Journal of Molecular Biology最新文献

{"title":"Consecutive Steps of Membrane Insertion of the Two-spanning MscL Protein by Insertase YidC","authors":"Philip Kauffman ,&nbsp;Haoze He ,&nbsp;Andreas Kuhn ,&nbsp;Ross E. Dalbey","doi":"10.1016/j.jmb.2025.169074","DOIUrl":"10.1016/j.jmb.2025.169074","url":null,"abstract":"<div><div>A fundamental problem in biology is understanding how membrane proteins are inserted and assembled into their three-dimensional structures. The YidC/Oxa1/Alb3 insertases, found in bacteria, mitochondria, and chloroplasts play crucial roles in membrane protein insertion. In this study, we investigated the YidC-mediated insertion of MscL, a 2-spanning membrane protein by analyzing a series of translational arrested intermediates and probing the interactions with YidC using thio-crosslinking. Our findings reveal that the first TM segment and the second TM segment of MscL interact cotranslationally with the YidC membrane-embedded greasy slide, although in a delayed manner. The translocation of the periplasmic loop in between the two TM segments only occurs after TM2 engages with the greasy slide of YidC, showing that full insertion occurs late during synthesis. Remarkably, TM2 does not displace TM1 from the slide, and the contact is maintained even when the full-length protein emerges from the ribosome. These results demonstrate a well-ordered sequence of events during the membrane insertion of multi-spanning membrane proteins, providing new insights into the mechanistic role of YidC in protein assembly.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 10","pages":"Article 169074"},"PeriodicalIF":4.7,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143584120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rethinking RNA Modifications: Therapeutic Strategies for Targeting Dysregulated RNA.","authors":"Isobel E Bowles, Esteban A Orellana","doi":"10.1016/j.jmb.2025.169046","DOIUrl":"https://doi.org/10.1016/j.jmb.2025.169046","url":null,"abstract":"<p><p>The vast array of cellular ribonucleic acid (RNA) modifications hold a crucial role in regulating RNA stability, folding, localization, and the accuracy of translation. Numerous diseases have been associated with mutations found in genes of RNA-modifying enzymes that can lead to truncated or misfolded proteins incapable of modifying their RNA substrates, causing downstream defects. In contrast, dysregulated levels of RNA-modifying enzymes and the resulting changes in RNA modifications on their substrates are increasingly linked to the activation of oncogenic pathways. This phenomenon has been especially studied through the lens of methyltransferases such as METTL1 and METTL3. The field has developed several small molecule inhibitors of RNA-modifying enzymes to mitigate their related diseases, including targeting the upregulation of METTL3 in cancer. However, increasing evidence suggests that RNA-modifying enzymes play essential roles in numerous cellular processes, including the immune response, neural health, and regeneration, among others. This could lead to off-target effects when treating proteins with small molecules, particularly when these enzymes are upregulated. We propose that developing treatments to specifically target the RNA substrates mis-regulated due to abnormal levels of RNA-modifying enzymes responsible for malignant hallmarks may offer an alternative strategy for treating diseases. We review current RNA-targeted therapies and the diseases they target, including advancements in oligonucleotide modalities and small molecules. We also identify gaps in knowledge that need to be addressed to enhance drug development in the epitranscriptome field to use these therapies to target mis-regulated RNA stemming from altered RNA-modifying enzyme levels.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169046"},"PeriodicalIF":4.7,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143584124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"5-Methyluridine is Ubiquitous in Pseudomonas aeruginosa tRNA and Modulates Antimicrobial Resistance and Virulence.","authors":"Jurairat Chittrakanwong, Ruixi Chen, Junzhou Wu, Michael S Demott, Jingjing Sun, Kamonwan Phatinuwat, Juthamas Jaroensuk, Sopapan Atichartpongkul, Skorn Mongkolsuk, Thomas Begley, Peter C Dedon, Mayuree Fuangthong","doi":"10.1016/j.jmb.2025.169020","DOIUrl":"https://doi.org/10.1016/j.jmb.2025.169020","url":null,"abstract":"<p><p>Building on decades of work in characterizing the dozens of RNA modifications in the microbial epitranscriptome, recent advances in analytical technology and genetics have revealed systems-level functions for many tRNA modifications. The tRNA (uracil-5-)-methyltransferase TrmA and its product, 5-methyl uridine (m⁵U) at position 54 in the T-loop, however, has not been linked to a specific phenotype. Here, we defined the functional and biological roles of TrmA in Pseudomonas aeruginosa (PA14), a major multidrug-resistant pathogen. Surprisingly, though TrmA was found to site-specifically catalyze m⁵U54 on all PA14 tRNAs, loss of TrmA had no effect on the levels of any of 36 tRNA modifications except m⁵U and had minimal effects on multiple phenotypic parameters, including growth rate, morphology, motility, and biofilm formation. However, loss of TrmA conferred a striking polymyxin antibiotic resistance. mRNA and tRNA profiling and proteomics analyses revealed that TrmA regulates the expression of codon-biased gene families at the level of translation, including components of a type III secretion system (T3SS). Loss of TrmA upregulated T3SS, leading to increased macrophage IL-1β in bacterial challenge tests. Altogether, these results revealed novel biological functions of TrmA and its roles in modulating gene expression at multiple levels in P. aeruginosa.