Poly(A) binding protein (PABP) homeostasis is mediated by the stability of its inhibitor, Paip2

The poly(A)-binding protein (PABP) is a unique translation initiation factor in that it binds to the mRNA 3' poly(A) tail and stimulates recruitment of the ribosome to the mRNA at the 5' end. PABP activity is tightly controlled by the PABP-interacting protein 2 (Paip2), which inhibits translation by displacing PABP from the mRNA. Here, we describe a close interplay between PABP and Paip2 protein levels in the cell. We demonstrate a mechanism for this co-regulation that involves an E3 ubiquitin ligase, EDD, which targets Paip2 for degradation. PABP depletion by RNA interference (RNAi) causes co-depletion of Paip2 protein without affecting Paip2 mRNA levels. Upon PABP knockdown, Paip2 interacts with EDD, which leads to Paip2 ubiquitination. Supporting a critical role for EDD in Paip2 degradation, knockdown of EDD expression by siRNA leads to an increase in Paip2 protein stability. Thus, we demonstrate that the turnover of Paip2 in the cell is mediated by EDD and is regulated by PABP. This mechanism serves as a homeostatic feedback to control the activity of PABP in cells.     

The poly(A)-binding protein and an mRNA stability protein jointly regulate an endoribonuclease activity

We previously identified a sequence-specific erythroid cell-enriched endoribonuclease (ErEN) activity involved in the turnover of the stable alpha-globin mRNA. We now demonstrate that ErEN activity is regulated by the poly(A) tail. The unadenylated alpha-globin 3' untranslated region (3'UTR) was an efficient substrate for ErEN cleavage, while the polyadenylated 3'UTR was inefficiently cleaved in an in vitro decay assay. The influence of the poly(A) tail was mediated through the poly(A)-binding protein (PABP) bound to the poly(A) tail, which can inhibit ErEN activity. ErEN cleavage of an adenylated alpha-globin 3'UTR was accentuated upon depletion of PABP from the cytosolic extract, while addition of recombinant PABP reestablished the inhibition of endoribonuclease cleavage. PABP inhibited ErEN activity indirectly through an interaction with the alphaCP mRNA stability protein. Sequestration of alphaCP resulted in an increase of ErEN cleavage activity, regardless of the polyadenylation state of the RNA. Using electrophoretic mobility shift assays, PABP was shown to enhance the binding efficiency of alphaCP to the alpha-globin 3'UTR, which in turn protected the ErEN target sequence. Conversely, the binding of PABP to the poly(A) tail was also augmented by alphaCP, implying that a stable higher-order structural network is involved in stabilization of the alpha-globin mRNA. Upon deadenylation, the interaction of PABP with alphaCP would be disrupted, rendering the alpha-globin 3'UTR more susceptible to endoribonuclease cleavage. The data demonstrated a specific role for PABP in protecting the body of an mRNA in addition to demonstrating PABP's well-characterized effect of stabilizing the poly(A) tail.     

iPABP, an inducible poly(A)-binding protein detected in activated human T cells.

The poly(A)-binding protein (PABP) binds to the poly(A) tail present at the 3' ends of most eukaryotic mRNAs. PABP is thought to play a role in both translation and mRNA stability. Here we describe the molecular cloning and characterization of an inducible PABP, iPABP, from a cDNA library prepared from activated T cells. iPABP shows 79% sequence identity to PABP at the amino acid level. The RNA binding domains of iPABP and PABP are nearly identical, while their C termini are more divergent. Like PABP, iPABP is primarily localized to the cytoplasm. iPABP is expressed at low levels in resting normal human T cells; following T-cell activation, however, iPABP mRNA levels are rapidly up-regulated. In contrast, PABP is constitutively expressed in both resting and activated T cells. iPABP mRNA was also expressed at much higher levels than PABP mRNA in heart and skeletal muscle tissue. These data suggest that the regulation of cytoplasmic poly(A)-binding activity is more complex than previously believed. In most tissues, poly(A)-binding activity is likely to be the result of the combined effects of constitutively expressed PABP and iPABP, whose expression is subject to more complex regulation.     

