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Small-molecule compounds that elicit mRNA-selective translation repression have attracted interest due to their potential for expansion of druggable space. However, only a limited number of examples have been reported to date. Here, we show that desmethyl desamino pateamine A (DMDA-PatA) represses translation in an mRNA-selective manner by clamping eIF4A, a DEAD-box RNA-binding protein, onto GNG motifs. By systematically comparing multiple eIF4A inhibitors by ribosome profiling, we found that DMDA-PatA has unique mRNA selectivity for translation repression. Unbiased Bind-n-Seq reveals that DMDA-PatA-targeted eIF4A exhibits a preference for GNG motifs in an ATP-independent manner. This unusual RNA binding sterically hinders scanning by 40S ribosomes. A combination of classical molecular dynamics simulations and quantum chemical calculations, and the subsequent development of an inactive DMDA-PatA derivative reveals that the positive charge of the tertiary amine on the trienyl arm induces G selectivity. Moreover, we identified that DDX3, another DEAD-box protein, is an alternative DMDA-PatA target with the same effects on eIF4A. Our results provide an example of the sequence-selective anchoring of RNA-binding proteins and the mRNA-selective inhibition of protein synthesis by small-molecule compounds. Here the authors report that DMDA-PatA, an anti-tumor compound, functions as a mRNA-selective translational inhibitor. This drug clamps the target eIF4A and DDX3 RNA-binding proteins on the GNG RNA motif, providing steric hindrance for scanning ribosomes. [ABSTRACT FROM AUTHOR]

The intraflagellar transport (IFT) machinery plays a crucial role in the bidirectional trafficking of components necessary for ciliary signaling, such as the Hedgehog, Wnt/PCR, and cAMP/PKA systems. Defects in some components of the IFT machinery cause dysfunction, leading to a wide range of human diseases and developmental disorders termed ciliopathies, such as nephronophthisis. The IFT machinery comprises three sub-complexes: BBsome, IFT-A, and IFT-B. The IFT protein 54 (IFT54) is an important component of the IFT-B sub-complex. In anterograde movement, IFT54 binds to active kinesin-II, walking along the cilia microtubule axoneme and carrying the dynein-2 complex in an inactive state, which works for retrograde movement. Several mutations in IFT54 are known to cause Senior-Loken syndrome, a ciliopathy. IFT54 possesses a divergent Calponin Homology (CH) domain termed as NN-CH domain at its N-terminus. However, several aspects of the function of the NN-CH domain of IFT54 are still obscure. Here, we report the 1H, 15N, and 13C resonance assignments of the NN-CH domain of human IFT54 and its solution structure. The NN-CH domain of human IFT54 adopts essentially the α1–α2–α3–α4–α5 topology as that of mouse IFT54, whose structure was determined by X-ray crystallographic study. The structural information and assignments obtained in this study shed light on the molecular function of the NN-CH domain in IFT54. [ABSTRACT FROM AUTHOR]

Ribosome biogenesis is a complicated, multistage process coordinated by ribosome assembly factors. Ribosome binding factor A (RbfA) is a bacterial one, which possesses a single structural type-II KH domain. By this domain, RbfA binds to a 16S rRNA precursor in small ribosomal subunits to promote its 5′-end processing. The human RbfA homolog, mtRbfA, binds to 12S rRNAs in the mitoribosomal small subunits and promotes its critical maturation process, the dimethylation of two highly conserved consecutive adenines, which differs from that of RbfA. However, the structural basis of the mtRbfA-mediated maturation process is poorly understood. Herein, we report the 1H, 15N, and 13C resonance assignments of the KH domain of mtRbfA and its solution structure. The mtRbfA domain adopts essentially the same α1–β1–β2–α2(kinked)–β3 topology as the type-II KH domain. Comparison with the RbfA counterpart showed structural differences in specific regions that function as a putative RNA-binding site. Particularly, the α2 helix of mtRbfA forms a single helix with a moderate kink at the Ser-Ala-Ala sequence, whereas the corresponding α2 helix of RbfA is interrupted by a distinct kink at the Ala-x-Gly sequence, characteristic of bacterial RbfA proteins, to adopt an α2-kink-α3 conformation. Additionally, the region linking α1 and β1 differs considerably in the sequence and structure between RbfA and mtRbfA. These findings suggest some variations of the RNA-binding mode between them and provide a structural basis for mtRbfA function in mitoribosome biogenesis. [ABSTRACT FROM AUTHOR]

