Inhibition of mRNA deadenylation by the nuclear cap binding complex (CBC)

Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3'-exoribonuclease. Here we have investigated how the cap binding complex (CBC) affects human PARN activity. We showed that CBC, via its 80-kDa subunit (CBP80), inhibited PARN, suggesting that CBC can regulate mRNA deadenylation. The CBC-mediated inhibition of PARN was cap-independent, and in keeping with this, the CBP80 subunit alone inhibited PARN. Our data suggested a new function for CBC, identified CBC as a potential regulator of PARN, and emphasized the importance of communication between the two extreme ends of the mRNA as a key strategy to regulate mRNA degradation. Based on our data, we have proposed a model for CBC-mediated regulation of PARN, which relies on an interaction between CBP80 and PARN. Association of CBC with PARN might have importance in the regulated recruitment of PARN to the nonsense-mediated decay pathway during the pioneer round of translation.     

Overexpression of poly(A) binding protein prevents maturation-specific deadenylation and translational inactivation in Xenopus oocytes.

The translational regulation of maternal mRNAs is the primary mechanism by which stage-specific programs of protein synthesis are executed during early development. Translation of a variety of maternal mRNAs requires either the maintenance or cytoplasmic elongation of a 3' poly(A) tail. Conversely, deadenylation results in translational inactivation. Although its precise function remains to be elucidated, the highly conserved poly(A) binding protein I (PABP) mediates poly(A)-dependent events in translation initiation and mRNA stability. Xenopus oocytes contain less than one PABP per poly(A) binding site suggesting that the translation of maternal mRNAs could be either limited by or independent of PABP. In this report, we have analyzed the effects of overexpressing PABP on the regulation of mRNAs during Xenopus oocyte maturation. Increased levels of PABP prevent the maturation-specific deadenylation and translational inactivation of maternal mRNAS that lack cytoplasmic polyadenylation elements. Overexpression of PABP does not interfere with maturation-specific polyadenylation, but reduces the recruitment of some mRNAs onto polysomes. Deletion of the C-terminal basic region and a single RNP motif from PABP significantly reduces both its binding to polyadenylated RNA in vivo and its ability to prevent deadenylation. In contrast to a yeast PABP-dependent poly(A) nuclease, PABP inhibits Xenopus oocyte deadenylase in vitro. These results indicate that maturation-specific deadenylation in Xenopus oocytes is facilitated by a low level of PABP consistent with a primary function for PABP to confer poly(A) stability.     

Characterization of deadenylation in trypanosome extracts and its inhibition by poly(A)-binding protein Pab1p

The stability of mRNAs is an important point in the regulation of gene expression in eukaryotes. The mRNA turnover pathways have been identified in yeast and mammals. However, mRNA turnover pathways in trypanosomes have not been widely studied. Deadenylation is the first step in the major mRNA turnover pathways of yeast and mammals. To better understand mRNA degradation processes in these organisms, we have developed an in vitro mRNA turnover system that is functional for deadenylation. In this system, addition of poly(A) homopolymer activates the deadenylation of poly(A) tails. The trypanosomal deadenylase activity is a 3'-->5' exonuclease specific for adenylate residues, generates 5'-AMP as a product, is magnesium dependent, and is inhibited by neomycin B sulfate. These characteristics suggest similarity with other eukaryotic deadenylases. Furthermore, this activity is cap independent, indicating a potential difference between the trypanosomal activity and PARN, but suggesting similarity to Ccr4p/Pop2p activities. Extracts immunodepleted of Pab1p required the addition of poly(A) competition to activate deadenylation. Trypanosomal Pab1p functions as an inhibitor of the activity under in vitro conditions. Pab1p appears to be one of several mRNA stability proteins in trypanosomal extracts.     

Suppression of cellular transformation by poly (A) binding protein interacting protein 2 (Paip2)

Controlling translation is crucial for the homeostasis of a cell. Its deregulation can facilitate the development and progression of many diseases including cancer. Poly (A) binding protein interacting protein 2 (Paip2) inhibits efficient initiation of translation by impairing formation of the necessary closed loop of mRNA. The over production of Paip2 in the presence of a constitutively active form of hRas(V12) can reduce colony formation in a semi-solid matrix and focus formation on a cell monolayer. The ability of Paip2 to bind to Pabp is required to suppress the transformed phenotype mediated by hRas(V12). These observations indicate that Paip2 is able to function as a tumor suppressor.     

