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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 1 H, 15 N, and 13 C 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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The pre-spliceosomal complex involves interactions between U1 and U2 snRNPs, where a ubiquitin-like domain (ULD) of SF3A1, a component of U2 snRNP, binds to the stem-loop 4 (SL4; the UUCG tetraloop) of U1 snRNA in U1 snRNP. Here, we reported the 1.80 Å crystal structure of human SF3A1 ULD (ULDSF3A1) complexed with SL4. The structural part of ULDSF3A1 (res. 704-785) adopts a typical β-grasp fold with a topology of β1-β2-α1-310a-β3-β4-310b-β5, closely resembling that of ubiquitin, except for the length and structure of the β1/β2 loop. A patch on the surface formed by three ULDSF3A1-specific residues, Lys756 (β3), Phe763 (β4) and Lys765 (following β4), contacts the canonical UUCG tetraloop structure. In contrast, the directly following C-terminal tail composed of 786KERGGRKK793 was essentially stretched. The main or side chains of all the residues interacted with the major groove of the stem helix; the RGG residues adopted a peculiar conformation for RNA recognition. These findings were confirmed by mutational studies using bio-layer interferometry. Collectively, a unique combination of the β-grasp fold and the C-terminal tail constituting ULDSF3A1 is required for the SL4-specific binding. This interaction mode also suggests that putative post-translational modifications, including ubiquitination in ULDSF3A1, directly inhibit SL4 binding., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

A 61-year-old patient with cystic bronchiectasis and bronchial artery hyperplasia in the left lung was diagnosed with polymyositis-related interstitial lung disease. After nine months of immunosuppressive therapy, he developed unilateral autoimmune pulmonary alveolar proteinosis (APAP) in the right lung with respiratory failure. After bronchial artery embolization to prevent massive hemoptysis, whole-lung lavage was performed using veno-venous extracorporeal membrane oxygenation. His respiratory condition improved, and he was discharged from the hospital with supplemental oxygen. Three reported cases of APAP with polymyositis-related interstitial lung disease, including the present case, were all positive for anti-glycyl tRNA synthetase antibody and were under immunosuppressive treatment.

SURP domains are exclusively found in splicing-related proteins in all eukaryotes. SF3A1, a component of the U2 snRNP, has two tandem SURP domains, SURP1, and SURP2. SURP2 is permanently associated with a specific short region of SF3A3 within the SF3A protein complex whereas, SURP1 binds to the splicing factor SF1 for recruitment of U2 snRNP to the early spliceosomal complex, from which SF1 is dissociated during complex conversion. Here, we determined the solution structure of the complex of SURP1 and the human SF1 fragment using nuclear magnetic resonance (NMR) methods. SURP1 adopts the canonical topology of α1-α2-3 10 -α3, in which α1 and α2 are connected by a single glycine residue in a particular backbone conformation, allowing the two α-helices to be fixed at an acute angle. A hydrophobic patch, which is part of the characteristic surface formed by α1 and α2, specifically contacts a hydrophobic cluster on a 16-residue α-helix of the SF1 fragment. Furthermore, whereas only hydrophobic interactions occurred between SURP2 and the SF3A3 fragment, several salt bridges and hydrogen bonds were found between the residues of SURP1 and the SF1 fragment. This finding was confirmed through mutational studies using bio-layer interferometry. The study also revealed that the dissociation constant between SURP1 and the SF1 fragment peptide was approximately 20 μM, indicating a weak or transient interaction. Collectively, these results indicate that the interplay between U2 snRNP and SF1 involves a transient interaction of SURP1, and this transient interaction appears to be common to most SURP domains, except for SURP2., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

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 1 H, 15 N, and 13 C 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., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Cryobiopsy enables us to obtain larger specimens than conventional forceps biopsy despite the caution regarding complications. This study aimed to evaluate the clinical utility of rapid on-site evaluation of touch imprint cytology (ROSE-TIC) during cryobiopsy of peripheral pulmonary lesions (PPLs). We retrospectively reviewed the data of consecutive patients who underwent cryobiopsy for solid PPLs between June 2020 and December 2021. ROSE-TIC was performed on the first specimen obtained via cryobiopsy and assessed using Diff-Quik staining. The results of ROSE-TIC for each patient were compared with the histological findings of the first cryobiopsy specimen. Sixty-three patients were enrolled in this study. Overall, 57 (90.5%) lesions were ≤30 mm in size and 37 (58.7%) had positive bronchus signs. The radial endobronchial ultrasound findings were located within and adjacent to the lesion in 46.0% and 54.0% of the cases, respectively. The sensitivity, specificity, and positive and negative predictive values of the ROSE results for histological findings of the corresponding specimens were 69.8%, 90.0%, 93.8%, and 58.1%, respectively. The concordance rate was 76.2%. In conclusion, ROSE-TIC, due to its high specificity and positive predictive value, may be a potential tool in deciding whether cryobiopsy sampling could be finished during bronchoscopy.

Although sotrovimab, one of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies has been shown to be effective in patients with mild-to-moderate coronavirus disease 2019 (COVID-19) with risk factors, their efficacy in mRNA COVID-19 vaccinated patients in omicron era is unknown. To evaluate the effectiveness of sotrovimab clinical data from both COVID-19 vaccinated and unvaccinated patients who were hospitalized and receiving sotrovimab at the Japanese Red Cross Medical Center were compared. The efficacy and adverse events were evaluated. Of the total 60 patients enrolled in this study, 45 had received the mRNA COVID-19 vaccine and 15 were unvaccinated. The clinical progression with low nasal cannula or face mask was not significantly different between groups (occurring in one patient in each group; p = 0.44), with no further progression in both groups. The duration of hospitalization was eight days for both groups (p = 0.90). Two patients in each group experienced adverse events (7%, p = 0.26). The results suggested that the efficacy and safety of sotrovimab against mild-to-moderate COVID-19 with risk factors in the omicron era might not be different regardless of the vaccination status. The results of the present study are encouraging; however, further randomized clinical studies are needed.

