FMRP Enhances the Translation of 4EBP2 mRNA during Neuronal Differentiation.

FMRP is a multifunctional protein encoded by the Fragile X Messenger Ribonucleoprotein 1 gene (FMR1). The inactivation of the FMR1 gene results in fragile X syndrome (FXS), a serious neurodevelopmental disorder. FMRP deficiency causes abnormal neurite outgrowth, which is likely to lead to abnormal learning and memory capabilities. However, the mechanism of FMRP in modulating neuronal development remains unknown. We found that FMRP enhances the translation of 4EBP2, a neuron-specific form of 4EBPs that inactivates eIF4E by inhibiting the interaction between eIF4E and eIF4G. Depletion of 4EBP2 results in abnormal neurite outgrowth. Moreover, the impairment of neurite outgrowth upon FMRP depletion was overcome by the ectopic expression of 4EBP2. These results suggest that FMRP controls neuronal development by enhancing 4EBP2 expression at the translational level. In addition, treatment with 4EGI-1, a chemical that blocks eIF4E activity, restored neurite length in FMRP-depleted and 4EBP2-depleted cells. In conclusion, we discovered that 4EBP2 functions as a key downstream regulator of FMRP activity in neuronal development and that FMRP represses eIF4E activity by enhancing 4EBP2 translation.

Structure-Guided Development of Bivalent Aptamers Blocking SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused devastation to human society through its high virulence, infectivity, and genomic mutations, which reduced the efficacy of vaccines. Here, we report the development of aptamers that effectively interfere with SARS-CoV-2 infection by targeting its spike protein, which plays a pivotal role in host cell entry of the virus through interaction with the viral receptor angiotensin-converting enzyme 2 (ACE2). To develop highly effective aptamers and to understand their mechanism in inhibiting viral infection, we determined the three-dimensional (3D) structures of aptamer/receptor-binding domain (RBD) complexes using cryogenic electron microscopy (cryo-EM). Moreover, we developed bivalent aptamers targeting two distinct regions of the RBD in the spike protein that directly interact with ACE2. One aptamer interferes with the binding of ACE2 by blocking the ACE2-binding site in RBD, and the other aptamer allosterically inhibits ACE2 by binding to a distinct face of RBD. Using the 3D structures of aptamer-RBD complexes, we minimized and optimized these aptamers. By combining the optimized aptamers, we developed a bivalent aptamer that showed a stronger inhibitory effect on virus infection than the component aptamers. This study confirms that the structure-based aptamer-design approach has a high potential in developing antiviral drugs against SARS-CoV-2 and other viruses.

PLK1-ELAVL1/HuR-miR-122 signaling facilitates hepatitis C virus proliferation.

The liver-specific microRNA, miR-122, plays an essential role in the propagation of hepatitis C virus (HCV) by binding directly to the 5'-end of its genomic RNA. Despite its significance for HCV proliferation, the host factors responsible for regulating miR-122 remain largely unknown. In this study, we identified the cellular RNA-binding protein, ELAVL1/HuR (embryonic lethal-abnormal vision-like 1/human antigen R), as critically contributing to miR-122 biogenesis by strong binding to the 3'-end of miR-122. The availability of ELAVL1/HuR was highly correlated with HCV proliferation in replicon, infectious, and chronically infected patient conditions. Furthermore, by screening a kinase inhibitor library, we identified rigosertib, an anticancer agent under clinical trials, as having both miR-122-modulating and anti-HCV activities that were mediated by its ability to target polo-like kinase 1 (PLK1) and subsequently modulate ELAVL1/HuR-miR-122 signaling. The expression of PLK1 was also highly correlated with HCV proliferation and the HCV positivity of HCC patients. ELAVL1/HuR-miR-122 signaling and its mediation of PLK1-dependent HCV proliferation were demonstrated by performing various rescue experiments and utilizing an HCV mutant with low dependency on miR-122. In addition, the HCV-inhibitory effectiveness of rigosertib was validated in various HCV-relevant conditions, including replicons, infected cells, and replicon-harboring mice. Rigosertib was highly effective in inhibiting the proliferation of not only wild-type HCVs, but also sofosbuvir resistance-associated substitution-bearing HCVs. Our study identifies PLK1-ELAVL1/HuR-miR-122 signaling as a regulatory axis that is critical for HCV proliferation, and suggests that a therapeutic approach targeting this host cell signaling pathway could be useful for treating HCV and HCV-associated diseases.

A Simple and Effective Method to Concentrate Hepatitis C Virus: Aqueous Two-Phase System Allows Highly Efficient Enrichment of Enveloped Viruses.

