Concerted action of ataxin-2 and PABPC1-bound mRNA poly(A) tail in the formation of stress granules.

Stress induces global stabilization of the mRNA poly(A) tail (PAT) and the assembly of untranslated poly(A)-tailed mRNA into mRNPs that accumulate in stress granules (SGs). While the mechanism behind stress-induced global PAT stabilization has recently emerged, the biological significance of PAT stabilization under stress remains elusive. Here, we demonstrate that stress-induced PAT stabilization is a prerequisite for SG formation. Perturbations in PAT length impact SG formation; PAT shortening, achieved by overexpressing mRNA deadenylases, inhibits SG formation, whereas PAT lengthening, achieved by overexpressing their dominant negative mutants or downregulating deadenylases, promotes it. PABPC1, which specifically binds to the PAT, is crucial for SG formation. Complementation analyses reveal that the PABC/MLLE domain of PABPC1, responsible for binding PAM2 motif-containing proteins, plays a key role. Among them, ataxin-2 is a known SG component. A dominant-negative approach reveals that the PAM2 motif of ataxin-2 is essential for SG formation. Notably, ataxin-2 increases stress sensitivity, lowering the threshold for SG formation, probably by promoting the aggregation of PABPC1-bound mRNA. The C-terminal region is responsible for the self-aggregation of ataxin-2. These findings underscore the critical roles of mRNA PAT, PABPC1 and ataxin-2 in SG formation and provide mechanistic insights into this process.

The impact of single-stranded RNAs on the dimerization of double-stranded RNA-dependent protein kinase PKR.

The RNA-binding protein PKR serves as a crucial antiviral innate immune factor that globally suppresses translation by sensing viral double-stranded RNA (dsRNA) and by phosphorylating the translation initiation factor eIF2α. Recent findings have unveiled that single-stranded RNAs (ssRNAs), including in vitro transcribed (IVT) mRNA, can also bind to and activate PKR. However, the precise mechanism underlying PKR activation by ssRNAs, remains incompletely understood. Here, we developed a NanoLuc Binary Technology (NanoBiT)-based in vitro PKR dimerization assay to assess the impact of ssRNAs on PKR dimerization. Our findings demonstrate that, akin to double-stranded polyinosinic:polycytidylic acid (polyIC), an encephalomyocarditis virus (EMCV) RNA, as well as NanoLuc luciferase (Nluc) mRNA, can induce PKR dimerization. Conversely, homopolymeric RNA lacking secondary structure fails to promote PKR dimerization, underscoring the significance of secondary structure in this process. Furthermore, adenovirus VA RNA 1, another ssRNA, impedes PKR dimerization by competing with Nluc mRNA. Additionally, we observed structured ssRNAs capable of forming G-quadruplexes induce PKR dimerization. Collectively, our results indicate that ssRNAs have the ability to either induce or inhibit PKR dimerization, thus representing potential targets for the development of antiviral and anti-inflammatory agents.

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, Kd = 1 nM). However, the Kd values of isolated RRM2 were 200 and 4 µM in their interactions with poly(A) and Paip2A, respectively; Kd 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.

Direct evidence that Ataxin-2 is a translational activator mediating cytoplasmic polyadenylation.

The RNA-binding protein Ataxin-2 binds to and stabilizes a number of mRNA sequences, including that of the transactive response DNA-binding protein of 43 kDa (TDP-43). Ataxin-2 is additionally involved in several processes requiring translation, such as germline formation, long-term habituation, and circadian rhythm formation. However, it has yet to be unambiguously demonstrated that Ataxin-2 is actually involved in activating the translation of its target mRNAs. Here we provide direct evidence from a polysome profile analysis showing that Ataxin-2 enhances translation of target mRNAs. Our recently established method for transcriptional pulse-chase analysis under conditions of suppressing deadenylation revealed that Ataxin-2 promotes post-transcriptional polyadenylation of the target mRNAs. Furthermore, Ataxin-2 binds to a poly(A)-binding protein PABPC1 and a noncanonical poly(A) polymerase PAPD4 via its intrinsically disordered region (amino acids 906-1095) to recruit PAPD4 to the targets. Post-transcriptional polyadenylation by Ataxin-2 explains not only how it activates translation but also how it stabilizes target mRNAs, including TDP-43 mRNA. Ataxin-2 is known to be a potent modifier of TDP-43 proteinopathies and to play a causative role in the neurodegenerative disease spinocerebellar ataxia type 2, so these findings suggest that Ataxin-2-induced cytoplasmic polyadenylation and activation of translation might impact neurodegeneration (i.e. TDP-43 proteinopathies), and this process could be a therapeutic target for Ataxin-2-related neurodegenerative disorders.