Background: This study aimed to investigate the clinical characteristics and prognosis of patients with pleuroparenchymal fibroelastosis (PPFE) and pulmonary hypertension (PH)., Methods: We retrospectively analyzed the data of patients who were diagnosed with PPFE and underwent transthoracic echocardiography (TTE) for the evaluation of their right heart systems within 3 months of their first visit between 2011 and 2018. Patients were divided into the PH and non-PH groups based on their peak tricuspid regurgitation velocity (TRV) on TTE (cutoff, 2.8 m/s). The clinical characteristics of PH and association between PH and survival among patients with PPFE were investigated., Results: In total, 83 patients were enrolled. Sixteen (19.3%) patients were included in the PH group. The PH group had a lower body mass index, percent predicted forced vital capacity (FVC), 6-min walk distance, and partial pressure of arterial oxygen than the non-PH group. There was no significant difference in the presence of usual interstitial pneumonia patterns in the lower lobes between the two groups. The survival period was significantly shorter in the PH group than in the non-PH group (median survival 16.3 versus 50.2 months, log-rank p < 0.001). The multivariate Cox proportional hazard model showed that male sex (hazard ratio [HR] = 4.83, p < 0.001), Krebs von den Lungen-6 (KL-6) > 550 U/mL (HR = 3.48, p = 0.005), %FVC < 50% (HR = 3.04, p = 0.028), and peak TRV > 2.8 m/s (HR = 3.26, p = 0.038) were independently associated with poor survival., Conclusions: PH was not rare in patients with PPFE. Male sex, increased KL-6, lower FVC, and PH were independently associated with poor survival in patients with PPFE., (Copyright © 2022 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

Introduction: Curative-intent surgery is the best therapeutic option for thymic malignancies. However, patients with advanced or recurrent thymic malignancies often require palliative-intent chemotherapy or radiotherapy. Since thymic malignancies are rare cancers, the efficacy and safety of treatments have been verified based on small Phase 2 trials or retrospective studies., Area Covered: We comprehensively reviewed the treatment strategies for thymic malignancies, including surgery, radiotherapy, and pharmacotherapy, including cytotoxic chemotherapy, molecular-targeted therapy, and immunotherapy. Additionally, we reviewed specific situations, such as pleural dissemination, central nervous system metastasis, and paraneoplastic syndrome., Expert Opinion: Cytotoxic chemotherapy remains the standard option in pharmacotherapy. However, multikinase inhibitors, such as sunitinib and lenvatinib, and immune checkpoint inhibitors including pembrolizumab have been developed to treat thymic carcinomas. Now, a Phase 2 study is evaluating whether lenvatinib plus pembrolizumab benefits patients with type B3 thymoma or thymic carcinoma. Phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin inhibitors may contribute to disease control and octreotide scan is only applicable to somatostatin analogues. Although the genomic characteristics of thymic malignancies have been analyzed, few actionable mutations have been detected in general. The development of a treatment strategy using combination pharmacotherapy is anticipated.

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 1 H, 15 N, and 13 C 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., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: The prolonged pandemic of coronavirus disease 2019 (COVID-19) has resulted in mental burden among healthcare workers (HCWs). This study aimed to conduct a repeated study to assess changes in psychological concerns among Japanese HCWs., Methods: This study is the second survey involving HCWs at the Japanese Red Cross Medical Center conducted between November 20, 2020 and December 4, 2020. The degree of symptoms of anxiety, depression, and resilience was assessed using the Japanese versions of the 7-item Generalized Anxiety Disorder Scale, Center for Epidemiologic Studies Depression Scale, and 10-item Connor-Davidson Resilience Scale, respectively., Results: The survey included 594 HCWs, comprising 95 physicians, 261 nurses, 150 other co-medical staff, and 88 office workers. Among them, 46 (7.7%) and 152 (25.6%) developed moderate-to-severe symptoms of anxiety and depression, respectively. Compared with those in the initial survey conducted 6 months earlier, the resilience score did not change, whereas the anxiety and depression scores improved significantly (P < 0.001, P = 0.033, respectively). However, the frequency of HCWs developing moderate-to-severe symptoms of anxiety or depression did not significantly improve. Multivariable logistic regression analysis showed that having higher anxiety symptoms was a risk factor for depression symptoms, while older HCWs and those with higher resilience were less likely to develop depression symptoms., Conclusions: Many HCWs still suffer from psychological concerns during the COVID-19 pandemic.

Background: Drug-induced hypersensitivity syndrome (DIHS)/drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome is an important adverse reaction caused by a few drugs. Reactivation of human herpesvirus 6 (HHV-6) is known to be associated with its pathogenesis. DIHS occasionally manifests as pulmonary lesions with a variety of imaging findings., Case Presentation: An 83-year-old woman started taking minodronic acid hydrate 5 years before admission. She noticed a generalized skin rash 44 days before admission and started oral betamethasone-d-chlorpheniramine maleate combination tablets for allergic dermatitis. She developed a fever and cough in addition to the rash, and was referred to our hospital. Laboratory data showed a high level of eosinophils and liver and biliary enzymes. Computed tomography (CT) studies revealed bilateral diffuse ground-glass opacities with ill-defined centrilobular nodules from the central to peripheral regions of the lungs. Transbronchial lung cryobiopsy specimens showed that lymphocyte infiltration was observed in the alveolar walls and fibrinous exudates and floating macrophages in the alveolar lumina. Immunohistochemistry of biopsy specimens showed more CD4 + lymphocytes than CD8 + lymphocytes, while few Foxp3 + lymphocytes were recognized. The serum anti-HHV-6 immunoglobulin G titer increased at 3 weeks after the first test. Based on these findings, we diagnosed her with DIHS. We continued care without using corticosteroids since there was no worsening of breathing or skin condition. Eventually, her clinical symptoms chest CT had improved. Minodronic acid hydrate was identified as the culprit drug based on the positive results of the patch test and drug-induced lymphocyte stimulation test., Conclusions: We described the first case of DIHS caused by minodronic acid hydrate. Lung lesions in DIHS can present with bilateral diffuse ground-glass opacities and ill-defined centrilobular nodules on a CT scan during the recovery phase. Clinicians should be aware of DIHS, even if patients are not involved with typical DIHS/DRESS-causing drugs., (© 2021. The Author(s).)