Matrin-3 is a multifunctional protein that binds to both DNA and RNA. Its DNA-binding activity is linked to the formation of the nuclear matrix and transcriptional regulation, while its RNA-binding activity is linked to mRNA metabolism including splicing, transport, stabilization, and degradation. Correspondingly, Matrin-3 has two zinc finger domains for DNA binding and two consecutive RNA recognition motif (RRM) domains for RNA binding. Matrin-3 has been reported to cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) when its disordered region contains pathogenic mutations. Simultaneously, it has been shown that the RNA-binding activity of Matrin-3 mediated by its RRM domains, affects the formation of insoluble cytoplasmic granules, which are related to the pathogenic mechanism of ALS/FTD. Thus, the effect of the RRM domains on the phase separation of condensed protein/RNA mixtures has to be clarified for a comprehensive understanding of ALS/FTD. Here, we report the 1H, 15N, and 13C resonance assignments of the two RNA binding domains and their solution structures. The resonance assignments and the solution structures obtained in this work will contribute to the elucidation of the molecular basis of Matrin-3 in the pathogenic mechanism of ALS and/or FTD. [ABSTRACT FROM AUTHOR]

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotide molecules on its substrate, unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. However, its precise mechanism has remained unclear. Here, our cryo-electron microscopy (cryo-EM) analysis reveals that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B retains nucleotide exchange activity even in the presence of eIF2(αP), in line with the cryo-EM structures of the eIF2B•SFSV NSs•unphosphorylated eIF2 complex. A genome-wide ribosome profiling analysis clarified that SFSV NSs expressed in cultured human cells attenuates the ISR triggered by thapsigargin, an endoplasmic reticulum stress inducer. Furthermore, SFSV NSs introduced in rat hippocampal neurons and human induced-pluripotent stem (iPS) cell-derived motor neurons exhibits neuroprotective effects against the ISR-inducing stress. Since ISR inhibition is beneficial in various neurological disease models, SFSV NSs may be a promising therapeutic ISR inhibitor. Here the authors show that a viral protein interferes with the binding of phosphorylated eIF2 to eIF2B, thereby suppressing the host integrated stress response (ISR). This suppression of the ISR abrogates translational changes of the host and ameliorates neurite degradation under stress. [ABSTRACT FROM AUTHOR]

In humans, YTH (YT521-B homology) domain containing protein 2 (YTHDC2) plays a crucial role in the phase-shift from mitosis to meiosis. YTH domains bind to methylated adenosine nucleotides such as m6A. In a phylogenic tree, the YTH domain of YTHDC2 (YTH2) and that of the YTH containing protein YTHDC1 (YTH1) belong to the same sub-group. However, the binding affinity of m6A differs between these proteins. Here, we report 1H, 13C and 15N resonance assignment of YTH2 and its solution structure to examine the difference of the structural architecture and the dynamic properties of YTH1 and YTH2. YTH2 adopts a β1–α1–β2–α2–β3–β4–β5–α3–β6–α4 topology, which was also observed in YTH1. However, the β4–β5 loops of YTH1 and YTH2 are distinct in length and amino acid composition. Our data revealed that, unlike in YTH1, the structure of m6A-binding pocket of YTH2 formed by the β4–β5 loop is stabilized by electrostatic interaction. This assignment and the structural information for YTH2 will provide the insight on the further functional research of YTHDC2. [ABSTRACT FROM AUTHOR]