The RNA-binding protein QKI-7 recruits the poly(A) polymerase GLD-2 for 3' adenylation and selective stabilization of microRNA-122.

MicroRNA-122 (miR-122) is highly expressed in hepatocytes, where it plays an important role in regulating cholesterol and fatty acid metabolism, and it is also a host factor required for hepatitis C virus replication. miR-122 is selectively stabilized by 3' adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2 (also known as PAPD4 or TENT2). However, it is unclear how GLD-2 specifically stabilizes miR-122. Here, we show that QKI7 KH domain-containing RNA binding (QKI-7), one of three isoforms of the QKI proteins, which are members of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins, is involved in miR-122 stabilization. QKI down-regulation specifically decreased the steady-state level of mature miR-122, but did not affect the pre-miR-122 level. We also found that QKI-7 uses its C-terminal region to interact with GLD-2 and its QUA2 domain to associate with the RNA-induced silencing complex protein Argonaute 2 (Ago2), indicating that the GLD-2-QKI-7 interaction recruits GLD-2 to Ago2. QKI-7 exhibited specific affinity to miR-122 and significantly promoted GLD-2-mediated 3' adenylation of miR-122 in vitro Taken together, our findings indicate that miR-122 binds Ago2-interacting QKI-7, which recruits GLD-2 for 3' adenylation and stabilization of miR-122.

DDX6 is a positive regulator of Ataxin-2/PAPD4 cytoplasmic polyadenylation machinery.

The RNA-binding protein Ataxin-2 regulates translation and mRNA stability through cytoplasmic polyadenylation of the targets. Here we newly identified DDX6 as a positive regulator of the cytoplasmic polyadenylation. Analysis of Ataxin-2 interactome using LC-MS/MS revealed prominent interaction with the DEAD-box RNA helicase DDX6. DDX6 interacted with components of the Ataxin-2 polyadenylation machinery; Ataxin-2, PABPC1 and PAPD4. As in the case for Ataxin-2 downregulation, DDX6 downregulation led to an increase in Ataxin-2 target mRNAs with short poly(A) tails as well as a reduction in their protein expression. In contrast, Ataxin-2 target mRNAs with short poly(A) tails were decreased by the overexpression of Ataxin-2, which was compromised by the DDX6 downregulation. However, polyadenylation induced by Ataxin-2 tethering was not affected by the DDX6 downregulation. Taken together, these results suggest that DDX6 positively regulates Ataxin-2-induced cytoplasmic polyadenylation to maintain poly(A) tail length of the Ataxin-2 targets provably through accelerating binding of Ataxin-2 to the target mRNAs.

Dom34 mediates targeting of exogenous RNA in the antiviral OAS/RNase L pathway.

The 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway is an innate immune system that protects hosts against pathogenic viruses and bacteria through cleavage of exogenous single-stranded RNA; however, this system's selective targeting mechanism remains unclear. Here, we identified an mRNA quality control factor Dom34 as a novel restriction factor for a positive-sense single-stranded RNA virus. Downregulation of Dom34 and RNase L increases viral replication, as well as half-life of the viral RNA. Dom34 directly binds RNase L to form a surveillance complex to recognize and eliminate the exogenous RNA in a manner dependent on translation. Interestingly, the feature detected by the surveillance complex is not the specific sequence of the viral RNA but the 'exogenous nature' of the RNA. We propose the following model for the selective targeting of exogenous RNA; OAS3 activated by the exogenous RNA releases 2'-5'-oligoadenylates (2-5A), which in turn converts latent RNase L to an active dimer. This accelerates formation of the Dom34-RNase L surveillance complex, and its selective localization to the ribosome on the exogenous RNA, thereby promoting degradation of the RNA. Our findings reveal that the selective targeting of exogenous RNA in antiviral defense occurs via a mechanism similar to that in the degradation of aberrant transcripts in RNA quality control.