Background: This study aimed to examine risk factors associated with critical coronavirus disease 19 (COVID-19) and to establish a risk predictive model for Japanese patients., Methods: We retrospectively assessed adult Japanese patients diagnosed with COVID-19 at the Japanese Red Cross Medical Center, Tokyo, Japan between February 1, 2020 and March 10, 2021. The patients were divided into critical and non-critical groups based on their condition during the clinical courses. Univariate and multivariate logistic regression analyses were performed to investigate the relationship between clinical characteristics and critical illness. Based on the results, we established a predictive model for the development of critical COVID-19., Results: In total, 300 patients were enrolled in this study. Among them, 86 were included in the critical group. Analyses revealed that age ≥65 y, hemodialysis, need for O 2 supplementation upon diagnosis, and an initial serum C-reactive protein level of ≥6.5 mg/dL were independently associated with the development of critical COVID-19. Next, a predictive model for the development of critical COVID-19 was created, and this included the following variables: age ≥65 y, male sex, diabetes, hemodialysis, need for O 2 supplementation upon diagnosis, and an initial serum C-reactive protein level of ≥6.5 mg/dL. The area under the receiver operating characteristic curve of the model was 0.86 (95% confidence interval, 0.81-0.90). Using a cutoff score of 12, the positive and negative predictive values of 74.0% and 80.4% were obtained, respectively., Conclusions: Upon diagnosis, the predictive model can be used to identify adult Japanese patients with COVID-19 who will require intensive treatment., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2021 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

Background: Coronavirus disease 2019 (COVID-19) has spread worldwide and is also an important disease in Japan. Thus, the optimal treatment strategy for severe COVID-19 should be established urgently. The effects of combination treatment with baricitinib-a Janus kinase inhibitor, remdesivir, and dexamethasone (BRD) are unknown., Methods: Patients who received combination therapy with BRD at the Japanese Red Cross Medical Center were enrolled in the study. All patients received baricitinib (≤14 d), remdesivir (≤10 d), and dexamethasone (≤10 d). The efficacy and adverse events were evaluated., Results: In total, 44 patients with severe COVID-19 were enrolled in this study. The 28-d mortality rate was low at 2.3% (1/44 patients). The need for invasive mechanical ventilation was avoided in most patients (90%, 17/19 patients). Patients who received BRD therapy had a median hospitalization duration of 11 d, time to recovery of 9 d, duration of intensive care unit stay of 6 d, duration of invasive mechanical ventilation of 5 d, and duration of supplemental oxygen therapy of 5 d. Adverse events occurred in 15 patients (34%). Liver dysfunction, thrombosis, iliopsoas hematoma, renal dysfunction, ventilator-associated pneumonia, infective endocarditis, and herpes zoster occurred in 11%, 11%, 2%, 2%, 2%, 2%, and 2% of patients, respectively., Conclusions: Combination therapy with BRD was effective in treating severe COVID-19, and the incidence rate of adverse events was low. The results of the present study are encouraging; however, further randomized clinical studies are needed., Competing Interests: Conflict of Interest The authors have no conflict of interest to declare in relation to this work., (Copyright © 2021 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

In hospitalized coronavirus disease 2019 (COVID-19) patients, anticoagulation therapy is administered to prevent thrombosis. However, anticoagulation sometimes causes bleeding complications. We herein report two Japanese cases of severe COVID-19 in which spontaneous muscle hematomas (SMH) developed under therapeutic anticoagulation with unfractionated heparin. Although the activated partial prothrombin time was within the optimal range, contrast-enhanced computed tomography (CECT) revealed SMH in the bilateral iliopsoas muscles in both cases, which required emergent transcatheter embolization. Close monitoring of the coagulation system and the early diagnosis of bleeding complications through CECT are needed in severe COVID-19 patients treated with anticoagulants.

Background: The coronavirus disease (COVID-19) has afflicted large populations worldwide. Although vaccines aroused great expectations, their side effects on Japanese people and the antibody titer transition after vaccination are unclear., Methods: The side effects of the BNT162b2 mRNA COVID-19 vaccine in participants who received vaccination at our center were investigated. Some participants were also surveyed for the antibody titer transition., Results: In this study, 983 and 798 Japanese participants responded to the first and second doses, respectively. Side effects occurred in 757 (77.0%) and 715 participants (90.0%) after the first and second doses, respectively. No Grade 4 side effects occurred. The second dose had significantly more side effects than the first dose (p < 0.001). Side effects occurred after the second dose in 571 female (92.1%) and 178 male participants (80.1%). Female participants had a higher incidence of side effects than the male participants (p < 0.001). A comparison among the age groups showed significant differences (p = 0.018), and the frequency of side effects decreased with age. Twenty-three individuals participated in the survey of antibody titer transition. After the second vaccine dose, the median antibody titers for IgG and IgM were 3.76 and 0.07 AU/mL, respectively. Both IgG and IgM titers showed a significant increase over the study period (p < 0.001)., Conclusions: The BNT162b2 mRNA COVID-19 vaccine might be safe for Japanese people, and the antibody titer increased with two doses of vaccination. Larger nationwide studies are warranted to verify these findings., Competing Interests: Conflict of Interest The authors have no conflicts of interest to declare regarding this study., (Copyright © 2021 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