Inhibition of poly(A) polymerase by aminoglycosides

Aminoglycosides are potent inhibitors of bacterial growth and are used clinically as antibiotics. However, their usage has declined in recent years due to the emergence of resistance and severe toxic side effects. Here we show that human poly(A) polymerase gamma (PAPgamma) is inhibited by aminoglycosides. The inhibition was pH dependent and could be released by Mg(II) ions in a competitive manner suggesting that electrostatic interactions are important for inhibition and that the binding sites for aminoglycosides overlap with Mg(II) ion binding sites. Kinetic analysis revealed that aminoglycosides of the neomycin and kanamycin families behaved as mixed non-competitive inhibitors for the PAPgamma substrates oligoA15 and ATP. Interestingly, sisomicin and 5-epi-sisomycin showed a competitive mechanism of inhibition for the oligoA15 whereas they inhibited the ATP substrate mixed non-competitive. This implies that different aminoglycosides bind in different ways to a common binding pocket and suggests that the binding sites for related aminoglycosides are not overlapping even if they may share molecular determinants. Our study emphasizes the possibility that aminoglycoside toxicity could be due to interference with housekeeping enzymes involved in breaking and forming phosphodiester bonds.     

Inhibition of poly(ADP-ribose)polymerase binding to DNA by thymidine dimer

The ability of poly(ADP-ribose)polymerase to bind damaged DNA was assessed by electrophoretic mobility shift assay. DNA binding domain of poly(ADP-ribose)polymerase (PARPDBD) binds to synthetic deoxyribonucleotide duplex 10-mer. However, the synthetic deoxyribonucleotide duplex containing cys-syn thymidine dimer which produces the unwinding of DNA helix structure lost its affinity to PARPDBD. It was shown that the binding of PARPDBD to the synthetic deoxyribonucleotide duplex was not affected by O6-Me-dG which causes only minor distortion of DNA helix structure. This study suggests that the stabilized DNA helix structure is important for poly(ADP-ribose)polymerase binding to DNA breaks, which are known to stimulate catalytic activity of poly(ADP-ribose)polymerase.     

Inhibition of poly(A) polymerase by rifamycin derivatives

The effect of several rifamycin derivatives on poly(A) synthesis in vitro was tested using purified rat liver mitochondrial poly(A) polymerase assayed with an exogenous primer. When used at a concentration of 300 μg/ml, derivatives AF/013, PR/19, AF/AETP, M/88 and AF/ABDP completely inhibited activity corresponding to 50 μg of enzyme protein. Under similar conditions, derivatives DMAO and AF/MO failed to inhibit enzyme activity. Studies with PR/19 showed that the drug interacted directly with the enzyme molecule and did not affect the enzyme-primer complex formation. The inhibition by the drug could be reversed by increasing the substrate (ATP) concentration. It is concluded that some rifamycin derivatives can specifically inhibit template-independent nucleotide chain elongation reactions.     

Identification of a novel Xenopus laevis poly (A) binding protein

Poly (A) binding proteins are intimately implicated in controlling a number of events in mRNA metabolism from nuclear polyadenylation to cytoplasmic translation and stability. The known poly(A) binding proteins can be divided into three distinct structural groups (prototypes PABP1, PABPN1/PABP2 and Nab2p) and two functional families, showing that similar functions can be accomplished by differing structural units. This has prompted us to perform a screen for novel poly(A) binding proteins using Xenopus laevis. A novel poly(A) binding protein of 32 kDa (p32) was identified. Sequence analysis showed that p32 has about 50% identity to the known nuclear poly(A) binding proteins (PABPN1) but is more closely related to a group of mammalian proteins of unknown function. The expression of Xenopus laevis ePABP2 is restricted to early embryos. Accordingly, we propose that p32 is the founder member of a novel class of poly(A) binding proteins named ePABP2.     

Stimulation of poly(A) polymerase through a direct interaction with the nuclear poly(A) binding protein allosterically regulated by RNA

During polyadenylation of mRNA precursors in metazoan cells, poly(A) polymerase is stimulated by the nuclear poly(A) binding protein PABPN1. We report that stimulation depends on binding of PABPN1 to the substrate RNA directly adjacent to poly(A) polymerase and results in an approximately 80-fold increase in the apparent affinity of poly(A) polymerase for RNA without significant effect on catalytic efficiency. PABPN1 associates directly with poly(A) polymerase either upon allosteric activation by oligo(A) or, in the absence of RNA, upon deletion of its N-terminal domain. The N-terminal domain of PABPN1 may function to inhibit undesirable interactions of the protein; the inhibition is relieved upon RNA binding. Tethering of poly(A) polymerase is mediated largely by the C-terminal domain of PABPN1 and is necessary but not sufficient for stimulation of the enzyme; an additional interaction dependent on a coiled-coil structure located within the N-terminal domain of PABPN1 is required for a productive interaction.     