A novel method for stabilizing microRNA mimics.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that negatively regulate gene expression at post-transcriptional level via translational repression and/or mRNA degradation. miRNAs are associated with many cellular processes, and down-regulation of miRNAs causes numerous diseases including cancer, neurological disorders, inflammation, and cardiovascular diseases, for which miRNA replacement therapy has emerged as a promising approach. This approach aims to restore down-regulated miRNAs using synthetic miRNA mimics. However, it remains a critical issue that miRNA mimics are unstable and transient in cells. Here, we first show that miRNA mimics are rapidly degraded by a mechanism different from Tudor-staphylococcal/micrococcal-like nuclease (TSN)-mediated miRNA decay, which degrades endogenous miRNAs, and newly identified 2'-5'-oligoadenylate synthetase (OAS)/RNase L as key factors responsible for the degradation of miRNA mimics in human cells. Our results suggest that the OAS1 recognizes miRNA mimics and produces 2'-5'-oligoadenylates (2-5A), which leads to the activation of latent endoribonuclease RNase L to degrade miRNA mimics. A small-molecule inhibitor that blocks RNase L can stabilize miRNA mimics. These findings provide a promising method for the stabilization of miRNA mimics, as well as for the efficient miRNA replacement therapy.

MicroRNP-mediated translational activation of nonadenylated mRNAs in a mammalian cell-free system.

MicroRNAs are small noncoding RNAs that regulate translation and mRNA stability by binding target mRNAs in complex with Argonaute (AGO) proteins. AGO interacts with a member of the TNRC6 family proteins to form a microRNP complex, which recruits the CCR4-NOT complex to accelerate deadenylation and inhibits translation. MicroRNAs primarily repress translation of target mRNAs but have been shown to enhance translation of a specific type of target reporter mRNAs in various experimental systems: G0 quiescent mammalian cells, Xenopus laevis oocytes, Drosophila embryo extracts, and HeLa cells. In all of the cases mentioned, a common feature of the activated target mRNAs is the lack of a poly(A) tail. Here, we show let-7-microRNP-mediated translational activation of nonadenylated target mRNAs in a mammalian cell-free system, which contains over-expressed AGO2, TNRC6B, and PAPD7 (TUTase5, TRF4-1). Importantly, translation of nonadenylated mRNAs was activated also by tethered TNRC6B silencing domain (SD), in the presence of PAPD7. Deletion of the poly(A)-binding protein (PABP) interacting motif (PAM2) from the TNRC6B-SD abolished the translational activation, suggesting the involvement of PABP in the process. Similar results were also obtained in cultured HEK293T cells. This work may provide novel insights into microRNP-mediated mRNA regulation.

Comparison of high-dose and low-dose corticosteroid therapy for refractory Mycoplasma pneumoniae pneumonia in children.

BACKGROUND: Mycoplasma pneumoniae pneumonia (MPP) is generally a self-limiting disease, but it may become refractory. It is thought that refractory MPP is linked to the excessive immunologic responses of the host. Consequently, the use of adjunctive systemic corticosteroids may have beneficial effects. In this study, we compared the effects of high- and low-dose corticosteroid therapy in a pediatric population with refractory MPP. METHODS: We retrospectively collected data from 91 pediatric MPP patients treated with adjunctive systemic corticosteroids between April 2014 and October 2016. The patients were divided into the following two groups: high-dose corticosteroid group (2 mg/kg/day or more of prednisolone equivalents; n = 38) and low-dose corticosteroid group (<2 mg/kg/day; n = 53). Additionally, we compared the number of febrile days post-corticosteroid administration. We used 25 paired patients in a propensity score matching analysis to correct for confounding factors both by age and by days (from onset till corticosteroid therapy initiation). RESULTS: We observed that in the high-dose corticosteroid group defervescence following corticosteroid therapy initiation was achieved significantly earlier and length of hospitalization was significantly shorter (0.8 ± 1.0 vs. 1.5 ± 1.4 days and 8.2 ± 2.4 vs. 10.7 ± 2.7 days, respectively). In the propensity score matching, we observed that significant differences in the length of fever following corticosteroid therapy initiation and hospitalization were still present. Further, neither of the groups developed corticosteroid-related adverse events. CONCLUSION: Our results suggest that patients with refractory MPP treated with high-dose corticosteroid could achieve defervescence earlier and have a shorter hospitalization.