To investigate the proliferation cycle of a virus, virus-host interaction, and pathogenesis of a virus, virion particles must be concentrated from the media of virus cell culture or the sera of virus-infected patients. Ultracentrifugation of the culture media is a standard method for concentrating virion particles. However, this method is time-consuming and requires special equipment (ultracentrifuge). Moreover, a large number of infectious viruses are lost during enrichment. We developed a new method of hepatitis C virus (HCV) concentration to overcome the issues associated with traditional methods of virus concentration. We used an aqueous two-phase system (ATPS) to concentrate the virus. HCV, which causes various liver diseases, such as liver fibrosis, cirrhosis, and hepatocellular carcinoma, was used as a model virus to test the efficacy and reliability of the ATPS. The efficiency of HCV concentration by the ATPS was approximately three times higher than that by ultracentrifugation. Moreover, the infectivity of the concentrated HCV, which is a labile virus, remained the same after concentration of the virus by the ATPS. Considering the simplicity and effectiveness of the ATPS, it is the method of choice for concentrating viruses.

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 Beginning of Ending Hepatitis C Virus: A Summary of the 26th International Symposium on Hepatitis C Virus and Related Viruses.

: Hepatitis C virus (HCV) infects ~71 million people worldwide, and 399,000 people die annually due to HCV-related liver cirrhosis and hepatocellular carcinoma. The use of direct-acting antivirals results in a sustained virologic response in >95% of patients with chronic HCV infection. However, several issues remain to be solved to eradicate HCV. At the 26th International Symposium on Hepatitis C Virus and Related Viruses (HCV2019) held in Seoul, South Korea, October 5-8, 2019, virologists, immunologists, and clinical scientists discussed these remaining issues and how we can achieve the elimination of HCV.

A cyclin-dependent kinase, CDK11/p58, represses cap-dependent translation during mitosis.

During mitosis, translation of most mRNAs is strongly repressed; none of the several explanatory hypotheses suggested can fully explain the molecular basis of this phenomenon. Here we report that cyclin-dependent CDK11/p58-a serine/threonine kinase abundantly expressed during M phase-represses overall translation by phosphorylating a subunit (eIF3F) of the translation factor eIF3 complex that is essential for translation initiation of most mRNAs. Ectopic expression of CDK11/p58 strongly repressed cap-dependent translation, and knockdown of CDK11/p58 nullified the translational repression during M phase. We identified the phosphorylation sites in eIF3F responsible for M phase-specific translational repression by CDK11/p58. Alanine substitutions of CDK11/p58 target sites in eIF3F nullified its effects on cell cycle-dependent translational regulation. The mechanism of translational regulation by the M phase-specific kinase, CDK11/p58, has deep evolutionary roots considering the conservation of CDK11 and its target sites on eIF3F from C. elegans to humans.

Structure-activity relationships of fluorene compounds inhibiting HCV variants.

Approximately 71 million people suffer from hepatitis C virus (HCV) infection worldwide. Persistent HCV infection causes liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, resulting in approximately 400,000 deaths annually. Effective direct-acting antiviral agents (DAAs) have been developed and are currently used for HCV treatment targeting the following three proteins: NS3/4A proteinase that cleaves the HCV polyprotein into various functional proteins, RNA-dependent RNA polymerase (designated as NS5B), and NS5A, which is required for the formation of double membrane vesicles serving as RNA replication organelles. At least one compound inhibiting NS5A is included in current HCV treatment regimens due to the high efficacy and low toxicity of drugs targeting NS5A. Here we report fluorene compounds showing strong inhibitory effects on GT 1b and 3a of HCV. Moreover, some compounds were effective against resistance-associated variants to DAAs. The structure-activity relationships of the compounds were analyzed. Furthermore, we investigated the molecular bases of the inhibitory activities of some compounds by the molecular docking method.

Virus-based SELEX (viro-SELEX) allows development of aptamers targeting knotty proteins.

It has been 100 years since the worst flu (Spanish flu) mankind has ever experienced. Rapid, accurate diagnosis and subtyping of flu are still an urgent unmet medical need. By using surrogate virus-based SELEX (viro-SELEX), we report here multiple advances incorporated into the field of flu diagnostics: (i) aptamers that can bind to the native virus well even though they cannot bind strongly to a recombinant protein (hemagglutinin); (ii) a couple of aptamers that can target a broad range of strains belonging to the H1N1 subtype and detect only the H1N1 subtype and nothing else; (iii) a highly sensitive lateral flow assay system (limit of detection is 0.08 HAU) using fluorescence-tagged aptamers. The viro-SELEX method of aptamer selection in conjunction with a fluorescent tag on aptamers is a very useful approach to develop highly sensitive, specific, portable, rapid, and quantitative point-of-care testing diagnostic tools for the future.