Translational control by the poly(A) binding protein: A check for mRNA integrity

The eukaryotic mRNA 3′ poly(A) tail and the 5′ cap cooperate to synergistically enhance translation. This interaction is mediated by a ribonucleoprotein network that contains, at a minimum, the poly(A) binding protein (PABP), the cap-binding protein eIF4E, and a scaffolding protein, eIF4G. eIF4G, in turn, contains binding sites for eIF4A and eIF3, a 40S ribosome-associated initiation factor. The combined cooperative interactions within this “closed loop” mRNA among other effects enhance the affinity of eIF4E for the 5′ cap, by lowering its dissociation rate and, ultimately, facilitate the formation of 48S and 80S ribosome initiation complexes. The PABP-poly(A) interaction also stimulates initiation driven by picornavirus’ internal ribosomal entry sites (IRESs), a process that requires eIF4G but not eIF4E. PABP, therefore, should be considered a canonical initiation factor, integral to the formation of the initiation complex. Poly(A)-mediated translation is subjected to regulation by the PABP-interacting proteins Paip1 and Paip2. Paip1 acts as a translational enhancer. In contrast, Paip2 strongly inhibits translation by promoting dissociation of PABP from poly(A) and by competing with eIF4G for binding to PABP. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 4, pp. 684–693. The article is published in the original.     

PAB1 self-association precludes its binding to poly(A), thereby accelerating CCR4 deadenylation in vivo

The mRNA deadenylation process, catalyzed by the CCR4 deadenylase, is known to be the major factor controlling mRNA decay rates in Saccharomyces cerevisiae. We have identified the proline-rich region and RRM1 domains of poly(A) binding protein (PAB1) as necessary for CCR4 deadenylation. Deletion of either of these regions but not other regions of PAB1 significantly reduced PAB1-PAB1 protein interactions, suggesting that PAB1 oligomerization is a required step for deadenylation. Moreover, defects in these two regions inhibited the formation of a novel, circular monomeric PAB1 species that forms in the absence of poly(A). Removal of the PAB1 RRM3 domain, which promoted PAB1 oligomerization and circularization, correspondingly accelerated CCR4 deadenylation. Circular PAB1 was unable to bind poly(A), and PAB1 multimers were severely deficient or unable to bind poly(A), implicating the PAB1 RNA binding surface as critical in making contacts that allow PAB1 self-association. These results support the model that the control of CCR4 deadenylation in vivo occurs in part through the removal of PAB1 from the poly(A) tail following its self-association into multimers and/or a circular species. Known alterations in the P domains of different PAB proteins and factors and conditions that affect PAB1 self-association would, therefore, be expected to be critical to controlling mRNA turnover in the cell.     

Paip1 interacts with poly(A) binding protein through two independent binding motifs

The 3' poly(A) tail of eukaryotic mRNAs plays an important role in the regulation of translation. The poly(A) binding protein (PABP) interacts with eukaryotic initiation factor 4G (eIF4G), a component of the eIF4F complex, which binds to the 5' cap structure. The PABP-eIF4G interaction brings about the circularization of the mRNA by joining its 5' and 3' termini, thereby stimulating mRNA translation. The activity of PABP is regulated by two interacting proteins, Paip1 and Paip2. To study the mechanism of the Paip1-PABP interaction, far-Western, glutathione S-transferase pull-down, and surface plasmon resonance experiments were performed. Paip1 contains two binding sites for PABP, PAM1 and PAM2 (for PABP-interacting motifs 1 and 2). PAM2 consists of a 15-amino-acid stretch residing in the N terminus, and PAM1 encompasses a larger C-terminal acidic-amino-acid-rich region. PABP also contains two Paip1 binding sites, one located in RNA recognition motifs 1 and 2 and the other located in the C-terminal domain. Paip1 binds to PABP with a 1:1 stoichiometry and an apparent K(d) of 1.9 nM.     

The RNA binding domains of the nuclear poly(A)-binding protein

The nuclear poly(A)-binding protein (PABPN1) is involved in the synthesis of the mRNA poly(A) tails in most eukaryotes. We report that the protein contains two RNA binding domains, a ribonucleoprotein-type RNA binding domain (RNP domain) located approximately in the middle of the protein sequence and an arginine-rich C-terminal domain. The C-terminal domain also promotes self-association of PABPN1 and moderately cooperative binding to RNA. Whereas the isolated RNP domain binds specifically to poly(A), the isolated C-terminal domain binds non-specifically to RNA and other polyanions. Despite this nonspecific RNA binding by the C-terminal domain, selection experiments show that adenosine residues throughout the entire minimal binding site of approximately 11 nucleotides are recognized specifically. UV-induced cross-links with oligo(A) carrying photoactivatable nucleotides at different positions all map to the RNP domain, suggesting that most or all of the base-specific contacts are made by the RNP domain, whereas the C-terminal domain may contribute nonspecific contacts, conceivably to the same nucleotides. Asymmetric dimethylation of 13 arginine residues in the C-terminal domain has no detectable influence on the interaction of the protein with RNA. The N-terminal domain of PABPN1 is not required for RNA binding but is essential for the stimulation of poly(A) polymerase.     