Proteolysis suppresses spontaneous prion generation in yeast.

Prions are infectious proteins that cause fatal neurodegenerative disorders including Creutzfeldt-Jakob and bovine spongiform encephalopathy (mad cow) diseases. The yeast [PSI+] prion is formed by the translation-termination factor Sup35, is the best-studied prion, and provides a useful model system for studying such diseases. However, despite recent progress in the understanding of prion diseases, the cellular defense mechanism against prions has not been elucidated. Here, we report that proteolytic cleavage of Sup35 suppresses spontaneous de novo generation of the [PSI+] prion. We found that during yeast growth in glucose media, a maximum of 40% of Sup35 is cleaved at its N-terminal prion domain. This cleavage requires the vacuolar proteases PrA-PrB. Cleavage occurs in a manner dependent on translation but independently of autophagy between the glutamine/asparagine-rich (Q/N-rich) stretch critical for prion formation and the oligopeptide-repeat region required for prion maintenance, resulting in the removal of the Q/N-rich stretch from the Sup35 N terminus. The complete inhibition of Sup35 cleavage, by knocking out either PrA (pep4Δ) or PrB (prb1Δ), increased the rate of de novo formation of [PSI+] prion up to ∼5-fold, whereas the activation of Sup35 cleavage, by overproducing PrB, inhibited [PSI+] formation. On the other hand, activation of the PrB pathway neither cleaved the amyloid conformers of Sup35 in [PSI+] strains nor eliminated preexisting [PSI+]. These findings point to a mechanism antagonizing prion generation in yeast. Our results underscore the usefulness of the yeast [PSI+] prion as a model system to investigate defense mechanisms against prion diseases and other amyloidoses.

Diagnosis of congenital hyperinsulinism: Biochemical profiles during hypoglycemia.

OBJECTIVES: To define the ranges of biochemical markers during hypoglycemia for the diagnosis of congenital hyperinsulinism (CHI), using high sensitivity insulin assays. SUBJECTS: A total of 298 patients with CHI and 58 control patients with non-hyperinsulinemic hypoglycemia, who were diagnosed after 2007. METHODS: The levels of biochemical markers (glucose, insulin, ß-hydroxybutyrate [BHB], free fatty acids [FFA], lactate, ammonia) at the time of hypoglycemia were analyzed along with the maximal glucose infusion rate (GIR) to maintain euglycemia and clinical outcomes. RESULTS: Median levels of blood glucose in patients with CHI and in controls were 30 and 46 mg/dL, while insulin levels were 9.90 and undetectable (<.5) µU/mL, respectively. Similarly, median levels of BHB were 17.5 and 3745 µmol/L, and those of FFA were 270.5 and 2660 µmol/L, respectively. For patients after 5 months, cutoffs of insulin >1.25 µU/mL, BHB < 2000 µmol/L, and FFA < 1248 µmol/L predicted CHI with sensitivities of 97.5, 96.2, and 95.2% and specificities of 84.2, 89.3, and 92.3%, respectively. Maximal GIR in the CHI groups tended to decrease with age. In addition, decreased gestational age, low birth weight, and elevated lactate at hypoglycemia were significantly more common in patients who were off treatment within 100 days without pancreatectomy. CONCLUSIONS: After introduction of high-sensitive assays, the diagnostic value of insulin was improved, allowing for more efficient cutoffs to be set for diagnosis of CHI. Premature birth, low birth weight and elevated lactate might be helpful in predicting early remission of hypoglycemia.

The STAR protein QKI-7 recruits PAPD4 to regulate post-transcriptional polyadenylation of target mRNAs.

Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A) tail has not been widely studied due to the difficulty in distinguishing whether any observed increase in length is due to the synthesis of new mRNA, reduced deadenylation or cytoplasmic polyadenylation. Here, we overcame this barrier by developing a method for transcriptional pulse-chase analysis under conditions where deadenylases are suppressed. This strategy was used to show that a member of the Star family of RNA binding proteins, QKI, promotes polyadenylation when tethered to a reporter mRNA. Although multiple RNA binding proteins have been implicated in cytoplasmic polyadenylation during early development, previously only CPEB was known to function in this capacity in somatic cells. Importantly, we show that only the cytoplasmic isoform QKI-7 promotes poly(A) tail extension, and that it does so by recruiting the non-canonical poly(A) polymerase PAPD4 through its unique carboxyl-terminal region. We further show that QKI-7 specifically promotes polyadenylation and translation of three natural target mRNAs (hnRNPA1, p27(kip1)and ß-catenin) in a manner that is dependent on the QKI response element. An anti-mitogenic signal that induces cell cycle arrest at G1 phase elicits polyadenylation and translation of p27(kip1)mRNA via QKI and PAPD4. Taken together, our findings provide significant new insight into a general mechanism for positive regulation of gene expression by post-transcriptional polyadenylation in somatic cells.

RNA Exosome Complex Regulates Stability of the Hepatitis B Virus X-mRNA Transcript in a Non-stop-mediated (NSD) RNA Quality Control Mechanism.

Hepatitis B virus (HBV) is a stealth virus, minimally inducing the interferon system required for efficient induction of both innate and adaptive immune responses. However, 90% of acutely infected adults can clear the virus, suggesting the presence of other, interferon-independent pathways leading to viral clearance. Given the known ability of helicases to bind viral nucleic acids, we performed a functional screening assay to identify helicases that regulate HBV replication. We identified the superkiller viralicidic activity 2-like (SKIV2L) RNA helicase (a homolog of the Saccharomyces cerevisiae Ski2 protein) on the basis of its direct and preferential interaction with HBV X-mRNA. This interaction was essential for HBV X-mRNA degradation at the RNA exosome. The degradation of HBV X-mRNA at the RNA exosome was also mediated by HBS1L (HBS1-like translational GTPase) protein, a known component of the host RNA quality control system. We found that the redundant HBV-precore translation initiation site present at the 3'-end of HBV X-mRNA (3' precore) is translationally active. The initiation of translation from this site without a proper stop codon was identified by the non-stop-mediated RNA decay mechanism leading to its degradation. Although 3' precore is present in the five main HBV-RNA transcripts, only X-mRNA lacks the presence of an upstream start codons for large, middle, and small (L, M, and S) HBV surface proteins. These upstream codons are in-frame with 3' precore translation initiation site, blocking its translation from the other HBV-mRNA transcripts. To our knowledge, this is the first demonstration of the anti-viral function of the non-stop-mediated RNA decay mechanism.

Anti-proliferative protein Tob negatively regulates CPEB3 target by recruiting Caf1 deadenylase.

Tob is a member of the anti-proliferative protein family, which functions in transcription and mRNA decay. We have previously demonstrated that Tob is involved in the general mechanism of mRNA decay by mediating mRNA deadenylation through interaction with Caf1 and a general RNA-binding protein, PABPC1. Here, we focus on the role of Tob in the regulation of specific mRNA. We show that Tob binds directly to a sequence-specific RNA-binding protein, cytoplasmic polyadenylation element-binding protein 3 (CPEB3). CPEB3 negatively regulates the expression of a target by accelerating deadenylation and decay of its mRNA, which it achieves by tethering to the mRNA. The carboxyl-terminal RNA-binding domain of CPEB3 binds to the carboxyl-terminal unstructured region of Tob. Tob then binds Caf1 deadenylase and recruits it to CPEB3 to form a ternary complex. The CPEB3-accelerated deadenylation was abrogated by a dominant-negative mutant of either Caf1 or Tob. Together, these results indicate that Tob mediates the recruitment of Caf1 to the target of CPEB3 and elicits deadenylation and decay of the mRNA. Our results provide an explanation of how Tob regulates specific biological processes.

Structural and functional changes in retinal vasculature induced by retinal ischemia-reperfusion in rats.