Our aim was to evaluate the association between ritodrine and magnesium sulfate (MgSO4) and the occurrence of neonatal hyperkalemia or hypoglycemia among late preterm infants in a retrospective cohort study. We used a nationwide obstetrical database from 2014. A total of 4,622 live preterm infants born at 32–36 gestational weeks participated. Fourteen risk factors based on both clinical relevance and univariate analysis were adjusted in multivariable logistic regression analyses. Neonatal hyperkalemia and hypoglycemia occurred in 7.6% (284/3,732) and 32.4% (1,458/4,501), respectively. Occurrence of hyperkalemia was associated with concomitant usage of ritodrine and MgSO4 compared with no usage (adjusted odds ratio [aOR] 1.53, 95% confidence interval [CI] 1.09–2.15). Occurrence of hypoglycemia was associated with ritodrine alone (aOR 2.58 [CI 2.21–3.01]) and with concomitant usage of ritodrine and MgSO4 (aOR 2.59 [CI 2.13–3.15]), compared with no usage, and was associated with long-term usage (≥ 48 hours) of ritodrine and cessation directly before delivery. In conclusion, in late preterm infants, usage of ritodrine together with MgSO4 was associated with occurrence of critical neonatal hyperkalemia, and long-term usage of ritodrine and cessation directly before delivery were associated with neonatal hypoglycemia. [ABSTRACT FROM AUTHOR]

To mitigate the damage caused by debris flows resulting from heavy precipitation and to aid in evacuation plan preparation, areas at risk should be mapped on a scale appropriate for affected individuals and communities. We tested the effectiveness of simply identifying debris-flow hazards through automated derivation of surface curvatures using LiDAR digital elevation models. We achieved useful correspondence between plan curvatures and areas of existing debris-flow damage in two localities in Japan using the analysis of digital elevation models (DEMs). We found that plan curvatures derived from 10m DEMs may be useful to indicate areas that are susceptible to debris flow in mountainous areas. In residential areas located on gentle sloping debris flow fans, the greatest damage to houses was found to be located in the elongated depressions that are connected to mountain stream valleys. Plan curvature derived from 5m DEM was the most sensitive indicators for susceptibility to debris flows. [ABSTRACT FROM AUTHOR]

Purpose: To describe the clinical features and surgical outcomes of macular dragging or tractional retinal detachment that occurred as a result of fibrovascular tissue (FT) progression toward the posterior retina in eyes with familial exudative vitreoretinopathy (FEVR). Methods: The medical records of 4 patients (2 girls, 2 boys) with FEVR were reviewed retrospectively. Results: All 4 patients had retinal dragging or radial retinal folds with FT in the peripheral retina in at least 1 eye at the initial visit. During the follow-up period, all the patients had FT that progressed toward the posterior pole and developed from the peripheral FT, resulting in macular dragging or posterior tractional retinal detachment. Vitrectomy with or without scleral buckling and laser photocoagulation were performed in all 4 cases, with removal of the FT in the posterior pole and segmentation of the FT between the posterior and peripheral FT. The traction resolved postoperatively and the retina extended in all 4 cases. Conclusions: In 4 cases with FEVR, the FT developed from the peripheral FT, progressed toward the posterior retina, and generated traction. Vitrectomy with focal removal and segmentation of the FT in the posterior pole might be a good surgical option in such eyes. [ABSTRACT FROM AUTHOR]