Equilibrium studies on the association of the nuclear poly(A) binding protein with poly(A) of different lengths

The nuclear poly(A) binding protein (PABPN1) binds the growing poly(A) tail during pre-mRNA 3'-end processing, stimulating its elongation and controlling its final length. Here we report binding studies of PABPN1 to poly(A) in solution. Quantitative fluorescence titration was used to determine the stoichiometry, intrinsic affinity, and cooperativity of binding to a series of size-fractionated poly(A). The intrinsic association constant K(i) was about 2 x 10(6) M(-1) for oligo(A) and all size classes of poly(A). The binding of PABPN1 to poly(A) was enhanced by protein-protein interactions which were, however, weak (cooperativity parameter omega < 50). No significant change of cooperativity could be detected with increasing polynucleotide length in the range of 140-450 nucleotides. An average binding site size n of 11-14 was found for all poly(A) lengths, which is close to the minimal site size m found for binding to oligo(A). The data are discussed with respect to the previous observation of two different forms of the poly(A)-PABPN1 complex.     

Expression of murine poly(A) binding protein II gene in testis

Insect defensin refers to a group of antibacterial peptides derived from a variety of insect species as well as from scorpion and possessing a three-dimensional structure highly similar to that of scorpion toxins. A full-length cDNA encoding an insect defensin-like peptide was isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch. The precursor, the overall organization of which is similar to that of insect defensins, consists of 61 amino acid residues with a putative signal peptide of 15 residues, a propeptide of 7 residues, and a mature peptide of 39 residues (named BmTXKS2). The positions of six cysteines and a conserved glycine in mature BmTXKS2 are the same as those in LqDef, the first defensin found in scorpions, which suggests these peptides should present a similar cysteine-stabilized alphabetamotif. Phylogenetic analysis further shows that the structure of BmTXKS2 is closer to that of ancient defensins (e.g., LqDef and AaDef, two insect defensins present in the scorpion hemolymph) than to scorpion toxins.     

Growth factor regulated expression of poly(A) binding protein messenger RNA

A 72,000 mol wt protein designated PABP binds to the poly(A)+ track of messenger RNAs with high affinity and has been suggested to play an important role in mRNA metabolism in eucaryotic cells. We have employed a human PABP cDNA probe to study the expression of this gene at the mRNA level in BALB/c3T3 mouse cells under different growth conditions and in exponentially growing HeLa cells throughout the cell division cycle. We describe experiments which establish that in BALB/c3T3 cells the expression of this gene is growth factor regulated. Moreover, the gene behaves like a primary response gene in that its induction in quiescent cells does not require the prior synthesis of other growth factor-regulated proteins. In exponentially growing HeLa cells PABP mRNA is expressed throughout the cell division cycle indicating that the expression of this gene is not limited to a specific phase of the cell cycle.     

Inhibition of DNA binding by the phosphorylation of poly ADP-ribose polymerase protein catalysed by protein kinase C.

Purified type II (beta) and type III (alpha) protein kinase C phosphorylates highly purified polyADP-ribose polymerase in vitro whereby 2 mols of phosphate are transferred from ATP to serine and threonine residues present in the 36 and 56 kDa polypeptide domains of the polymerase protein. Calf thymus DNA was a non-competitive inhibitor of the protein kinase C catalyzed phosphorylation of polyADP-ribose polymerase. Coincidental with the phosphorylation of the protein the polymerase activity and DNA binding capacity of polyADP-ribose polymerase were inhibited. These in vitro findings may have possible cell biological significance in cellular signal transduction.     

Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP)

Picornavirus infectivity is dependent on the RNA poly(A) tail, which binds the poly(A) binding protein (PABP). PABP was reported to stimulate viral translation and RNA synthesis. Here, we studied encephalomyocarditis virus (EMCV) and poliovirus (PV) genome expression in Krebs-2 and HeLa cell-free extracts that were drastically depleted of PABP (96%-99%). Although PABP depletion markedly diminished EMCV and PV internal ribosome entry site (IRES)-mediated translation of a polyadenylated luciferase mRNA, it displayed either no (EMCV) or slight (PV) deleterious effect on the translation of the full-length viral RNAs. Moreover, PABP-depleted extracts were fully competent in supporting EMCV and PV RNA replication and virus assembly. In contrast, removing the poly(A) tail from EMCV RNA dramatically reduced RNA synthesis and virus yields in cell-free reactions. The advantage conferred by the poly(A) tail to EMCV synthesis was more pronounced in untreated than in nuclease-treated extract, indicating that endogenous cellular mRNAs compete with the viral RNA for a component(s) of the RNA replication machinery. These results suggest that the poly(A) tail functions in picornavirus replication largely independent of PABP.