Objectives: Japan's healthcare expenditures, especially on oncology, are rapidly growing; however, there are scant data on actual costs and cost-effectiveness in the real world. The aim was to assess the medical costs and outcomes of patients with advanced lung cancer., Methods: We retrospectively investigated all patients who were diagnosed with advanced lung cancer at the Japanese Red Cross Medical Center between 1 January 2008 and 31 December 2018. Patients were classified into three cohorts according to the year of diagnosis-Cohort 1: 2008-2010, Cohort 2: 2011-2014 and Cohort 3: 2015-2018-and assessed for medical costs and outcome. Medical costs were divided into outpatient and inpatient costs and were calculated on a monthly basis., Results: Ninety-five patients with small cell lung cancer (SCLC) and 330 with nonsmall cell lung cancer (NSCLC) were included. There was a trend toward increased costs during the first two years after diagnosis in NSCLC patients, without changes in monthly costs, reflecting improved survival. Compared to Cohort 1, Cohort 3 patients with NSCLC had longer survival (median: 24 versus 12 months, P < 0.001), with a median incremental cost of Japanese Yen 6 million during the initial two years. The proportion of outpatient costs increased over time, especially for NSCLC patients (P < 0.001). No changes in costs or survival were observed in SCLC patients., Conclusions: In NSCLC patients, medical costs increased with prolonged survival during the last decade. The costs on a monthly basis did not change. The proportion of outpatient costs increased., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Objective: The medical costs associated with cancer treatment have increased rapidly in Japan; however, little data exist on actual costs, especially for end-of-life care. Therefore, this study aimed to examine the medical costs of lung cancer patients during the last 3 months before death and to compare the costs with those of initial anticancer treatment., Methods: We retrospectively evaluated all patients who died from lung cancer at the Japanese Red Cross Medical Center between 1 January 2008 and 31 August 2019. Patients were classified into three cohorts (2008-2011, 2012-2015 and 2016-2019) according to the year of death; the medical costs were evaluated for each cohort. Costs were then divided into outpatient and inpatient costs and calculated per month., Results: Seventy-nine small cell lung cancer and 213 non-small cell lung cancer patients were included. For small cell lung cancer and non-small cell lung cancer patients, most end-of-life medical costs were inpatient costs across all cohorts. The median monthly medical costs for the last 3 months among both small cell lung cancer and non-small cell lung cancer patients did not differ significantly among the cohorts, but the mean monthly costs for non-small cell lung cancer tended to increase. The monthly medical costs for the last 3 months were significantly higher than those for the first year in SCLC (P = 0.013) and non-small cell lung cancer (P < 0.001) patients and those for the first 3 months in non-small cell lung cancer patients (P = 0.005)., Conclusions: The medical costs during the end-of-life period for lung cancer were high and surpassed those for initial treatment., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

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 m 6 A. 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 m 6 A differs between these proteins. Here, we report 1 H, 13 C and 15 N 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 m 6 A-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.

Background: Radial endobronchial ultrasonography (R-EBUS) has been used in conjunction with transbronchial lung cryobiopsy (TBLC) to diagnose diffuse parenchymal lung disease (DPLD) and to decrease the risk of bleeding complications. The diagnostic utility of different R-EBUS signs, however, remains unknown., Objectives: This study aimed to determine whether different R-EBUS signs could be used to more accurately diagnose DPLD and whether bronchial bleeding could be prevented with use of R-EBUS during TBLC., Method: Eighty-seven patients with DPLD were included in this multicentre prospective study, with 49 patients undergoing R-EBUS. R-EBUS signals were characterised as displaying either dense or blizzard signs. Pathological confidence of specimens obtained from TBLC was compared between patients with dense versus blizzard signs, and severity of bronchial bleeding was determined based on whether R-EBUS was performed or not., Results: All patients with dense signs on R-EBUS showed consolidation on high-resolution CT (HRCT) imaging. Pathological confidence of lung specimens was significantly higher in patients with dense signs versus those with blizzard signs (p<0.01) and versus those who did not undergo R-EBUS (p<0.05). Patients who underwent TBLC with R-EBUS were more likely to experience no or mild bronchial bleeding than patients who did not undergo R-EBUS (p<0.01), with shorter procedure times (p<0.01)., Conclusions: The dense R-EBUS sign corresponded with consolidation on HRCT. High-quality lung specimens may be obtainable when the dense sign is observed on R-EBUS, and R-EBUS combined with TBLC may reduce risk of bronchial bleeding and shorten procedure times., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Most lung cancer patients present with lesions in both lung fields and lymphadenopathy. Thus, transbronchial lung cryobiopsy (TBLC) and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) are commonly performed for diagnosing lung cancer. However, the adequacy of these samples for next-generation sequencing (NGS) analysis remains unclear. This study aimed to compare the adequacy between TBLC and EBUS-TBNA samples for NGS analysis., Methods: This retrospective cohort study included patients whose lung samples were collected via TBLC or EBUS-TBNA and analyzed using NGS. Out of 46 genes, the number of genes in TBNA and TBLC samples that could not be assessed via NGS analysis was mainly evaluated., Results: A total of 37 patients were included and classified into two groups (TBLC group, n = 18 and TBNA group, n = 19). The mean number of genes that could not be evaluated via NGS analysis was significantly lower in the TBLC group than in the TBNA group (0.9 vs. 10.3, P = 0.024). The median total area of tumor cells in TBLC samples was significantly greater than that in TBNA samples (6.3 [1.6-4.2] vs. 2.6 [0.2-17.3] mm 2 , P < 0.01). In the TBNA group, there were two fully inadequate samples for NGS analysis with a high degree of cell crush or low tumor content, while there was no fully inadequate sample in the TBLC group., Conclusions: TBLC is more effective in obtaining adequate samples for NGS analysis than EBUS-TBNA. TBLC should be performed to obtain adequate samples for NGS analysis in lung cancer patients with target lesions in lung fields, even if they have lymphadenopathy., Key Points: Significant findings of the study The mean number of genes that could not be evaluated was significantly lower in TBLC samples than in EBUS-TBNA samples (0.9 vs. 10.3, P = 0.024). TBLC could obtain adequate samples with a high concentration of uncrushed tumor cells for NGS. What this study adds To obtain samples for NGS analysis, the use of TBLC should be aggressively considered in lung-cancer patients with target lesions located in lung fields, even if they have lymphadenopathy., (© 2020 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Background: Coronavirus disease 2019 (COVID-19) is currently spreading worldwide. This study examined whether serum Krebs von den Lungen-6 (KL-6) level is a useful biomarker for evaluating the severity of COVID-19., Methods: We retrospectively examined patients diagnosed with COVID-19 at the Japanese Red Cross Medical Center between February 1, 2020, and May 15, 2020. Patients were divided into four categories based on clinical and radiological findings: mild, moderate, severe, and critical. Patients who presented with a mild or moderate illness and patients who started with or worsened to a severe or critical illness were classified as the non-severe and severe groups, respectively. The two groups were compared for patient characteristics, including serum KL-6 levels. Receiver operating characteristic curves were used to define the optimum cut-off value of serum KL-6 level to evaluate COVID-19 severity., Results: A total of 54 patients were enrolled, including 33 in the non-severe group and 21 in the severe group, of which four died. Compared with those in the non-severe group, more patients in the severe group were significantly older and had comorbidities. Serum KL-6 levels were significantly higher in the severe group than in the non-severe group both at diagnosis (median, 338 U/mL) and at peak levels within one week after diagnosis (median, 781 U/mL) (both p < 0.001). Serum KL-6 value at peak level (371 U/mL) was used as the optimal cut-off to evaluate disease severity (sensitivity, 85.7%; specificity, 96.6%)., Conclusions: Serum KL-6 levels were significantly elevated in severe COVID-19 and is useful for evaluating its severity., Competing Interests: Conflict of interest The authors declare no conflicts of interest., (Copyright © 2020 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

