Identification and characterization of proteins that interact with the carboxy terminus of poly(A)-binding protein and inhibit translation in vitro

Poly(A)-binding proteins (PABPs) are multifunctional proteins that play important roles in mRNA stability and protein translation. Two cucumber ( Cucumis sativus L.) proteins, PCI6 (PABP-CT-interacting) and PCI243 were identified based on ability to interact with the carboxy terminus (CT) of PABP in yeast two-hybrid and in vitro binding assays. PCI6 and PCI243 share a conserved amino acid domain (SxLnpnApxFxP) in common with human PABP-CT interactors, and with Arabidopsis ERD15 (early-responsive to dehydration). Deletion analysis and point mutations indicate that presence of this domain is necessary for the interaction, and tests with ERD15 demonstrate that it is predictive of interaction. Other plant proteins possessing this domain fall into two categories: small, acidic proteins like PCI6, PCI243 and ERD15, and larger neutral proteins that also include an RNA recognition motif. PCI6 is expressed in a range of tissues, e.g., leaves, roots, stems and flowers, and follows a diurnal pattern of expression, increasing during light hours and declining overnight. In wheat germ and mouse ascites Krebs-2 in vitro translation systems, PCI6 inhibited translation whereas the non-interacting mutant, PCI6-23A, did not or had a greatly reduced effect. The activity of PCI6, therefore, is reminiscent of that of human PABP-interacting protein 2 (Paip2). These results demonstrate a novel interaction between PABP and several plant proteins sharing a SxLnpxApxFxP motif, with possible implications for translational regulation.     

Degradation of poly(A)-binding protein in apoptotic cells and linkage to translation regulation

We have recently shown that poly(A)-binding protein (PABP) is cleaved during poliovirus and Coxsackievirus infection by viral 3Cprotease and that 3Cprotease modification of a subset of PABP can result in significant translation inhibition. During apoptosis, translation undergoes significant down-regulation that correlates with caspase-3 mediated cleavage of several translation factors, including eIF4G, 4EBP1 and eIF2alpha. The fate of PABP in apoptotic cells has not yet been examined. Here we show that PABP levels decline significantly via proteolytic degradation in apoptotic HeLa, Jurkat and MCF7 cells. The degradation of PABP correlated with translation inhibition but lagged behind cleavage of eIF4GI. In apoptotic MCF7 cells translation inhibition occurred without modification of most translation factors and correlated with PABP degradation. PABP was not cleaved during incubation with several caspases, yet caspase 3 induced weak PABP degradative activity in cells lysates. Both the caspase inhibitor zVAD and calpain inhibitors blocked PABP cleavage in vivo, while the proteosome inhibitor MG132 induced PABP degradation. Protease(s) activated during apoptosis preferentially degraded PABP associated with ribosomes and translation factors, but not PABP in other cellular compartments. The data suggest that targeted degradation of PABP contributes to translation inhibition in apoptotic cells.     

Splicing factor poly(rC)-binding protein 1 is a novel and distinctive tumor suppressor

A lot of evidence has been found on the link between tumorigenesis and the aberrant expression of splicing factors. A number of splicing factors have been reported to be either oncogenic or overexpressed in cancer cells. However, splicing factors can also play negative roles in tumorigenesis. In the current review, we focus on splicing factor poly(rC)-binding protein 1 (PCBP1), a novel tumor suppressor that is characterized by downregulation in many cancer types and shows inhibition of tumor formation and metastasis. Notably, the messenger RNA levels of PCBP1 are not significantly decreased in most cancer types. In fact, PCBP1 protein is often degraded or shows a loss-of-function through phosphorylation in cancer cells. PCBP1 is highly homologous to its family member, PCBP2. Interestingly, PCBP2 appears to be an oncogenic splicing factor. A growing body of evidence has shown that PCBP1 regulates alternative splicing, translation, and RNA stability of many cancer-related genes. Taking together, PCBP1 has distinctive tumor suppressive functions, and increasing PCBP1 expression may represent a new approach for cancer treatment.     

Solution structure of the C-terminal domain from poly(A)-binding protein in Trypanosoma cruzi: a vegetal PABC domain

PABC is a phylogenetically conserved peptide-binding domain primarily found within the C terminus of poly(A)-binding proteins (PABPs). This domain recruits a series of translation factors including poly(A)-interacting proteins (Paip1 and Paip2) and release factor 3 (RF3/GSPT) to the initiation complex on mRNA. Here, we determine the solution structure of the Trypanosoma cruzi PABC domain (TcPABC), a representative of the vegetal class of PABP proteins. TcPABC is similar to human PABC (hPABC) and consists of five alpha-helices, in contrast to the four helices observed in PABC domains from yeast (yPABC) and hyper plastic disk proteins (hHYD). A mobile N-terminal helix is observed in TcPABC that does not pack against the core of the protein, as found in hPABC. Characteristic to all PABC domains, the last four helices of TcPABC fold into a right-handed super coil. TcPABC demonstrates high-affinity binding to PABP interacting motif-2 (PAM-2) and reveals a peptide-binding surface homologous to that of hPABC. Our results demonstrate the last four helices in TcPABC are sufficient for peptide recognition and we predict a similar binding mode in PABC domains. Furthermore, these results point to the presence of putative PAM-2 site-containing proteins in trypanosomes.     