RACK1 mediates rewiring of intracellular networks induced by hepatitis C virus infection.

Hepatitis C virus (HCV) is a positive-strand RNA virus replicating in a membranous replication organelle composed primarily of double-membrane vesicles (DMVs) having morphological resemblance to autophagosomes. To define the mechanism of DMV formation and the possible link to autophagy, we conducted a yeast two-hybrid screening revealing 32 cellular proteins potentially interacting with HCV proteins. Among these was the Receptor for Activated Protein C Kinase 1 (RACK1), a scaffolding protein involved in many cellular processes, including autophagy. Depletion of RACK1 strongly inhibits HCV RNA replication without affecting HCV internal ribosome entry site (IRES) activity. RACK1 is required for the rewiring of subcellular membranous structures and for the induction of autophagy. RACK1 binds to HCV nonstructural protein 5A (NS5A), which induces DMV formation. NS5A interacts with ATG14L in a RACK1 dependent manner, and with the ATG14L-Beclin1-Vps34-Vps15 complex that is required for autophagosome formation. Both RACK1 and ATG14L are required for HCV DMV formation and viral RNA replication. These results indicate that NS5A participates in the formation of the HCV replication organelle through interactions with RACK1 and ATG14L.

Development of a Subtype-Specific Diagnostic System for Influenza Virus H3N2 Using a Novel Virus-Based Systematic Evolution of Ligands by Exponential Enrichment (Viro-SELEX).

Aptamers are oligonucleotide molecules that bind to specific target molecules generated by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have high future potential for use in diagnostics and therapeutics as molecular probes that recognize target molecules. To develop aptamers against a target protein using a SELEX process, it is necessary to purify the target protein. Purifying a membrane protein, however, is usually a challenging task. Here, we report a novel approach to developing aptamers against membrane proteins. Surrogate viruses containing target proteins on the surface of an enveloped virus (e.g., baculovirus), instead of purified proteins, were used in a new SELEX process. We designated this new SELEX process as "surrogate virus-based SELEX (viro-SELEX)." Using viro-SELEX, we developed a pair of aptamers that specifically interact with the hemagglutinin protein of influenza subtype H3N2. Using the aptamer pair and a lateral flow assay system, we developed a very sensitive point-of-care diagnostic system for specifically detecting influenza virus subtype H3N2.

The RNA-binding protein hnRNP Q represses translation of the clock gene Bmal1 in murine cells.

Most living creatures have a circadian rhythm that is generated by a precisely regulated transcriptional-translational feedback loop of clock genes. Brain and muscle ARNT-like 1 (BMAL1) is one of the core clock genes and transcription factors that represents a positive arm of this autoregulatory circadian clock system. Despite the indispensable role of BMAL1 in the circadian rhythm, the molecular mechanisms underlying translational control of BMAL1 are largely unknown. Here, using murine NIH-3T3 cells, gene constructs, and a variety of biochemical approaches, including RNAi- and luciferase reporter gene-based assays, along with immunoblotting, in vitro transcription, quantitative real-time PCR, and real-time bioluminescence experiments, we show that translation of Bmal1 is negatively regulated by an RNA-binding protein, heterogeneous nuclear ribonucleoprotein Q (hnRNP Q). Interestingly, we found that hnRNP Q rhythmically binds to a specific region of the Bmal1 mRNA 5' UTR and controls its time-dependent expression. Moreover, we demonstrate that knockdown of hnRNP Q modulates BMAL1 protein oscillation amplitude without affecting mRNA rhythmic patterns. Furthermore, hnRNP Q depletion increases the mRNA oscillation amplitudes of BMAL1-regulated target genes. Together, our results suggest that hnRNP Q plays a pivotal role in both Bmal1 translation and BMAL1-regulated gene expression.

Upf1 regulates neurite outgrowth and branching by transcriptional and post-transcriptional modulation of Arc.

