Paip2A inhibits translation by competitively binding to the RNA recognition motifs of PABPC1 and promoting its dissociation from the poly(A) tail

Eukaryotic mRNAs possess a poly(A) tail at their 3′-end, to which poly(A)-binding protein C1 (PABPC1) binds and recruits other proteins that regulate translation. Enhanced poly(A)-dependent translation, which is also PABPC1 dependent, promotes cellular and viral proliferation. PABP-interacting protein 2A (Paip2A) effectively represses poly(A)-dependent translation by causing the dissociation of PABPC1 from the poly(A) tail; however, the underlying mechanism remains unknown. This study was conducted to investigate the functional mechanisms of Paip2A action by characterizing the PABPC1–poly(A) and PABPC1–Paip2A interactions. Isothermal titration calorimetry and NMR analyses indicated that both interactions predominantly occurred at the RNA recognition motif (RRM)2–RRM3 regions of PABPC1, which have comparable affinities for poly(A) and Paip2A (dissociation constant, K_d = 1 nM). However, the K_d values of isolated RRM2 were 200 and 4 μM in their interactions with poly(A) and Paip2A, respectively; K_d values of 5 and 1 μM were observed for the interactions of isolated RRM3 with poly(A) and Paip2A, respectively. NMR analyses also revealed that Paip2A can bind to the poly(A)-binding interfaces of the RRM2 and RRM3 regions of PABPC1. Based on these results, we propose the following functional mechanism for Paip2A: Paip2A initially binds to the RRM2 region of poly(A)-bound PABPC1, and RRM2-anchored Paip2A effectively displaces the RRM3 region from poly(A), resulting in dissociation of the whole PABPC1 molecule. Together, our findings provide insight into the translation repression effect of Paip2A and may aid in the development of novel anticancer and/or antiviral drugs.     

Quantitative Characterization of Tob Interactions Provides the Thermodynamic Basis for Translation Termination-coupled Deadenylase Regulation

Translation termination-coupled deadenylation is the first and often the rate-limiting step of eukaryotic mRNA decay in which two deadenylases, Ccr4-Caf1 and Pan2, play key roles. One of the deadenylases, Caf1, associates with Tob, which recruits Caf1 to the poly(A) tail through interactions with a cytoplasmic poly(A)-binding protein 1 (PABPC1). We previously proposed that the competition between Tob and eRF3 (a translation termination factor that interacts with PABPC1) is responsible for the regulation of deadenylase activity. However, the molecular mechanism of the regulation should be addressed by investigating the binding affinity and the cellular levels of these proteins. In this work, we characterized the human Tob interactions with Caf1 and a C-terminal domain of PABPC1 (PABC). Nuclear magnetic resonance (NMR) and Western blot analyses revealed that Tob consists of a structured N-terminal BTG-Tob domain and an unstructured C-terminal region with two conserved PAM2 (PABPC1-interacting motif 2) motifs. The BTG-TOB domain associates with Caf1, whereas the C-terminal PAM2 motif binds to PABC, with a K_d value of 20 μm. Furthermore, we demonstrated that the levels of eRF3 and Tob in HeLa cells are 4–5 μm and less than 0.2 μm, respectively. On the basis of these results, we propose a thermodynamic mechanism for the translation termination-coupled deadenylation mediated by the Tob-Caf1 complex.     

Dual-domain, dual-targeting organellar protein presequences in Arabidopsis can use non-AUG start codons

The processes accompanying endosymbiosis have led to a complex network of interorganellar protein traffic that originates from nuclear genes encoding mitochondrial and plastid proteins. A significant proportion of nucleus-encoded organellar proteins are dual targeted, and the process by which a protein acquires the capacity for both mitochondrial and plastid targeting may involve intergenic DNA exchange coupled with the incorporation of sequences residing upstream of the gene. We evaluated targeting and sequence alignment features of two organellar DNA polymerase genes from Arabidopsis thaliana. Within one of these two loci, protein targeting appeared to be plastidic when the 5' untranslated leader region (UTR) was deleted and translation could only initiate at the annotated ATG start codon but dual targeted when the 5' UTR was included. Introduction of stop codons at various sites within the putative UTR demonstrated that this region is translated and influences protein targeting capacity. However, no ATG start codon was found within this upstream, translated region, suggesting that translation initiates at a non-ATG start. We identified a CTG codon that likely accounts for much of this initiation. Investigation of the 5' region of other nucleus-encoded organellar genes suggests that several genes may incorporate upstream sequences to influence targeting capacity. We postulate that a combination of intergenic recombination and some relaxation of constraints on translation initiation has acted in the evolution of protein targeting specificity for those proteins capable of functioning in both plastids and mitochondria.     