Biological role of the two overlapping poly(A)-binding protein interacting motifs 2 (PAM2) of eukaryotic releasing factor eRF3 in mRNA decay

Eukaryotic releasing factor GSPT/eRF3 mediates translation termination-coupled mRNA decay via interaction with a cytosolic poly(A)-binding protein (PABPC1). A region of eRF3 containing two overlapping PAM2 (PABPC1-interacting motif 2) motifs is assumed to bind to the PABC domain of PABPC1, on the poly(A) tail of mRNA. PAM2 motifs are also found in the major deadenylases Caf1–Ccr4 and Pan2–Pan3, whose activities are enhanced upon PABPC1 binding to these motifs. Their deadenylase activities are regulated by eRF3, in which two overlapping PAM2 motifs competitively prevent interaction with PABPC1. However, it is unclear how these overlapping motifs recognize PABC and regulate deadenylase activity in a translation termination-coupled manner. We used a dominant-negative approach to demonstrate that the N-terminal PAM2 motif is critical for eRF3 binding to PABPC1 and that both motifs are required for function. Isothermal titration calorimetry (ITC) and NMR analyses revealed that the interaction is in equilibrium between the two PAM2–PABC complexes, where only one of the two overlapping PAM2 motifs is PABC-bound and the other is PABC-unbound and partially accessible to the other PABC. Based on these results, we proposed a biological role for the overlapping PAM2 motifs in the regulation of deadenylase accessibility to PABPC1 at the 3′ end of poly(A).     

Interaction of rat poly(A)-binding protein with poly(A)- and non-poly(A) sequences is preferentially mediated by RNA recognition motifs 3+4

Vasopressin (VP) mRNA and the non-coding BC200 RNA are sorted to neuronal dendrites. Among proteins interacting specifically with both RNAs is the multifunctional poly(A)-binding protein (PABP) consisting of four RNA recognition motifs (RRMs) and a C-terminal auxiliary domain. The protein/RNA interaction studies presented here reveal that PABPs association with VP- and BC200 RNA is exclusively mediated by RRMs 3+4. Quantitative binding studies with PABP deletion mutants demonstrate preferential binding of RRMs 3+4 even to poly(A)-homopolymers, while RRMs 1+2 exhibit a lower affinity for those sequences. An optimal interaction with both poly(A)- and non-poly(A) sequences is only achieved by full-size PABP.     

The translation of the messenger for the poly(A)-binding protein-associated with translated mRNA is suppressed. A case of cytoplasmic repression in duck erythroblasts

In vivo protein synthesis in duck erythroblasts was compared to in vitro translation of polyribosomal and free cytoplasmic mRNA. The in vivo study showed the absence of de novo synthesis of the Mr 73 000 poly(A)-binding protein found associated with all polyribosomal mRNA. In vitro translation demonstrated that the mRNA for this protein is absent from the polyribosomal mRNA fraction but constitutes a medium frequency messenger among the repressed free mRNA. This result confirms the existence of a qualitative translational control in terminal differentiating duck erythroblasts leading eventually to the arrest of the protein synthesizing machinery.     

Proximity of the poly(A)-binding protein to a premature termination codon inhibits mammalian nonsense-mediated mRNA decay

mRNA surveillance pathways selectively clear defective mRNAs from the cell. As such, these pathways serve as important modifiers of genetic disorders. Nonsense-mediated decay (NMD), the most intensively studied surveillance pathway, recognizes mRNAs with premature termination codons (PTCs). In mammalian systems the location of a PTC more than 50 nucleotides 5' to the terminal exon-exon junction is a critical determinant of NMD. However, mRNAs with nonsense codons that fulfill this requirement but are located very early in the open reading frame can effectively evade NMD. The unexpected resistance of such mRNAs with AUG-proximal PTCs to accelerated decay suggests that important determinants of NMD remain to be identified. Here, we report that an NMD-sensitive mRNA can be stabilized by artificially tethering the cytoplasmic poly(A) binding protein 1, PABPC1, at a PTC-proximal position. Remarkably, the data further suggest that NMD of an mRNA with an AUG-proximal PTC can also be repressed by PABPC1, which might be brought into proximity with the PTC during cap-dependent translation and 43S scanning. These results reveal a novel parameter of NMD in mammalian cells that can account for the stability of mRNAs with AUG-proximal PTCs. These findings serve to expand current mechanistic models of NMD and mRNA translation.     

The size of poly(A)-containing RNAs in Drosophila melanogaster embryos

The size range of poly(A)-containing RNA from Drosophila melanogaster embryos has been estimated by hybridization with ³H-labeled poly(U) and subsequent fractionation on sucrose gradients. The median size of nuclear poly(A)-containing RNA is about 30 S (6000 nucleotides), and the median size of cytoplasmic poly(A)-containing RNA is about 17 S (1800 nucleotides). The relationship of these sizes to messenger RNA needed to code for protein and to the length of DNA contained in a chromomere is discussed.Research grant support was provided by NIH (6M35558; HD-00266) and NSF (GB-30600).     