The accurate exclusion of introns by RNA splicing is critical for the production of mature mRNA. U2AF1 binds specifically to the 3´ splice site, which includes an essential AG dinucleotide. Even a single amino acid mutation of U2AF1 can cause serious disease such as certain cancers or myelodysplastic syndromes. Here, we describe the first crystal structures of wild-type and pathogenic mutant U2AF1 complexed with target RNA, revealing the mechanism of 3´ splice site selection, and how aberrant splicing results from clinically important mutations. Unexpected features of this mechanism may assist the future development of new treatments against diseases caused by splicing errors.

Sirtuin 7 (SIRT7) is a NAD+ (nicotinamide adenine dinucleotide) dependent deacetylase that is reported to contribute to tumour growth and invasion by selectively acting on histone H3K18. It is overexpressed in several cancers including hepatocellular carcinoma (HCC). In this study, we investigated the relationship between SIRT7 expression, proliferation (Ki-67 index) in human HCC tissues, and patient prognosis. We analysed 219 HCC samples obtained retrospectively, for clinicopathological features, and with immunohistochemistry. SIRT7 overexpression was observed in 73 cases (33%) and correlated with vascular invasion and poor differentiation of HCC. Ki-67 labelling index was observed to be significantly higher in SIRT7 overexpressing cases. Interestingly, the Ki-67 labelling index was higher in SIRT7 overexpressing cases regardless of the differentiation status of HCC. Multivariate analysis demonstrated SIRT7 overexpression as an independent factor predictive of poor prognosis (p=0.016). In vitro, SIRT7 knockdown led to reduced growth in cells and resulted in a lower percentage of G0/G1 cells compared to controls. In addition, the ratio of apoptotic cells following sorafenib treatment was significantly higher in SIRT7 knockdown cells than control cells (p=0.040), implying that SIRT7 knockdown potentiated the effect of sorafenib. In conclusion, our study showed that overexpression of SIRT7 was associated with increased proliferative activity in HCC and predictive of poor prognosis. In addition, our in vitro model showed that SIRT7 knockdown was associated with reduced proliferation, and suggested abrogation of SIRT7 may potentiate the effect of sorafenib. Therefore, we propose that SIRT7 expression by HCC may be used as a prognostic biomarker, and that SIRT7 may be a potential target for new therapeutic modalities., (Copyright © 2020 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Objective Coronavirus disease 2019 (COVID-19) is spreading around the world. The aim of this study was to assess the degree of anxiety, depression, resilience, and other psychiatric symptoms among healthcare workers in Japan during the COVID-19 pandemic. Methods This survey involved medical healthcare workers at the Japanese Red Cross Medical Center (Tokyo, Japan) between April 22 and May 15, 2020. The degree of symptoms of anxiety, depression, and resilience was assessed using the Japanese versions of the 7-item Generalized Anxiety Disorder Scale (GAD-7), Center for Epidemiologic Studies Depression Scale (CES-D), and 10-item Connor-Davidson Resilience Scale. Furthermore, we added original questionnaires comprising three factors: (i) anxiety and fear of infection and death; (ii) isolation and unreasonable treatment; and (iii) motivation and escape behavior at work. Results In total, 848 healthcare workers participated in this survey: 104 doctors, 461 nurses, 184 other co-medical staff, and 99 office workers. Among all participants, 85 (10.0%) developed moderate-to-severe anxiety disorder, and 237 (27.9%) developed depression. Problems with anxiety and fear of infection and death, isolation and unreasonable treatment, and motivation and escape from work were higher in the depression group than in the non-depression group (total CES-D score ≥ 16 points). Being a nurse and high total GAD-7 scores were risk factors of depression. Older workers and those with higher resilience were less likely to develop depression than others. Conclusion During the COVID-19 epidemic, many healthcare workers suffered from psychiatric symptoms. Psychological support and interventions for protecting the mental health of them are needed.

The spliceosomal protein SF3b49, a component of the splicing factor 3b (SF3b) protein complex in the U2 small nuclear ribonucleoprotein, contains two RNA recognition motif (RRM) domains. In yeast, the first RRM domain (RRM1) of Hsh49 protein (yeast orthologue of human SF3b49) reportedly interacts with another component, Cus1 protein (orthologue of human SF3b145). Here, we solved the solution structure of the RRM1 of human SF3b49 and examined its mode of interaction with a fragment of human SF3b145 using NMR methods. Chemical shift mapping showed that the SF3b145 fragment spanning residues 598-631 interacts with SF3b49 RRM1, which adopts a canonical RRM fold with a topology of β1-α1-β2-β3-α2-β4. Furthermore, a docking model based on NOESY measurements suggests that residues 607-616 of the SF3b145 fragment adopt a helical structure that binds to RRM1 predominantly via α1, consequently exhibiting a helix-helix interaction in almost antiparallel. This mode of interaction was confirmed by a mutational analysis using GST pull-down assays. Comparison with structures of all RRM domains when complexed with a peptide found that this helix-helix interaction is unique to SF3b49 RRM1. Additionally, all amino acid residues involved in the interaction are well conserved among eukaryotes, suggesting evolutionary conservation of this interaction mode between SF3b49 RRM1 and SF3b145., (© 2016 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.)