Recent studies have shown retinal blood vessel damage in experimental models of retinal degeneration. The present study aimed to provide a detailed description of the structural and functional changes in retinal vasculature induced by retinal ischemia-reperfusion (I/R) in rats. Retinal ischemia was induced for 60 min by raising the intraocular pressure to 130 mmHg. Morphological changes in vascular components (endothelial cells, pericytes, and basement membranes), the patency and perfusion of blood vessels, and expression of vascular endothelial growth factor (VEGF) were assessed in the retinas at 2, 7, and 14 days after I/R. Significant reductions in vascular densities were observed at 7 and 14 days after I/R. Pericyte loss occurred after the appearance of endothelial cell degeneration, whereas the vascular basement membranes remained unchanged. Some vessels showed no perfusion in damaged retina. A decrease in the immunoreactivity of VEGF in the region extending from the ganglion cell layer to the outer plexiform layer was evident 2 days after I/R. In retinal I/R model, retinal ganglion cells are rapidly (<2 day) damaged following reperfusion, therefore, the current results suggest that neuronal cell damage precedes capillary degeneration, and neuronal cells may play an important role in maintaining vascular structure and function through the production and release of endothelial cell survival factors, including VEGF. Neuronal cell damage could be an additional cause of progression of ischemic retinal damage by reducing blood supply to the retinal neurons due to the destruction of the blood vessel network.

The Hbs1-Dom34 protein complex functions in non-stop mRNA decay in mammalian cells.

In yeast, aberrant mRNAs lacking in-frame termination codons are recognized and degraded by the non-stop decay (NSD) pathway. The recognition of non-stop mRNAs involves a member of the eRF3 family of GTP-binding proteins, Ski7. Ski7 is thought to bind the ribosome stalled at the 3'-end of the mRNA poly(A) tail and recruit the exosome to degrade the aberrant message. However, Ski7 is not found in mammalian cells, and even the presence of the NSD mechanism itself has remained enigmatic. Here, we show that unstable non-stop mRNA is degraded in a translation-dependent manner in mammalian cells. The decay requires another eRF3 family member (Hbs1), its binding partner Dom34, and components of the exosome-Ski complex (Ski2/Mtr4 and Dis3). Hbs1-Dom34 binds to form a complex with the exosome-Ski complex. Also, the elimination of aberrant proteins produced from non-stop transcripts requires the RING finger protein listerin. These findings demonstrate that the NSD mechanism exists in mammalian cells and involves Hbs1, Dom34, and the exosome-Ski complex.

Arsenite inhibits mRNA deadenylation through proteolytic degradation of Tob and Pan3.

The poly(A) tail of mRNAs plays pivotal roles in the posttranscriptional control of gene expression at both translation and mRNA stability. Recent findings demonstrate that the poly(A) tail is globally stabilized by some stresses. However, the mechanism underlying this phenomenon has not been elucidated. Here, we show that arsenite-induced oxidative stress inhibits deadenylation of mRNA primarily through downregulation of Tob and Pan3, both of which mediate the recruitment of deadenylases to mRNA. Arsenite selectively induces the proteolytic degradation of Tob and Pan3, and siRNA-mediated knockdown of Tob and Pan3 recapitulates stabilization of the mRNA poly(A) tail observed during arsenite stress. Although arsenite also inhibits translation by activating the eIF2α kinase HRI, arsenite-induced mRNA stabilization can be observed under HRI-depleted conditions. These results highlight the essential role of Tob and Pan3 in the stress-induced global stabilization of mRNA.

The processed isoform of the translation termination factor eRF3 localizes to the nucleus to interact with the ARF tumor suppressor.

The eukaryotic releasing factor eRF3 is a multifunctional protein that plays pivotal roles in translation termination as well as the initiation of mRNA decay. eRF3 also functions in the regulation of apoptosis; eRF3 is cleaved at Ala73 by an as yet unidentified protease into processed isoform of eRF3 (p-eRF3), which interacts with the inhibitors of apoptosis proteins (IAPs). The binding of p-eRF3 with IAPs leads to the release of active caspases from IAPs, which promotes apoptosis. Although full-length eRF3 is localized exclusively in the cytoplasm, p-eRF3 localizes in the nucleus as well as the cytoplasm. We here focused on the role of p-eRF3 in the nucleus. We identified leptomycin-sensitive nuclear export signal (NES) at amino acid residues 61-71 immediately upstream of the cleavage site Ala73. Thus, the proteolytic cleavage of eRF3 into p-eRF3 leads to release an amino-terminal fragment containing NES to allow the relocalization of eRF3 into the nucleus. Consistent with this, p-eRF3 more strongly interacted with the nuclear ARF tumor suppressor than full-length eRF3. These results suggest that while p-eRF3 interacts with IAPs to promote apoptosis in the cytoplasm, p-eRF3 also has some roles in regulating cell death in the nucleus.

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).