An essential cytoplasmic function for the nuclear poly(A) binding protein, PABP2, in poly(A) tail length control and early development in Drosophila

Translational control of maternal mRNA through regulation of poly(A) tail length is crucial during early development. The nuclear poly(A) binding protein, PABP2, was identified biochemically from its role in nuclear polyadenylation. Here, we analyze the in vivo function of PABP2 in Drosophila. PABP2 is required in vivo for polyadenylation, and Pabp2 function, including poly(A) polymerase stimulation, is essential for viability. We also demonstrate an unanticipated cytoplasmic function for PABP2 during early development. In contrast to its role in nuclear polyadenylation, cytoplasmic PABP2 acts to shorten the poly(A) tails of specific mRNAs. PABP2, together with the deadenylase CCR4, regulates the poly(A) tails of oskar and cyclin B mRNAs, both of which are also controlled by cytoplasmic polyadenylation. Both Cyclin B protein levels and embryonic development depend upon this regulation. These results identify a regulator of maternal mRNA poly(A) tail length and highlight the importance of this mode of translational control.     

A complex containing the CCR4 and CAF1 proteins is involved in mRNA deadenylation in Drosophila

The CCR4-NOT complex is the major enzyme catalyzing mRNA deadenylation in Saccharomyces cerevisiae. We have identified homologs for almost all subunits of this complex in the Drosophila genome. Biochemical fractionation showed that the two likely catalytic subunits, CCR4 and CAF1, were associated with each other and with a poly(A)-specific 3' exonuclease activity. In Drosophila, the CCR4 and CAF1 proteins were ubiquitously expressed and present in cytoplasmic foci. Individual knock-down of several potential subunits of the Drosophila CCR4-NOT complex by RNAi in tissue culture cells led to a lengthening of bulk mRNA poly(A) tails. Knock-down of two individual subunits also interfered with the rapid deadenylation of Hsp70 mRNA during recovery from heat shock. Similarly, ccr4 mutant flies had elongated bulk poly(A) and a defect in Hsp70 mRNA deadenylation. A minor increase in bulk poly(A) tail length was also observed in Rga mutant flies, which are affected in the NOT2 subunit. The data show that the CCR4-NOT complex is conserved in Drosophila melanogaster and plays a role in general and regulated mRNA deadenylation.     

Regulation of the nuclear poly(A)-binding protein by arginine methylation in fission yeast

Two structurally different poly(A)-binding proteins (PABP) bind the poly(A) tract of mRNAs in most mammalian cells: PABPC in the cytoplasm and PABP2/PABPN1 in the nucleus. Whereas yeast orthologs of the cytoplasmic PABP are characterized, a gene product homologous to mammalian PABP2 has not been identified in yeast. We report here the identification of a homolog of PABP2 as an arginine methyltransferase 1 (RMT1)-associated protein in fission yeast. The product of the Schizosaccharomyces pombe pab2 gene encodes a nonessential nuclear protein and demonstrates specific poly(A) binding in vitro. Consistent with a functional role in poly(A) tail metabolism, mRNAs from pab2-null cells displayed hyperadenylated 3'-ends. We also show that arginine residues within the C-terminal arginine-rich domain of Pab2 are modified by RMT1-dependent methylation. Whereas the arginine methylated and unmethylated forms of Pab2 behaved similarly in terms of subcellular localization, poly(A) binding, and poly(A) tail length control; Pab2 oligomerization levels were markedly increased when Pab2 was not methylated. Significantly, Pab2 overexpression reduced growth rate, and this growth inhibitory effect was exacerbated in rmt1-null cells. Our results indicate that the main cellular function of Pab2 is in poly(A) tail length control and support a biological role for arginine methylation in the regulation of Pab2 oligomerization.     

Identification of a human cytoplasmic poly(A) nuclease complex stimulated by poly(A)-binding protein

The poly(A) tail shortening in mRNA, called deadenylation, is the first rate-limiting step in eukaryotic mRNA turnover, and the polyadenylate-binding protein (PABP) appears to be involved in the regulation of this step. However, the precise role of PABP remains largely unknown in higher eukaryotes. Here we identified and characterized a human PABP-dependent poly(A) nuclease (hPAN) complex consisting of catalytic hPan2 and regulatory hPan3 subunits. hPan2 has intrinsically a 3' to 5' exoribonuclease activity and requires Mg2+ for the enzyme activity. On the other hand, hPan3 interacts with PABP to simulate hPan2 nuclease activity. Interestingly, the hPAN nuclease complex has a higher substrate specificity to poly(A) RNA upon its association with PABP. Consistent with the roles of hPan2 and hPan3 in mRNA decay, the two subunits exhibit cytoplasmic co-localization. Thus, the human PAN complex is a poly(A)-specific exoribonuclease that is stimulated by PABP in the cytoplasm.     