A large number of neuronal proteins must show correct spatiotemporal localization in order to carry out their critical functions. The mRNA transcript for the somatodendritic protein activity-regulated cytoskeleton-associated protein (Arc; also known as Arg3.1) contains two conserved introns in the 3' untranslated region (UTR), and was proposed to be a natural target for nonsense-mediated mRNA decay (NMD). However, a well-known NMD component Upf1 has differential roles in transcriptional and translational regulation of Arc gene expression. Specifically, Upf1 suppresses Arc transcription by enhancing destabilization of mRNAs encoding various transcription factors, including Mef2a. Upf1 also binds to the Arc 3'UTR, resulting in suppression of translation. Surprisingly, the Arc transcript escapes from Upf1-mediated NMD by binding to Ago2 (also known as miRISC), which blocks NMD and further suppresses Arc mRNA translation. Upf1 knockdown triggered sustained Arc expression, which contributes to Cofilin (also known as Cfl1) hyperphosphorylation and abnormal neuronal outgrowth and branching. Collectively, these data reveal that multiple levels of Upf1-mediated inhibition of Arc gene expression may allow neurons to more effectively respond to changes in neuronal activity.

Prefoldin 6 mediates longevity response from heat shock factor 1 to FOXO in C. elegans.

Heat shock factor 1 (HSF-1) and forkhead box O (FOXO) are key transcription factors that protect cells from various stresses. In Caenorhabditis elegans, HSF-1 and FOXO together promote a long life span when insulin/IGF-1 signaling (IIS) is reduced. However, it remains poorly understood how HSF-1 and FOXO cooperate to confer IIS-mediated longevity. Here, we show that prefoldin 6 (PFD-6), a component of the molecular chaperone prefoldin-like complex, relays longevity response from HSF-1 to FOXO under reduced IIS. We found that PFD-6 was specifically required for reduced IIS-mediated longevity by acting in the intestine and hypodermis. We showed that HSF-1 increased the levels of PFD-6 proteins, which in turn directly bound FOXO and enhanced its transcriptional activity. Our work suggests that the prefoldin-like chaperone complex mediates longevity response from HSF-1 to FOXO to increase the life span in animals with reduced IIS.

The mechanistic insight of a specific interaction between 15d-Prostaglandin-J2 and eIF4A suggests an evolutionary conserved role across species.

15-deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) is an anti-inflammatory/anti-neoplastic prostaglandin that functions through covalent binding to cysteine residues of various target proteins. We previously showed that 15d-PGJ2 mediated anti-inflammatory responses are dependent on the translational inhibition through its interaction with eIF4A (Kim et al., 2007). Binding of 15d-PGJ2 to eIF4A specifically blocks the interaction between eIF4G and eIF4A, which leads to the formation of stress granules (SGs), which then cluster mRNAs with inhibited translation. Here, we show that the binding between 15d-PGJ2 and eIF4A specifically blocks the interaction between the MIF4G domain of eIF4G and eIF4A. To reveal the mechanism of this interaction, we used computational simulation-based docking studies and identified that the carboxyl tail of 15d-PGJ2 could stabilize the binding of 15d-PGJ2 to eIF4A through arginine 295 of eIF4A, which is the first suggestion that the 15d-PGJ2 tail plays a physiological role. Interestingly, the putative 15d-PGJ2 binding site on eiF4A is conserved across many species, suggesting a biological role. Our data propose that studying 15d-PGJ2 and its targets may uncover new therapeutic approaches in anti-inflammatory drug discovery.

eIF2A, an initiator tRNA carrier refractory to eIF2α kinases, functions synergistically with eIF5B.

The initiator tRNA (Met-tRNA i Met ) at the P site of the small ribosomal subunit plays an important role in the recognition of an mRNA start codon. In bacteria, the initiator tRNA carrier, IF2, facilitates the positioning of Met-tRNA i Met on the small ribosomal subunit. Eukarya contain the Met-tRNA i Met carrier, eIF2 (unrelated to IF2), whose carrier activity is inhibited under stress conditions by the phosphorylation of its α-subunit by stress-activated eIF2α kinases. The stress-resistant initiator tRNA carrier, eIF2A, was recently uncovered and shown to load Met-tRNA i Met on the 40S ribosomal subunit associated with a stress-resistant mRNA under stress conditions. Here, we report that eIF2A interacts and functionally cooperates with eIF5B (a homolog of IF2), and we describe the functional domains of eIF2A that are required for its binding of Met-tRNA i Met , eIF5B, and a stress-resistant mRNA. The results indicate that the eukaryotic eIF5B-eIF2A complex functionally mimics the bacterial IF2 containing ribosome-, GTP-, and initiator tRNA-binding domains in a single polypeptide.

Translational control of phloem development by RNA G-quadruplex-JULGI determines plant sink strength.