Induction of expression and co-localization of heat shock polypeptides with the polyalanine expansion mutant of poly(A)-binding protein N1 after chemical stress

Formation of nuclear inclusions consisting of aggregates of a polyalanine expansion mutant of nuclear poly(A)-binding protein (PABPN1) is the hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant disease. Patients with this disorder exhibit progressive swallowing difficulty and drooping of their eye lids, which starts around the age of 50. Previously we have shown that treatment of cells expressing the mutant PABPN1 with a number of chemicals such as ibuprofen, indomethacin, ZnSO₄, and 8-hydroxy-quinoline induces HSP70 expression and reduces PABPN1 aggregation. In these studies we have shown that expression of additional HSPs including HSP27, HSP40, and HSP105 were induced in mutant PABPN1 expressing cells following exposure to the chemicals mentioned above. Furthermore, all three additional HSPs were translocated to the nucleus and probably helped to properly fold the mutant PABPN1 by co-localizing with this protein.     

Inhibitory effect of naked neural BC1 RNA or BC200 RNA on eukaryotic in vitro translation systems is reversed by poly(A)-binding protein (PABP)

Regulated protein biosynthesis in dendrites of neurons might be a key mechanism underlying learning and memory. Neuronal dendritic BC1 RNA and BC200 RNA and similar small untranslated RNAs inhibit protein translation in vitro systems, such as rabbit reticulocyte lysate. Likewise, co-transfection of these RNAs with reporter mRNA suppressed translation levels in HeLa cells. The oligo(A)-rich region of all active small RNAs were identified as the RNA domains chiefly responsible for the inhibitory effects. Addition of recombinant human poly(A)-binding protein (PABP) significantly compensated the inhibitory effect of the small oligo(A)-rich RNA. In vivo, all BC1 RNA appears to be complexed with PABP. Nevertheless, in the micro-environment of dendritic spines of neuronal cells, BC1 RNPs or BC200 RNPs might mediate regulatory functions by differential interactions with locally limited PABP and/or directly or indirectly, with other translation initiation factors.     

Cytoplasmic targeting of mutant poly(A)-binding protein nuclear 1 suppresses protein aggregation and toxicity in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by a polyalanine expansion from 10 to 12-17 residues, located at the N-terminus of the poly(A)-binding protein nuclear 1 (PABPN1). A distinct pathological hallmark of OPMD is the presence of filamentous intranuclear aggregates in patients' skeletal muscle cells. Wildtype PABPN1 protein is expressed ubiquitously and was shown to be mostly concentrated in discrete nuclear domains called 'speckles'. Using an established cell- culture model, we show that most mutant PABPN1- positive (alanine expanded form) intranuclear aggregates are structures distinct from intranuclear speckles. In contrast, the promyelocytic leukaemia protein, a major component of nuclear bodies, strongly colocalized to intranuclear aggregates of mutant PABPN1. Wildtype PABPN1 can freely shuttle between the nucleus and cytoplasm. We determined whether the nuclear environment is necessary for mutant PABPN1 inclusion formation and cellular toxicity. This was achieved by inactivating the mutant PABPN1 nuclear localization signal and by generating full-length mutant PABPN1 fused to a strong nuclear export sequence. A green fluorescence protein tag inserted at the N-terminus of both wildtype PABPN1 (ala10) and mutant PABPN1 (ala17) proteins allowed us to visualize their subcellular localization. Targeting mutant PABPN1 to the cytoplasm resulted in a significant suppression of both intranuclear aggregates formation and cellular toxicity, two histological consequences of OPMD. Our results indicate that the nuclear localization of mutant PABPN1 is crucial to OPMD pathogenesis.     

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.     

The fertilization—induced Ca^2＋ oscillation in mouse oocytes is cytoplasmic maturation dependent

Mature eggs (at metaphase II stage) produce a series of Ca^2 oscillation at fertilization.To define whether the fertilization-induced Ca^2 oscillation is restrict to the metaphase II eggs and cell cycle dependent,mouse oocytes at prophase I (arrested at germinal vesicle stage),metaphase I,metaphase II,as well as the pronuclear embryos at interphase of the first mitotic division derived from fertilization of parthenogenetic activation were inseminated after removal of zona pellucida,The results show that the fertilization-induced Ca^2 oscillation is not specific to metaphase II eggs.This is supported by the fact that immature oocytes generated the Ca^2 oscillations at fertilization regardless of their nuclear progression from prophase I to metaphase I (in vitro matured) stage.More interestingly,it was first found that pronuclear embryos at interphase derived from parthenogenetic activation showed Ca^2 oscillations in response to fertilization while the zygotes at interphase did not after reinsemination or intracytoplasmic injection of sperm extracts which induce Ca^2 oscillations in MII eggs.This suggests that the ability of oocytes to generate Ca^2 oscillation in response to sperm penetration is not regulated in a cell cycle dependent manner but dependent on the cytoplasmic maturation.