Interplay between Polyadenylate-Binding Protein 1 and Kaposi's Sarcoma-Associated Herpesvirus ORF57 in Accumulation of Polyadenylated Nuclear RNA,a Viral Long Noncoding RNA

Polyadenylate-binding protein cytoplasmic 1 (PABPC1) is a cytoplasmic-nuclear shuttling protein important for protein translation initiation and both RNA processing and stability. We report that PABPC1 forms a complex with the Kaposi''s sarcoma-associated herpesvirus (KSHV) ORF57 protein, which allows ORF57 to interact with a 9-nucleotide (nt) core element of KSHV polyadenylated nuclear (PAN) RNA, a viral long noncoding RNA (lncRNA), and increase PAN stability. The N-terminal RNA recognition motifs (RRMs) of PABPC1 are necessary for the direct interaction with ORF57. During KSHV lytic infection, the expression of viral ORF57 leads to a substantial decrease in overall PABPC1 expression, along with a shift in the cellular distribution of the remaining PABPC1 to the nucleus. Interestingly, PABPC1 and ORF57 have opposing functions in modulating PAN steady-state accumulation. The suppressive effect of PABPC1 specific to PAN expression is alleviated by small interfering RNA knockdown of PABPC1 or by overexpression of ORF57. Conversely, ectopic PABPC1 reduces ORF57 steady-state protein levels and induces aberrant polyadenylation of PAN and thereby indirectly inhibits ORF57-mediated PAN accumulation. However, E1B-AP5 (heterogeneous nuclear ribonucleoprotein U-like 1), which interacts with a region outside the 9-nt core to stimulate PAN expression, does not interact or even colocalize with ORF57. Unlike PABPC1, the nuclear distribution of E1B-AP5 remains unchanged by viral lytic infection or overexpression of ORF57. Together, these data indicate that PABPC1 is an important cellular target of viral ORF57 to directly upregulate PAN accumulation during viral lytic infection, and the ability of host PABPC1 to disrupt ORF57 expression is a strategic host counterbalancing mechanism.     

A viral nuclear noncoding RNA binds re-localized poly(A) binding protein and is required for late KSHV gene expression

During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV) expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN) RNA avidly binds host poly(A)-binding protein C1 (PABPC1), which normally functions in the cytoplasm to bind the poly(A) tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX) protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A) tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.     

A stimulatory role for the La-related protein 4B in translation

La-related proteins (LARPs) belong to an evolutionarily conserved family of factors with predicted roles in RNA metabolism. Here, we have analyzed the cellular interactions and function of LARP4B, a thus far uncharacterized member of the LARP family. We show that LARP4B is a cytosolic protein that accumulates upon arsenite treatment in cellular stress granules. Biochemical experiments further uncovered an interaction of LARP4B with the cytosolic poly(A) binding protein 1 (PABPC1) and the receptor for activated C Kinase (RACK1), a component of the 40S ribosomal subunit. Under physiological conditions, LARP4B co-sedimented with polysomes in cellular extracts, suggesting a role in translation. In agreement with this notion, overexpression of LARP4B stimulated protein synthesis, whereas knockdown of the factor by RNA interference impaired translation of a large number of cellular mRNAs. In sum, we identified LARP4B as a stimulatory factor of translation. We speculate that LARP4B exerts its function by bridging mRNA factors of the 3′ end with initiating ribosomes.     