The crystal structure of Schizosaccharomyces pombe guanine nucleotide exchange factor eIF2B, providing a structural framework for the eIF2B-mediated mechanism of stress-induced translational control.Translation initiation factor 2B structure: The guanine nucleotide exchange factor, eIF2B, is key to eukaryotic translation initiation, acting on the γ subunit of eIF2. During stress, the α subunit of eIF2 is phosphorylated; this inhibits the activity of eIF2B, resulting in downregulation of general translation. Shigeyuki Yokoyama and colleagues have now solved the structure of the ten-subunit eIF2B complex of fission yeast. The regulatory hexameric core is bounded on opposite faces by dimeric catalytic subcomplexes. The sites of interaction with the eIF2α and eIF2γ subunits are identified. Modelling reveals how phosophorylation of the α subunit affects its binding to eIF2B, and how assembly on the non-productive eIF2–eIF2B complex inhibits nucleotide exchange on eIF2γ.Eukaryotic cells restrict protein synthesis under various stress conditions, by inhibiting the eukaryotic translation initiation factor 2B (eIF2B)1,2. eIF2B is the guanine nucleotide exchange factor for eIF2, a heterotrimeric G protein consisting of α-, β- and γ-subunits. eIF2B exchanges GDP for GTP on the γ-subunit of eIF2 (eIF2γ), and is inhibited by stress-induced phosphorylation of eIF2α. eIF2B is a heterodecameric complex of two copies each of the α-, β-, γ-, δ- and ε-subunits3; its α-, β- and δ-subunits constitute the regulatory subcomplex4, while the γ- and ε-subunits form the catalytic subcomplex5. The three-dimensional structure of the entire eIF2B complex has not been determined. Here we present the crystal structure of Schizosaccharomyces pombe eIF2B with an unprecedented subunit arrangement, in which the α2β2δ2 hexameric regulatory subcomplex binds two γε dimeric catalytic subcomplexes on its opposite sides. A structure-based in vitro analysis by a surface-scanning site-directed photo-cross-linking method identified the eIF2α-binding and eIF2γ-binding interfaces, located far apart on the regulatory and catalytic subcomplexes, respectively. The eIF2γ-binding interface is located close to the conserved ‘NF motif’, which is important for nucleotide exchange. A structural model was constructed for the complex of eIF2B with phosphorylated eIF2α, which binds to eIF2B more strongly than the unphosphorylated form. These results indicate that the eIF2α phosphorylation generates the ‘nonproductive’ eIF2–eIF2B complex5, which prevents nucleotide exchange on eIF2γ, and thus provide a structural framework for the eIF2B-mediated mechanism of stress-induced translational control. [ABSTRACT FROM AUTHOR]

Tissue-specific alternative pre-mRNA splicing is often cooperatively regulated by multiple splicing factors, but the structural basis of cooperative RNA recognition is poorly understood. In Caenorhabditis elegans, ligand binding specificity of fibroblast growth factor receptors (FGFRs) is determined by mutually exclusive alternative splicing of the sole FGFR gene, egl-15. Here we determined the solution structure of a ternary complex of the RNA-recognition motif (RRM) domains from the RBFOX protein ASD-1, SUP-12 and their target RNA from egl-15. The two RRM domains cooperatively interact with the RNA by sandwiching a G base to form the stable complex. Multichromatic fluorescence splicing reporters confirmed the requirement of the G and the juxtaposition of the respective cis elements for effective splicing regulation in vivo. Moreover, we identified a new target for the heterologous complex through an element search, confirming the functional significance of the intermolecular coordination. [ABSTRACT FROM AUTHOR]

Antigen-specific cancer immunotherapy is a promising strategy for improving cancer treatment. Recently, many tumor-associated antigens and their epitopes recognized by cytotoxic T lymphocytes (CTLs) have been identified. However, the density of endogenously presented antigen-derived peptides on tumor cells is generally sparse, resulting in the inability of antigen-specific CTLs to work effectively. We hypothesize that increasing the density of an antigen-derived peptide would enhance antigen-specific cancer immunotherapy. Here, we demonstrated that intratumoral peptide injection leads to additional peptide loading onto major histocompatibility complex class I molecules of tumor cells, enhancing tumor cell recognition by antigen-specific CTLs. In in vitro studies, human leukocyte antigen (HLA)-A*02:01-restricted glypican-3 (FVGEFFTDV) and cytomegalovirus (NLVPMVATV) peptide-specific CTLs showed strong activity against all peptide-pulsed cell lines, regardless of whether the tumor cells expressed the antigen. In in vivo studies using immunodeficient mice, glypican-3 and cytomegalovirus peptides injected into a solid mass were loaded onto HLA class I molecules of tumor cells. In a peptide vaccine model and an adoptive cell transfer model using C57BL/6 mice, intratumoral injection of ovalbumin peptide (SIINFEKL) was effective for tumor growth inhibition and survival against ovalbumin-negative tumors without adverse reactions. Moreover, we demonstrated an antigen-spreading effect that occurred after intratumoral peptide injection. Intratumoral peptide injection enhances tumor cell antigenicity and may be a useful option for improvement in antigen-specific cancer immunotherapy against solid tumors. [ABSTRACT FROM AUTHOR]