interacts not only with the 3′ end of the mRNA but also with its 5′ end as PARN contains an RRM domain that specifically binds both the poly(A) tail and the 7-methylguanosine (m⁷G) cap. The interaction of PARN with the 5′ cap of mRNAs stimulates the deadenylation activity and enhances the processivity of this reaction. We have determined the crystal structure of the PARN-RRM domain with a bound m⁷G triphosphate nucleotide, revealing a novel binding mode for the m⁷G cap. The structure of the m⁷G binding pocket is located outside of the canonical RNA-binding surface of the RRM domain and differs significantly from that of other m⁷G-cap-binding proteins. The crystal structure also shows a remarkable conformational flexibility of the RRM domain, leading to a perfect exchange of two α-helices with an adjacent protein molecule in the crystal lattice.     

Poly(A) polymerase and the regulation of cytoplasmic polyadenylation

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. Data presented here shows that 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. We discuss the hypothesis that CPSF is required for all polyadenylation reactions, but that 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 catalyzed polyadenylation and stimulated translation approximately 40-fold; stimulation was exclusively cis-acting, but was independent of a CPE and AAUAAA. Both polyadenylation and translational stimulation required PAPs catalytic core, but did 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.     

Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro.

We have partially purified a poly(A) polymerase (PAP) from HeLa cell nuclear extract which is involved in the 3'-end formation of polyadenylated mRNA. PAP had a molecular weight of approximately 50 to 60 kilodaltons. In the presence of manganese ions, PAP was able to polyadenylate RNA nonspecifically. However, in the presence of magnesium ions PAP required the addition of a cleavage and polyadenylation factor to specifically polyadenylate pre-mRNAs that contain an intact AAUAAA sequence and end at the poly(A) addition site (precleaved RNA substrates). The purified fraction containing PAP was also required in combination with a cleavage and polyadenylation factor and a cleavage factor for the correct cleavage at the poly(A) site of pre-mRNAs. Since the two activities of the PAP fractions, PAP and cleavage activity, could not be separated by extensive purification, we concluded that the two activities are contained in a single component, a PAP that is also required for the specific cleavage preceding the polyadenylation of pre-mRNA.     

Poly(A) polymerase specifically implicated in the mechanism of chemotherapeutic drug action during cell apoptosis

It has recently been established that most anticancer drugs act through the mechanism of apoptosis. It has also been clinically confirmed that drug combinations are more effective than single drugs and various chemotherapeutic strategies have therefore been developed. The experiments described here concern the induction of apoptosis with dimethylsulfoxide (DMSO), a substance with multiple activity especially as an inducer of differentiation, and interferon (IFN), a cytokine well known for its antiviral and antineoplastic effects; they are used alone or in combination. Apoptosis may be regulated at all levels of gene expression including the addition of the poly(A) tail to the 3' end of mRNAs. Poly(A) polymerase (PAP) [EC.2.7.7.19], the enzyme that catalyzes the addition of the poly(A) tail to mRNAs, changes in the process of development, differentiation, transformation and apoptosis. In the present study the induction of HeLa cells to apoptosis (65%) with a DMSO/rIFN-alpha combination resulted in pronounced PAP dephosphorylation and activity reduction. HeLa cells induced to apoptosis (35%) with DMSO gave lower levels of PAP dephosphorylation and reduction of activity and cells induced to apoptosis (18%) with rIFN-alpha gave only limited PAP dephosphorylation and reduction of activity. The implications of these observations for chemotherapeutic drug action at the level of mRNA polyadenylation point to the possible use of PAP as a biological marker for the evaluation of this action.     

Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase

In mammals, polyadenylation of mRNA precursors (pre-mRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF). CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U-rich upstream sequence elements on the pre-mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal. Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF. hFip1 interacts with PAP and has an arginine-rich RNA-binding motif that preferentially binds to U-rich sequence elements on the pre-mRNA. Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U-rich element-dependent manner. hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA. These results show that hFip1 significantly contributes to CPSF-mediated stimulation of PAP activity.     

Poly(A) polymerase contains multiple functional domains.

Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS.     

Poly(A) polymerase (PAP) diversity in gene expression – Star-PAP vs canonical PAP

Almost all eukaryotic mRNAs acquire a poly(A) tail at the 3′-end by a concerted RNA processing event: cleavage and polyadenylation. The canonical PAP, PAPα, was considered the only nuclear PAP involved in general polyadenylation of mRNAs. A phosphoinositide-modulated nuclear PAP, Star-PAP, was then reported to regulate a select set of mRNAs in the cell. In addition, several non-canonical PAPs have been identified with diverse cellular functions. Further, canonical PAP itself exists in multiple isoforms thus illustrating the diversity of PAPs. In this review, we compare two nuclear PAPs, Star-PAP and PAPα with a general overview of PAP diversity in the cell. Emerging evidence suggests distinct niches of target pre-mRNAs for the two PAPs and that modulation of these PAPs regulates distinct cellular functions.     