Importin alpha-mediated nuclear import of cytoplasmic poly(A) binding protein occurs as a direct consequence of cytoplasmic mRNA depletion

Recent studies have found the cytoplasmic poly(A) binding protein (PABPC) to have opposing effects on gene expression when concentrated in the cytoplasm versus in the nucleus. PABPC is predominantly cytoplasmic at steady state, where it enhances protein synthesis through simultaneous interactions with mRNA and translation factors. However, it accumulates dramatically within the nucleus in response to various pathogenic and nonpathogenic stresses, leading to an inhibition of mRNA export. The molecular events that trigger relocalization of PABPC and the mechanisms by which it translocates into the nucleus to block gene expression are not understood. Here, we reveal an RNA-based mechanism of retaining PABPC in the cytoplasm. Expression either of viral proteins that promote mRNA turnover or of a cytoplasmic deadenylase drives nuclear relocalization of PABPC in a manner dependent on the PABPC RNA recognition motifs (RRMs). Using multiple independent binding sites within its RRMs, PABPC interacts with importin α, a component of the classical import pathway. Finally, we demonstrate that the direct association of PABPC with importin α is antagonized by the presence of poly(A) RNA, supporting a model in which RNA binding masks nuclear import signals within the PABPC RRMs, thereby ensuring efficient cytoplasmic retention of this protein in normal cells. These findings further suggest that cells must carefully calibrate the ratio of PABPC to mRNA, as events that offset this balance can dramatically influence gene expression.     

The “tale” of poly(A) binding protein: The MLLE domain and PAM2-containing proteins

The cytoplasmic poly(A) binding protein 1 (PABPC1) is an essential eukaryotic translational initiation factor first described over 40 years ago. Most studies of PABPC1 have focused on its N-terminal RRM domains, which bind the mRNA 3′ poly(A) tail and 5′ translation complex eIF4F via eIF4G; however, the protein also contains a C-terminal MLLE domain that binds a peptide motif, termed PAM2, found in many proteins involved in translation regulation and mRNA metabolism. Studies over the past decade have revealed additional functions of PAM2-containing proteins (PACs) in neurodegenerative diseases, circadian rhythms, innate defense, and ubiquitin-mediated protein degradation. Here, we summarize functional and structural studies of the MLLE/PAM2 interaction and discuss the diverse roles of PACs.     

Mitochondrial and cytosolic isoforms of yeast fumarase are derivatives of a single translation product and have identical amino termini

We have previously proposed that a single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae and that all fumarase translation products are targeted and processed in mitochondria before distribution. Alternative models for fumarase distribution have been proposed that require more than one translation product. In the current work (i) we show by using sequential Edman degradation and mass spectrometry that fumarase cytosolic and mitochondrial isoenzymes have an identical amino terminus that is formed by cleavage by the mitochondrial processing peptidase, (ii) we have generated fumarase mutants in which the second potential translation initiation codon (Met-24) has been substituted, yet the protein is processed efficiently and retains its ability to be distributed between the cytosol and mitochondria, and (iii) we show that although a signal peptide is required for fumarase targeting to mitochondria the specific fumarase signal peptide and the sequence immediately downstream to the cleavage site are not required for the dual distribution phenomenon. Our results are discussed in light of our model of fumarase targeting and distribution that suggests rapid folding into an import-incompetent state and retrograde movement of the processed protein back to the cytosol through the translocation pore.     

Parkin-independent mitophagy via Drp1-mediated outer membrane severing and inner membrane ubiquitination

Here, we report that acute reduction in mitochondrial translation fidelity (MTF) causes ubiquitination of the inner mitochondrial membrane (IMM) proteins, including TRAP1 and CPOX, which occurs selectively in mitochondria with a severed outer mitochondrial membrane (OMM). Ubiquitinated IMM recruits the autophagy machinery. Inhibiting autophagy leads to increased accumulation of mitochondria with severed OMM and ubiquitinated IMM. This process occurs downstream of the accumulation of cytochrome c/CPOX in a subset of mitochondria heterogeneously distributed throughout the cell (“mosaic distribution”). Formation of mosaic mitochondria, OMM severing, and IMM ubiquitination require active mitochondrial translation and mitochondrial fission, but not the proapoptotic proteins Bax and Bak. In contrast, in Parkin-overexpressing cells, MTF reduction does not lead to the severing of the OMM or IMM ubiquitination, but it does induce Drp1-independent ubiquitination of the OMM. Furthermore, high–cytochrome c/CPOX mitochondria are preferentially targeted by Parkin, indicating that in the context of reduced MTF, they are mitophagy intermediates regardless of Parkin expression. In sum, Parkin-deficient cells adapt to mitochondrial proteotoxicity through a Drp1-mediated mechanism that involves the severing of the OMM and autophagy targeting ubiquitinated IMM proteins.     