p54(nrb)/NonO is a nuclear RNA-binding protein involved in many cellular events such as pre-mRNA processing, transcription, and nuclear retention of hyper-edited RNAs. In particular, it participates in the splicing process by directly binding the 5' splice site of pre-mRNAs. The protein also concentrates in a nuclear body called paraspeckle by binding a G-rich segment of the ncRNA NEAT1. The N-terminal section of p54(nrb)/NonO contains tandem RNA recognition motifs (RRMs) preceded by an HQ-rich region including a threonine residue (Thr15) whose phosphorylation inhibits its RNA binding ability, except for G-rich RNAs. In this work, our goal was to understand the rules that characterize the binding of the p54(nrb)/NonO RRMs to their RNA target. We have done in vitro RNA binding experiments which revealed that only the first RRM of p54(nrb)/NonO binds to the 5' splice site RNA. We have then determined the structure of the p54(nrb)/NonO RRM1 by liquid-state NMR which revealed the presence of a canonical fold (β1α1β2β3α2β4) and the conservation of aromatic amino acids at the protein surface. We also investigated the dynamics of this domain by NMR. The p54(nrb)/NonO RRM1 displays some motional properties that are typical of a well-folded protein with some regions exhibiting more flexibility (loops and β-strands). Furthermore, we determined the affinity of p54(nrb)/NonO RRM1 interaction to the 5' splice site RNA by NMR and fluorescence quenching and mapped its binding interface by NMR, concluding in a classical nucleic acid interaction. This study provides an improved understanding of the molecular basis (structure and dynamics) that governs the binding of the p54(nrb)/NonO RRM1 to one of its target RNAs.

The pre-mRNA splicing reaction of eukaryotic cells has to be carried out extremely accurately, as failure to recognize the splice sites correctly causes serious disease. The small subunit of the U2AF heterodimer is essential for the determination of 3' splice sites in pre-mRNA splicing, and several single-residue mutations of the U2AF small subunit cause severe disorders such as myelodysplastic syndromes. However, the mechanism of RNA recognition is poorly understood. Here we solved the crystal structure of the U2AF small subunit (U2AF23) from fission yeast, consisting of an RNA recognition motif (RRM) domain flanked by two conserved CCCH-type zinc fingers (ZFs). The two ZFs are positioned side by side on the β sheet of the RRM domain. Further mutational analysis revealed that the ZFs bind cooperatively to the target RNA sequence, but the RRM domain acts simply as a scaffold to organize the ZFs and does not itself contact the RNA directly. This completely novel and unexpected mode of RNA-binding mechanism by the U2AF small subunit sheds light on splicing errors caused by mutations of this highly conserved protein., (© 2015 Yoshida et al.; Published by Cold Spring Harbor Laboratory Press.)

D-Serine is an essential coagonist with glutamate for stimulation of N-methyl-D-aspartate (NMDA) glutamate receptors. Although astrocytic metabolic processes are known to regulate synaptic glutamate levels, mechanisms that control D-serine levels are not well defined. Here we show that d-serine production in astrocytes is modulated by the interaction between the D-serine synthetic enzyme serine racemase (SRR) and a glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In primary cultured astrocytes, glycolysis activity was negatively correlated with D-serine level. We show that SRR interacts directly with GAPDH, and that activation of glycolysis augments this interaction. Biochemical assays using mutant forms of GAPDH with either reduced activity or reduced affinity to SRR revealed that GAPDH suppresses SRR activity by direct binding to GAPDH and through NADH, a product of GAPDH. NADH allosterically inhibits the activity of SRR by promoting the disassociation of ATP from SRR. Thus, astrocytic production of D-serine is modulated by glycolytic activity via interactions between GAPDH and SRR. We found that SRR is expressed in astrocytes in the subiculum of the human hippocampus, where neurons are known to be particularly vulnerable to loss of energy. Collectively, our findings suggest that astrocytic energy metabolism controls D-serine production, thereby influencing glutamatergic neurotransmission in the hippocampus.

We developed fluorescent turn-on probes containing a fluorescent nucleoside, 5-(benzofuran-2-yl)deoxyuridine (dU(BF)) or 5-(3-methylbenzofuran-2-yl)deoxyuridine (dU(MBF)), for the detection of single-stranded DNA or RNA by utilizing DNA triplex formation. Fluorescence measurements revealed that the probe containing dU(MBF) achieved superior fluorescence enhancement than that containing dU(BF). NMR and fluorescence analyses indicated that the fluorescence intensity increased upon triplex formation partly as a consequence of a conformational change at the bond between the 3-methylbenzofuran and uracil rings. In addition, it is suggested that the microenvironment around the 3-methylbenzofuran ring contributed to the fluorescence enhancement. Further, we developed a method for detecting RNA by rolling circular amplification in combination with triplex-induced fluorescence enhancement of the oligonucleotide probe containing dU(MBF)., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The family of cytoplasmic polyadenylation element binding proteins CPEB1, CPEB2, CPEB3, and CPEB4 binds to the 3'-untranslated region (3'-UTR) of mRNA, and plays significant roles in mRNA metabolism and translation regulation. They have a common domain organization, involving two consecutive RNA recognition motif (RRM) domains followed by a zinc finger domain in the C-terminal region. We solved the solution structure of the first RRM domain (RRM1) of human CPEB3, which revealed that CPEB3 RRM1 exhibits structural features distinct from those of the canonical RRM domain. Our structural data provide important information about the RNA binding ability of CPEB3 RRM1., (© 2014 Wiley Periodicals, Inc.)

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.