Analysis of an essential requirement for the poly(A) binding protein function using cross-species complementation

Poly(A) binding protein (PABP) is an essential, well-conserved, multifunctional protein involved in translational initiation, mRNA biogenesis, and degradation [1--5]. We have used a cross-species complementation approach to address the nature of the essential requirement for PABP in yeast. The expression of Pab3p, a member of the Arabidopsis thaliana PABP multigene family, rescues the lethal phenotype associated with the loss of the yeast Pab1p. However, Pab3p neither protects the mRNA 5' cap from premature removal, nor does it support poly(A)-dependent translational initiation or the synergistic enhancement of translation by the poly(A) tail and 5' cap in yeast. However, Pab3p corrects the temporal lag prior to the entry of the mRNA into the degradation pathway characteristic of pab1 Delta yeast strains. Furthermore, this lag correction by Pab3p requires Pan3p, a subunit of poly(A) nuclease, an enzyme involved in the mRNA 3'-end processing. Importantly, the substitution of Pab3p for the yeast Pab1p is synthetically lethal with the PAN3 gene deletion. These results show that the function of PABP in mRNA biogenesis alone could be sufficient to support cell viability in yeast.     

mRNA with a <20-nt poly(A) tail imparted by the poly(A)-limiting element is translated as efficiently in vivo as long poly(A) mRNA

The poly(A)-limiting element (PLE) is a conserved sequence that restricts the length of the poly(A) tail to <20 nt. This study compared the translation of PLE-containing short poly(A) mRNA expressed in cells with translation in vitro of mRNAs with varying length poly(A) tails. In transfected cells, PLE-containing mRNA had a <20-nt poly(A) and accumulated to a level 20% higher than a matching control without a PLE. It was translated as well as the matching control mRNA with long poly(A) and showed equivalent binding to polysomes. Translation in a HeLa cell cytoplasmic extract was used to examine the impact of the PLE in the context of varying length poly(A) tails. Here the overall translation of +PLE mRNA was less than control mRNA with the same length poly(A), and the PLE did not overcome the effect of a short poly(A) tail. Because poly(A)-binding protein (PABP) is a dominant effector of poly(A)-dependent translation we reasoned excess PABP in our extract might overwhelm PLE regulation of translation. This was confirmed by experiments where PABP was inactivated with poly(rA) or Paip2, and the effect of both treatments was reversed by addition of recombinant PABP. These data indicate that the PLE functionally substitutes for bound PABP to stimulate translation of short poly(A) mRNA.     

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.     

The flip-flop configuration of the PABP-dimer leads to switching of the translation function

Poly(A)-binding protein (PABP) is a translation initiation factor that interacts with the poly(A) tail of mRNAs. PABP bound to poly(A) stimulates translation by interacting with the eukaryotic initiation factor 4G (eIF4G), which brings the 3′ end of an mRNA close to its 5′ m⁷G cap structure through consecutive interactions of the 3′-poly(A)–PABP-eIF4G-eIF4E-5′ m⁷G cap. PABP is a highly abundant translation factor present in considerably larger quantities than mRNA and eIF4G in cells. However, it has not been elucidated how eIF4G, present in limited cellular concentrations, is not sequestered by mRNA-free PABP, present at high cellular concentrations, but associates with PABP complexed with the poly(A) tail of an mRNA. Here, we report that RNA-free PABPs dimerize with a head-to-head type configuration of PABP, which interferes in the interaction between PABP and eIF4G. We identified the domains of PABP responsible for PABP–PABP interaction. Poly(A) RNA was shown to convert the PABP–PABP complex into a poly(A)–PABP complex, with a head-to-tail-type configuration of PABP that facilitates the interaction between PABP and eIF4G. Lastly, we showed that the transition from the PABP dimer to the poly(A)–PABP complex is necessary for the translational activation function.     

The nuclear poly(A) binding protein, PABP2, forms an oligomeric particle covering the length of the poly(A) tail

The mammalian nuclear poly(A) binding protein, PABP2, controls the length of the newly synthesized poly(A) tail on messenger RNAs. To gain a better understanding of the mechanism of length control, we have investigated the structure of the PABP2.poly(A) complex. Electron microscopy and scanning force microscopy studies reveal that PABP2, when bound to poly(A), forms both linear filaments and discrete-sized, compact, oligomeric particles. The maximum diameter of the filament is 7 nm; the maximum diameter of the particle is 21(+/-2) nm. Maximum particle size is realized when the PABP2. poly(A) complex is formed with poly(A) molecules 200-300 nt long, which corresponds to the average length of the newly synthesized poly(A) tail in vitro and in vivo. The equilibrium between filaments and particles is highly sensitive to ionic strength; filaments are favored at low ionic strength, while particles predominate at moderate to high ionic strength. Nitrocellulose filter binding and gel mobility shift assays indicate that the PABP2.poly(A) particle formed on A(300) is not significantly more stable than complexes formed with smaller species of poly(A). These results are discussed in the context of the proposed functions for PABP2.     