Facilitated leaky scanning and atypical ribosome shunting direct downstream translation initiation on the tricistronic S1 mRNA of avian reovirus

The S1 mRNA of avian reovirus is functionally tricistronic, encoding three unrelated proteins, p10, p17 and σC, from three sequential, partially overlapping open reading frames (ORFs). The mechanism of translation initiation at the 3′-proximal σC ORF is currently unknown. Transient RNA transfections using Renilla luciferase reporter constructs revealed only a modest reduction in reporter expression upon optimization of either the p10 or p17 start sites. Insertion of multiple upstream AUG (uAUG) codons in a preferred start codon sequence context resulted in a substantial retention of downstream translation initiation on the S1 mRNA, but not on a heterologous mRNA. The S1 mRNA therefore facilitates leaky scanning to promote ribosome access to the σC start codon. Evidence also indicates that σC translation is mediated by a second scanning-independent mechanism capable of bypassing upstream ORFs. This alternate mechanism is cap-dependent and requires a sequence-dependent translation enhancer element that is complementary to 18S rRNA. Downstream translation initiation of the tricistronic S1 mRNA is therefore made possible by two alternate mechanisms, facilitated leaky scanning and an atypical form of ribosome shunting. This dual mechanism of downstream translation initiation ensures sufficient expression of the σC cell attachment protein that is essential for infectious progeny virus production.     

Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.     

Mutation in PNPT1, which Encodes a Polyribonucleotide Nucleotidyltransferase,Impairs RNA Import into Mitochondria and Causes Respiratory-Chain Deficiency

Multiple-respiratory-chain deficiency represents an important cause of mitochondrial disorders. Hitherto, however, mutations in genes involved in mtDNA maintenance and translation machinery only account for a fraction of cases. Exome sequencing in two siblings, born to consanguineous parents, with severe encephalomyopathy, choreoathetotic movements, and combined respiratory-chain defects allowed us to identify a homozygous PNPT1 missense mutation (c.1160A>G) that encodes the mitochondrial polynucleotide phosphorylase (PNPase). Blue-native polyacrylamide gel electrophoresis showed that no PNPase complex could be detected in subject fibroblasts, confirming that the substitution encoded by c.1160A>G disrupts the trimerization of the protein. PNPase is predominantly localized in the mitochondrial intermembrane space and is implicated in RNA targeting to human mitochondria. Mammalian mitochondria import several small noncoding nuclear RNAs (5S rRNA, MRP RNA, some tRNAs, and miRNAs). By RNA hybridization experiments, we observed a significant decrease in 5S rRNA and MRP-related RNA import into mitochondria in fibroblasts of affected subject 1. Moreover, we found a reproducible decrease in the rate of mitochondrial translation in her fibroblasts. Finally, overexpression of the wild-type PNPT1 cDNA in fibroblasts of subject 1 induced an increase in 5S rRNA import in mitochondria and rescued the mitochondrial-translation deficiency. In conclusion, we report here abnormal RNA import into mitochondria as a cause of respiratory-chain deficiency.     

Structure and function of poly(A) binding proteins

Poly (A) tails are found at the 3' ends of almost all eukaryotic mRNAs. They are bound by two different poly (A) binding proteins, PABPC in the cytoplasm and PABPN1 in the nucleus. PABPC functions in the initiation of translation and in the regulation of mRNA decay. In both functions, an interaction with the m7G cap at the 5' end of the message plays an important role. PABPN1 is involved in the synthesis of poly (A) tails, increasing the processivity of poly (A) polymerase and contributing to defining the length of a newly synthesized poly (A) tail.