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169020"},"PeriodicalIF":4.7,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143584118","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Yeast Knowledge Graphs Database for Exploring Saccharomyces Cerevisiae and Schizosaccharomyces Pombe","authors":"Mani R. Kumar ,&nbsp;Karthick Raja Arulprakasam ,&nbsp;An-Nikol Kutevska ,&nbsp;Marek Mutwil ,&nbsp;Guillaume Thibault","doi":"10.1016/j.jmb.2025.169072","DOIUrl":"10.1016/j.jmb.2025.169072","url":null,"abstract":"<div><div>Biomedical literature contains an extensive wealth of information on gene and protein function across various biological processes and diseases. However, navigating this vast and often restricted-access data can be challenging, making it difficult to extract specific insights efficiently. In this study, we introduce a high-throughput pipeline that leverages OpenAI’s Generative Pre-Trained Transformer Model (GPT) to automate the extraction and analysis of gene function information. We applied this approach to 84,427 publications on <em>Saccharomyces cerevisiae</em> and 6,452 publications on <em>Schizosaccharomyces pombe</em>, identifying 3,432,749 relationships for budding yeast and 421,198 relationships for <em>S. pombe</em>. This resulted in a comprehensive, searchable online Knowledge Graph database, available at <u>yeast.connectome.tools</u> and <u>spombe.connectome.tools</u>, which offers users extensive access to various interactions and pathways. Our analysis underscores the power of integrating artificial intelligence with bioinformatics, as demonstrated through key insights into important nodes like Hsp104 and Atg8 proteins. This work not only facilitates efficient data extraction in yeast research but also presents a scalable model for similar studies in other biological systems.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 10","pages":"Article 169072"},"PeriodicalIF":4.7,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143584126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimized Preparation of Segmentally Labeled RNAs for NMR Structure Determination","authors":"Brian D. Grossman,&nbsp;Bethel G. Beyene,&nbsp;Bersabel Tekle,&nbsp;William Sakowicz,&nbsp;Xinjie Ji,&nbsp;Joshua Miguele Camacho,&nbsp;Nandini Vaishnav,&nbsp;Amina Ahmed,&nbsp;Naman Bhandari,&nbsp;Kush Desai,&nbsp;Josiah Hardy,&nbsp;Nele M. Hollman,&nbsp;Jan Marchant,&nbsp;Michael F. Summers","doi":"10.1016/j.jmb.2025.169073","DOIUrl":"10.1016/j.jmb.2025.169073","url":null,"abstract":"<div><div>RNA structures are significantly underrepresented in public repositories (∼ 100-fold compared to proteins) despite their importance for mechanistic understanding and for development of structure prediction/validation tools. A substantial portion of deposited RNA structures have been determined by NMR (∼30%), but most comprise fewer than 60 nucleotides due to complications associated with NMR signal overlap. A promising approach for applying NMR to larger RNAs involves use of a mutated DNA polymerase (TGK) that can extend “primer” RNA strands generated independently by synthetic or enzymatic methods [Haslecker et al., <em>Nature Commun.</em> 2023]. In attempts to employ this technology, we uncovered sequence- and enzyme-dependent complications for most constructs examined that prohibited preparation of homogeneous samples. By using TGK extension efficiency and NMR as guides, we identified non-templated run-on by wild-type T7-RNA polymerase (RNAP^WT) as the primary source of product heterogeneity. Use of 2′-O-methylated DNA templates did not prevent RNAP^WT run-on for most constructs examined. However, primer RNAs with appropriate 3′ end homogeneity were obtained in high yield using a recently described T7 RNAP mutant designed for improved immunogenic behavior. Minor spectral heterogeneity sometimes observed for 3′ residues, caused by partial premature TGK termination, could be moved to sites downstream of the RNA region of interest by employing extended template DNAs that encode additional non-interacting 3′ nucleotides. We additionally present an approach for large-scale synthesis of homogeneous template DNA required for TGK extension. With these modifications, segmentally labeled RNAs appropriate for high resolution structural studies are now routinely obtainable.