A nuclear 3'-5' exonuclease involved in mRNA degradation interacts with Poly(A) polymerase and the hnRNA protein Npl3p

Inactivation of poly(A) polymerase (encoded by PAP1) in Saccharomyces cerevisiae cells carrying the temperature-sensitive, lethal pap1-1 mutation results in reduced levels of poly(A)(+) mRNAs. Genetic selection for suppressors of pap1-1 yielded two recessive, cold-sensitive alleles of the gene RRP6. These suppressors, rrp6-1 and rrp6-2, as well as a deletion of RRP6, allow growth of pap1-1 strains at high temperature and partially restore the levels of poly(A)(+) mRNA in a manner distinct from the cytoplasmic mRNA turnover pathway and without slowing a rate-limiting step in mRNA decay. Subcellular localization of an Rrp6p-green fluorescent protein fusion shows that the enzyme residues in the nucleus. Phylogenetic analysis and the nature of the rrp6-1 mutation suggest the existence of a highly conserved 3'-5' exonuclease core domain within Rrp6p. As predicted, recombinant Rrp6p catalyzes the hydrolysis of a synthetic radiolabeled RNA in a manner consistent with a 3'-5' exonucleolytic mechanism. Genetic and biochemical experiments indicate that Rrp6p interacts with poly(A) polymerase and with Npl3p, a poly(A)(+) mRNA binding protein implicated in pre-mRNA processing and mRNA nuclear export. These findings suggest that Rrp6p may interact with the mRNA polyadenylation system and thereby play a role in a nuclear pathway for the degradation of aberrantly processed precursor mRNAs.     

ERK is a novel regulatory kinase for poly(A) polymerase

Poly(A) polymerase (PAP), which adds poly(A) tails to the 3′ end of mRNA, can be phosphorylated at several sites in the C-terminal domain. Phosphorylation often mediates regulation by extracellular stimuli, suggesting PAP may be regulated by such stimuli. In this study, we found that phosphorylation of PAP was increased upon growth stimulation and that the mitogen-activated protein kinase ERK was responsible for the increase in phosphorylation. We identified serine 537 of PAP as a unique phosphorylation site by ERK. PAP phosphorylation of serine 537 by ERK increased its nonspecific polyadenylation activity in vitro. This PAP activity was also activated by stimulation of ERK with phorbol-12-myristate-13-acetate in vivo. These data suggest that ERK is a novel regulatory kinase for PAP and further, that PAP activity could be regulated by extracellular stimuli through an ERK-dependent signaling pathway(s).     

Poly(A) dependent translation in rabbit reticulocyte lysate

Poly(A) tail has been known to enhance mRNA translation in eukaryotic cells. However, the effect of poly(A) tail in vitro is rather small. Rabbit reticulocyte lysate (RRL) is widely used for studying translation in vitro. Translation in RRL is typically performed in nuclease-treated lysate in which most of the endogenous mRNA have been removed. In this condition, the difference in the translational efficiency between poly(A)⁺ and poly(A)⁻ mRNAs is about two-fold. We studied the effect of poly(A) tail on luciferase mRNA translation in nuclease uncreated reticulocyte lysate, in which endogenous globin mRNAs were actively translated. In the case of capped mRNAs. stimulation of translation by poly(A) addition was about 1.5- to 1.6-fold and the effect of the poly(A) length was small. However, in the case of uncapped mRNAs, the addition of poly(A) tail increased luciferase expression over 10-fold. The effect of the poly(A) tail was dependent on its length. The difference in the translational efficiency was not due to the change of mRNA stability. These data indicate that RRL has the potential to translate mRNA in a poly(A) dependent manner.     

Poly(A) polymerase in Dormant Embryos of Triticum durum

Triticum durum‘Cappelli’ has a ‘relative’dormancy which can be broken by dry after-ripening at room temperature.The breakage of dormancy in the embryos of T. durum , is accompaniedby a decline in content and a different degree of synthesisof poly(A)⁺RNA. This work studies the activity of poly(A) polymerase(E.C. 2.7.7.19), the enzyme which permits polyadenylation. Anincrease in the activity of this enzyme in parallel with theenhanced rate of germination is revealed. Since poly(A) polymeraseactivity is the same in dormant and non-dormant dry embryos,it seems that the activity of the enzyme is not involved inthe breakage of dormancy. The use of cycloheximide and cordycepinshows the presence of enzymes with different origins: a storedenzyme and one bound to a long lived mRNA, present in dormantand non-dormant embryos, plus an enzyme bound to newly synthesizedmRNA which is mainly active in non-dormant embryos. Since dormancycould be the result of an interaction between hormones, thiswork analyses the effects of GA₃and ABA on poly(A) polymerase.GA₃enhanced poly(A) polymerase activity only in dormant embryoswhile ABA inhibited this activity only in non-dormant embryos.Cycloheximide applied to excised wheat embryos represses thestimulatory and inhibitory effects of GA₃and ABA, respectively.The hormone action on poly(A) polymerase activity is thus dependenton de novo protein synthesis. Results using cordycepin suggestthe presence of a stored mRNA for poly(A) polymerase, togetherwith hormonal regulation of enzyme activity at a translationallevel. Copyright 1999 Annals of Botany Company Triticum durum , wheat, dormancy breakage, poly(A) polymerase, GA₃, ABA, germination.