Anaphase-promoting complex or cyclosome (APC/C) is a multisubunit ubiquitin ligase E3 that targets cell-cycle regulators. Cdc20 is required for full activation of APC/C in M phase, and mediates substrate recognition. In vertebrates, Emi2/Erp1/FBXO43 inhibits APC/C-Cdc20, and functions as a cytostatic factor that causes long-term M phase arrest of mature oocytes. In this study, we found that a fragment corresponding to the zinc-binding region (ZBR) domain of Emi2 inhibits cell-cycle progression, and impairs the association of Cdc20 with the APC/C core complex in HEK293T cells. Furthermore, we revealed that the ZBR fragment of Emi2 inhibits in vitro ubiquitin chain elongation catalyzed by the APC/C cullin-RING ligase module, the ANAPC2-ANAPC11 subcomplex, in combination with the ubiquitin chain-initiating E2, E2C/UBE2C/UbcH10. Structural analyses revealed that the Emi2 ZBR domain uses different faces for the two mechanisms. Thus, the double-faced ZBR domain of Emi2 antagonizes the APC/C function by inhibiting both the binding with the coactivator Cdc20 and ubiquitylation mediated by the cullin-RING ligase module and E2C. In addition, the tail region between the ZBR domain and the C-terminal RL residues [the post-ZBR (PZ) region] interacts with the cullin subunit, ANAPC2. In the case of the ZBR fragment of the somatic paralogue of Emi2, Emi1/FBXO5, these inhibitory activities against cell division and ubiquitylation were not observed. Finally, we identified two sets of key residues in the Emi2 ZBR domain that selectively exert each of the dual Emi2-specific modes of APC/C inhibition, by their mutation in the Emi2 ZBR domain and their transplantation into the Emi1 ZBR domain.

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant β-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS.

RNA helicase A (RHA) is a multifunctional protein that regulates gene expression. RHA has two double-stranded RNA-binding domains (dsRBDs) that serve as modules for highly structured RNA binding and protein-protein interactions. Using the dsRBDs, RHA binds to cellular and viral mRNAs, exports them from the nucleus, and regulates splicing as well as translational initiation. The RHA dsRBDs also reportedly mediate interactions with small RNAs and other dsRBD-containing proteins, and altogether form a processing complex involved in RNA silencing pathways. In addition, the RHA dsRBDs bridge RNA polymerase II with several transcription factors. Here we report the (1)H, (13)C, and (15)N chemical shift assignments of the dsRBDs of RHA. The resonance assignments obtained in this work will contribute to the elucidation of the interactions between RHA and transcriptional or post-transcriptional gene regulators.

Molecular interactions between the tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins play an important role in the transcription-independent apoptosis of p53. The p53 transactivation domain (p53TAD) contains two conserved ΦXXΦΦ motifs (Φ indicates a bulky hydrophobic residue and X is any other residue) referred to as p53TAD1 (residues 15-29) and p53TAD2 (residues 39-57). We previously showed that p53TAD1 can act as a binding motif for anti-apoptotic Bcl-2 family proteins. In this study, we have identified p53TAD2 as a binding motif for anti-apoptotic Bcl-2 family proteins by using NMR spectroscopy, and we calculated the structures of Bcl-X(L)/Bcl-2 in complex with the p53TAD2 peptide. NMR chemical shift perturbation data showed that p53TAD2 peptide binds to diverse members of the anti-apoptotic Bcl-2 family independently of p53TAD1, and the binding between p53TAD2 and p53TAD1 to Bcl-X(L) is competitive. Refined structural models of the Bcl-X(L)·p53TAD2 and Bcl-2·p53TAD2 complexes showed that the binding sites occupied by p53TAD2 in Bcl-X(L) and Bcl-2 overlap well with those occupied by pro-apoptotic BH3 peptides. Taken together with the mutagenesis, isothermal titration calorimetry, and paramagnetic relaxation enhancement data, our structural comparisons provided the structural basis of p53TAD2-mediated interaction with the anti-apoptotic proteins, revealing that Bcl-X(L)/Bcl-2, MDM2, and cAMP-response element-binding protein-binding protein/p300 share highly similar modes of binding to the dual p53TAD motifs, p53TAD1 and p53TAD2. In conclusion, our results suggest that the dual-site interaction of p53TAD is a highly conserved mechanism underlying target protein binding in the transcription-dependent and transcription-independent apoptotic pathways of p53.

The WWE domain is often identified in proteins associated with ubiquitination or poly-ADP-ribosylation. Structural information about WWE domains has been obtained for the ubiquitination-related proteins, such as Deltex and RNF146, but not yet for the poly-ADP-ribose polymerases (PARPs). Here we determined the solution structures of the WWE domains from PARP11 and PARP14, and compared them with that of the RNF146 WWE domain. NMR perturbation experiments revealed the specific differences in their ADP-ribose recognition modes that correlated with their individual biological activities. The present structural information sheds light on the ADP-ribose recognition modes by the PARP WWE domains., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

RNA helicase A (RHA) is a highly conserved protein with multifaceted functions in the gene expression of cellular and viral mRNAs. RHA recognizes highly structured nucleotides and catalytically rearranges the various interactions between RNA, DNA, and protein molecules to provide a platform for the ribonucleoprotein complex. We present the first solution structures of the double-stranded RNA-binding domains (dsRBDs), dsRBD1 and dsRBD2, from mouse RHA. We discuss the binding mode of the dsRBDs of RHA, in comparison with the known dsRBD structures in their complexes. Our structural data provide important information for the elucidation of the molecular reassembly mediated by RHA., (Copyright © 2012 Wiley Periodicals, Inc.)

Multi-targeting therapy is an emerging strategy of drug discovery to improve therapeutic efficacy, safety and resistance profiles. In this study, we monitored the binding of a potent MDM2 inhibitor Nutlin-3 with anti-apoptotic Bcl-2 family proteins using NMR spectroscopy. Our results showed the universal binding of Nutlin-3 with diverse anti-apoptotic Bcl-2 family proteins. Taken together with the binding data for Nutlin-3 analogs, the structural model of the Bcl-X(L)/Nutlin-3 complex showed that the binding mode of Nutlin-3 resembles that of the Bcl-X(L)/Bcl-2 inhibitors, suggesting the molecular mechanism of transcription-independent mitochondrial apoptosis by Nutlin-3. Finally, our structural comparison provides structural insights into the dual-targeting mechanism of how Nutlin-3 can bind to two different target proteins, MDM2 and anti-apoptotic Bcl-2 family proteins in a similar manner., (Copyright © 2012 Elsevier Inc. All rights reserved.)