The emergence of a plant vascular system was a prerequisite for the colonization of land; however, it is unclear how the photosynthate transporting system was established during plant evolution. Here, we identify a novel translational regulatory module for phloem development involving the zinc-finger protein JULGI (JUL) and its targets, the 5' untranslated regions (UTRs) of the SUPPRESSOR OF MAX2 1-LIKE4/5 (SMXL4/5) mRNAs, which is exclusively conserved in vascular plants. JUL directly binds and induces an RNA G-quadruplex in the 5' UTR of SMXL4/5, which are key promoters of phloem differentiation. We show that RNA G-quadruplex formation suppresses SMXL4/5 translation and restricts phloem differentiation. In turn, JUL deficiency promotes phloem formation and strikingly increases sink strength per seed. We propose that the translational regulation by the JUL/5' UTR G-quadruplex module is a major determinant of phloem establishment, thereby determining carbon allocation to sink tissues, and that this mechanism was a key invention during the emergence of vascular plants.

Sulfur(vi) fluoride exchange as a key reaction for synthesizing biaryl sulfate core derivatives as potent hepatitis C virus NS5A inhibitors and their structure-activity relationship studies.

Extremely potent, new hepatitis C virus (HCV) nonstructural 5A (NS5A) featuring substituted biaryl sulfate core structures was designed and synthesized. Based on the previously reported novel HCV NS5A inhibitors featuring biaryl sulfate core structures which exhibit two-digit picomolar half-maximal effective concentration (EC50) values against HCV genotype 1b and 2a, the new inhibitors equipped with the sulfate core structures containing diversely substituted aryl groups were explored. In this study, highly efficient, chemoselective coupling reactions between an arylsulfonyl fluoride and an aryl silyl ether, known as the sulfur(vi) fluoride exchange (SuFEx) reaction, were utilized. Among the inhibitors prepared based on the SuFEx chemistry, compounds 14, 15 and 29 exhibited two-digit picomolar EC50 values against GT-1b and single digit or sub nanomolar activities against the HCV GT-2a strain. Nonsymmetrical inhibitors containing an imidazole and amide moieties on each side of the sulfate core structures were also synthesized. In addition, a biotinylated probe targeting NS5A protein was prepared for labeling using the same synthetic methodology.

The bent conformation of poly(A)-binding protein induced by RNA-binding is required for its translational activation function.

A recent study revealed that poly(A)-binding protein (PABP) bound to poly(A) RNA exhibits a sharply bent configuration at the linker region between RNA-recognition motif 2 (RRM2) and RRM3, whereas free PABP exhibits a highly flexible linear configuration. However, the physiological role of the bent structure of mRNA-bound PABP remains unknown. We investigated a role of the bent structure of PABP by constructing a PABP variant that fails to form the poly(A)-dependent bent structure but maintains its poly(A)-binding activity. We found that the bent structure of PABP/poly(A) complex is required for PABP's efficient interaction with eIF4G and eIF4G/eIF4E complex. Moreover, the mutant PABP had compromised translation activation function and failed to augment the formation of 80S translation initiation complex in an in vitro translation system. These results suggest that the bent conformation of PABP, which is induced by the interaction with 3' poly(A) tail, mediates poly(A)-dependent translation by facilitating the interaction with eIF4G and the eIF4G/eIF4E complex. The preferential binding of the eIF4G/eIF4E complex to the bent PABP/poly(A) complex seems to be a mechanism discriminating the mRNA-bound PABPs participating in translation from the idling mRNA-unbound PABPs.

An mRNA-specific tRNAi carrier eIF2A plays a pivotal role in cell proliferation under stress conditions: stress-resistant translation of c-Src mRNA is mediated by eIF2A.

c-Src, a non-receptor protein tyrosine kinase, activates NF-κB and STAT3, which in turn triggers the transcription of anti-apoptosis- and cell cycle-related genes. c-Src protein regulates cell proliferation, cell motility and programmed cell death. And the elevated level of activated c-Src protein is related with solid tumor generation. Translation of c-Src mRNA is directed by an IRES element which mediates persistent translation under stress conditions when translation of most mRNAs is inhibited by a phosphorylation of the alpha subunit of eIF2 carrying the initiator tRNA (tRNAi) to 40S ribosomal subunit under normal conditions. The molecular basis of the stress-resistant translation of c-Src mRNA remained to be elucidated. Here, we report that eIF2A, an alternative tRNAi carrier, is responsible for the stress-resistant translation of c-Src mRNA. eIF2A facilitates tRNAi loading onto the 40S ribosomal subunit in a c-Src mRNA-dependent manner. And a direct interaction between eIF2A and a stem-loop structure (SL I) in the c-Src IRES is required for the c-Src IRES-dependent translation under stress conditions but not under normal conditions. Finally, we showed that the eIF2A-dependent translation of c-Src mRNA plays a pivotal role in cell proliferation under stress conditions.