Evidence that poly(A) binding protein has an evolutionarily conserved function in facilitating mRNA biogenesis and export

Eukaryotic poly(A) binding protein (PABP) is a ubiquitous, essential cellular factor with well-characterized roles in translational initiation and mRNA turnover. In addition, there exists genetic and biochemical evidence that PABP has an important nuclear function. Expression of PABP from Arabidopsis thaliana, PAB3, rescues an otherwise lethal phenotype of the yeast pab1Delta mutant, but it neither restores the poly(A) dependent stimulation of translation, nor protects the mRNA 5' cap from premature removal. In contrast, the plant PABP partially corrects the temporal lag that occurs prior to the entry of mRNA into the decay pathway in the yeast strains lacking Pab1p. Here, we examine the nature of this lag-correction function. We show that PABP (both PAB3 and the endogenous yeast Pab1p) act on the target mRNA via physically binding to it, to effect the lag correction. Furthermore, substituting PAB3 for the yeast Pab1p caused synthetic lethality with rna15-2 and gle2-1, alleles of the genes that encode a component of the nuclear pre-mRNA cleavage factor I, and a factor associated with the nuclear pore complex, respectively. PAB3 was present physically in the nucleus in the complemented yeast strain and was able to partially restore the poly(A) tail length control during polyadenylation in vitro, in a poly(A) nuclease (PAN)-dependent manner. Importantly, PAB3 in yeast also promoted the rate of entry of mRNA into the translated pool, rescued the conditional lethality, and alleviated the mRNA export defect of the nab2-1 mutant when overexpressed. We propose that eukaryotic PABPs have an evolutionarily conserved function in facilitating mRNA biogenesis and export.     

Regulation of poly(A) binding protein function in translation: Characterization of the Paip2 homolog, Paip2B

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs act synergistically to enhance translation. This synergy is mediated via interactions between eIF4G (a component of the eIF4F cap binding complex) and poly(A) binding protein (PABP). Paip2 (PABP-interacting protein 2) binds PABP and inhibits translation both in vitro and in vivo by decreasing the affinity of PABP for polyadenylated RNA. Here, we describe the functional characteristics of Paip2B, a Paip2 homolog. A full-length brain cDNA of Paip2B encodes a protein that shares 59% identity and 80% similarity with Paip2 (Paip2A), with the highest conservation in the two PABP binding domains. Paip2B acts in a manner similar to Paip2A to inhibit translation of capped and polyadenylated mRNAs both in vitro and in vivo by displacing PABP from the poly(A) tail. Also, similar to Paip2A, Paip2B does not affect the translation mediated by the internal ribosome entry site (IRES) of hepatitis C virus (HCV). However, Paip2A and Paip2B differ with respect to both mRNA and protein distribution in different tissues and cell lines. Paip2A is more highly ubiquitinated than is Paip2B and is degraded more rapidly by the proteasome. Paip2 protein degradation may constitute a primary mechanism by which cells regulate PABP activity in translation.     

Autoregulation of poly(A)-binding protein synthesis in vitro.

The poly(A)-binding protein (PABP), in a complex with the 3'poly(A) tail of eukaryotic mRNAs, plays important roles in the control of translation and message stability. All known examples of PABP mRNAs contain an extensive A-rich sequence in their 5' untranslated regions. Studies in mammalian cells undergoing growth stimulation or terminal differentiation indicate that PABP expression is regulated at the translational level. Here we examine the hypothesis that synthesis of the PABP is autogenously controlled. We show that the endogenous inactive PABP mRNA in rabbit reticulocytes can be specifically stimulated by addition of low concentrations of poly(A) and that this stimulation is also observed with in vitro transcribed human PABP mRNA. By deleting the A-rich region from the leader of human PABP mRNA and adding it upstream of the initiator AUG in a reporter mRNA we show that the adenylate tract is sufficient and necessary for mRNA repression and poly(A)-mediated activation in the reticulocyte cell-free system. UV cross-linking experiments demonstrate that the leader adenylate tract binds PABP. Furthermore, addition of recombinant GST-PABP to the cell-free system represses translation of mRNAs containing the A-rich sequence in their 5'UTR, but has no effect on control mRNA. We thus conclude that in vitro PABP binding to the A-rich sequence in the 5' UTR of PABP mRNA represses its own synthesis.