'RNA recognition motifs (RRMs)' are common domain-folds composed of 80-90 amino-acid residues in eukaryotes, and have been identified in many cellular proteins. At first they were known as RNA binding domains. Through discoveries over the past 20 years, however, the RRMs have been shown to exhibit versatile molecular recognition activities and to behave as molecular Lego building blocks to construct biological systems. Novel RNA/protein recognition modes by RRMs are being identified, and more information about the molecular recognition by RRMs is becoming available. These RNA/protein recognition modes are strongly correlated with their biological significance. In this review, we would like to survey the recent progress on these versatile molecular recognition modules., (Copyright © 2012 John Wiley & Sons, Ltd.)

Human Transformer2-β (hTra2-β) is an important member of the serine/arginine-rich protein family, and contains one RNA recognition motif (RRM). It controls the alternative splicing of several pre-mRNAs, including those of the calcitonin/calcitonin gene-related peptide (CGRP), the survival motor neuron 1 (SMN1) protein and the tau protein. Accordingly, the RRM of hTra2-β specifically binds to two types of RNA sequences [the CAA and (GAA)(2) sequences]. We determined the solution structure of the hTra2-β RRM (spanning residues Asn110-Thr201), which not only has a canonical RRM fold, but also an unusual alignment of the aromatic amino acids on the β-sheet surface. We then solved the complex structure of the hTra2-β RRM with the (GAA)(2) sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the β-sheet surface. Further NMR experiments revealed that the hTra2-β RRM recognizes the CAA sequence when it is integrated in the stem-loop structure. This study indicates that the hTra2-β RRM recognizes two types of RNA sequences in different RNA binding modes.

The TAR RNA-binding Protein (TRBP) is a double-stranded RNA (dsRNA)-binding protein, which binds to Dicer and is required for the RNA interference pathway. TRBP consists of three dsRNA-binding domains (dsRBDs). The first and second dsRBDs (dsRBD1 and dsRBD2, respectively) have affinities for dsRNA, whereas the third dsRBD (dsRBD3) binds to Dicer. In this study, we prepared the single domain fragments of human TRBP corresponding to dsRBD1 and dsRBD2 and solved the crystal structure of dsRBD1 and the solution structure of dsRBD2. The two structures contain an α-β-β-β-α fold, which is common to the dsRBDs. The overall structures of dsRBD1 and dsRBD2 are similar to each other, except for a slight shift of the first α helix. The residues involved in dsRNA binding are conserved. We examined the small interfering RNA (siRNA)-binding properties of these dsRBDs by isothermal titration colorimetry measurements. The dsRBD1 and dsRBD2 fragments both bound to siRNA, with dissociation constants of 220 and 113 nM, respectively. In contrast, the full-length TRBP and its fragment with dsRBD1 and dsRBD2 exhibited much smaller dissociation constants (0.24 and 0.25 nM, respectively), indicating that the tandem dsRBDs bind simultaneously to one siRNA molecule. On the other hand, the loop between the first α helix and the first β strand of dsRBD2, but not dsRBD1, has a Trp residue, which forms hydrophobic and cation-π interactions with the surrounding residues. A circular dichroism analysis revealed that the thermal stability of dsRBD2 is higher than that of dsRBD1 and depends on the Trp residue.

The zinc finger CW (zf-CW) domain is a motif of about 60 residues that is frequently found in proteins involved in epigenetic regulation. Here, we determined the NMR solution structure of the zf-CW domain of the human zf-CW and PWWP domain containing protein 1 (ZCWPW1). The zf-CW domain adopts a new fold in which a zinc ion is coordinated tetrahedrally by four conserved Cys ligand residues. The tertiary structure of the zf-CW domain partially resembles that adopted by the plant homeo domain (PHD) finger bound to the histone tail, suggesting that the zf-CW domain and the PHD finger have similar functions. The solution structure of the complex of the zf-CW domain with the histone H3 tail peptide (1-10) with trimethylated K4 clarified its binding mode. Our structural and biochemical studies have identified the zf-CW domain as a member of the histone modification reader modules for epigenetic regulation., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The Notch signaling pathway is critical for many developmental processes and requires complex trafficking of both Notch receptor and its ligands, Delta and Serrate. In Drosophila melanogaster, the endocytosis of Delta in the signal-sending cell is essential for Notch receptor activation. The Neuralized protein from D. melanogaster (Neur) is a ubiquitin E3 ligase, which binds to Delta through its first neuralized homology repeat 1 (NHR1) domain and mediates the ubiquitination of Delta for endocytosis. Tom, a Bearded protein family member, inhibits the Neur-mediated endocytosis through interactions with the NHR1 domain. We have identified the domain boundaries of the novel NHR1 domain, using a screening system based on our cell-free protein synthesis method, and demonstrated that the identified Neur NHR1 domain had binding activity to the 20-residue peptide corresponding to motif 2 of Tom by isothermal titration calorimetry experiments. We also determined the solution structure of the Neur NHR1 domain by heteronuclear NMR methods, using a (15)N/(13)C-labeled sample. The Neur NHR1 domain adopts a characteristic beta-sandwich fold, consisting of a concave five-stranded antiparallel beta-sheet and a convex seven-stranded antiparallel beta-sheet. The long loop (L6) between the beta6 and beta7 strands covers the hydrophobic patch on the concave beta-sheet surface, and the Neur NHR1 domain forms a compact globular fold. Intriguingly, in spite of the slight, but distinct, differences in the topology of the secondary structure elements, the structure of the Neur NHR1 domain is quite similar to those of the B30.2/SPRY domains, which are known to mediate specific protein-protein interactions. Further NMR titration experiments of the Neur NHR1 domain with the 20-residue Tom peptide revealed that the resonances originating from the bottom area of the beta-sandwich (the L3, L5, and L11 loops, as well as the tip of the L6 loop) were affected. In addition, a structural comparison of the Neur NHR1 domain with the first NHR domain of the human KIAA1787 protein, which is from another NHR subfamily and does not bind to the 20-residue Tom peptide, suggested the critical amino acid residues for the interactions between the Neur NHR1 domain and the Tom peptide. The present structural study will shed light on the role of the Neur NHR1 domain in the Notch signaling pathway.

Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B'. Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.