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 10","pages":"Article 169073"},"PeriodicalIF":4.7,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143584121","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"AIUPred - Binding: Energy Embedding to Identify Disordered Binding Regions.","authors":"Gábor Erdős, Norbert Deutsch, Zsuzsanna Dosztányi","doi":"10.1016/j.jmb.2025.169071","DOIUrl":"https://doi.org/10.1016/j.jmb.2025.169071","url":null,"abstract":"<p><p>Intrinsically disordered regions (IDRs) play critical roles in various cellular processes, often mediating interactions through disordered binding regions that transition to ordered states. Experimental characterization of these functional regions is highly challenging, underscoring the need for fast and accurate computational tools. Despite their importance, predicting disordered binding regions remains a significant challenge due to limitations in existing datasets and methodologies. In this study, we introduce AIUPred-binding, a novel prediction tool leveraging a high dimensional mathematical representation of structural energies - we call energy embedding - and pathogenicity scores from AlphaMissense. By employing a transfer learning approach, AIUPred-binding demonstrates improved accuracy in identifying functional sites within IDRs. Our results highlight the tool's ability to discern subtle features within disordered regions, addressing biases and other challenges associated with manually curated datasets. We present AIUPred-binding integrated into the AIUPred web framework as a versatile and efficient resource for understanding the functional roles of IDRs. AIUPred-binding is freely accessible at https://aiupred.elte.hu.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169071"},"PeriodicalIF":4.7,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143708014","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Dual-Selection System for Enhanced Efficiency and Fidelity of Circular RNA Overexpression","authors":"Dawei Liu ,&nbsp;Xing Wang ,&nbsp;Yali Zhang ,&nbsp;Shiyi Zuo ,&nbsp;Bradley Chereda ,&nbsp;Philip A. Gregory ,&nbsp;Chun-Xia Zhao ,&nbsp;Gregory J. Goodall","doi":"10.1016/j.jmb.2025.169064","DOIUrl":"10.1016/j.jmb.2025.169064","url":null,"abstract":"<div><div>Circular RNAs (circRNAs) are essential regulators of cellular processes, but are challenging to study using traditional methods. Overexpression approaches, such as the use of linearized plasmids and viral vectors, often result in high rates of false-positive clones, where cells retain selection markers without expressing the target circRNA. This study addresses this limitation by developing a dual-selection circRNA system designed to enhance the accuracy and reliability of circRNA overexpression. Our system integrates a fluorescent reporter gene upstream of the circRNA expression cassette, under a shared promoter, and a downstream antibiotic resistance marker, allowing for both antibiotic selection and flow cytometric cell-sorting to identify and enrich cells with genuine circRNA expression. We successfully incorporated this system into an inducible lentiviral vector for controlled overexpression in various cell types. The dual-selection circRNA system offers a significant advance for circRNA research and studies of other RNA species where accurate and reliable overexpression is essential.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 10","pages":"Article 169064"},"PeriodicalIF":4.7,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143571744","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Evolving Landscape of Protein Allostery: From Computational and Experimental Perspectives.","authors":"Srinivasan Ekambaram, Grigor Arakelov, Nikolay V Dokholyan","doi":"10.1016/j.jmb.2025.169060","DOIUrl":"10.1016/j.jmb.2025.169060","url":null,"abstract":"<p><p>Protein allostery is a fundamental biological regulatory mechanism that allows communication between distant locations within a protein, modifying its function in response to signals. Experimental techniques, such as NMR spectroscopy and cryo-electron microscopy (cryo-EM), are critical validation tools for computational predictions and provide valuable insights into dynamic conformational changes. Combining these approaches has greatly improved our understanding of classical conformational allostery and complex dynamic coupling mechanisms. Recent advances in machine learning and enhanced sampling methods have broadened the scope of allostery research, identifying cryptic allosteric sites and directing new drug discovery approaches. Despite progress, bridging static structural data with dynamic functional states remains challenging. This review underscores the importance of combining experimental and computational approaches to comprehensively understand protein allostery and its diverse applications in biology and medicine.</p>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":" ","pages":"169060"},"PeriodicalIF":4.7,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565557","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DDX37 and DDX50 Maintain Genome Stability by Preventing Transcription-dependent R-loop Formation","authors":"Yeray Hernández-Reyes ,&nbsp;Cintia Fonseca-Rodríguez ,&nbsp;Raimundo Freire ,&nbsp;Veronique A.J. Smits","doi":"10.1016/j.jmb.2025.169061","DOIUrl":"10.1016/j.jmb.2025.169061","url":null,"abstract":"<div><div>R-loops consist of an RNA-DNA hybrid and a displaced single-stranded DNA strand that play a central role in several biological processes. However, as the presence of aberrant R-loops forms a significant threat to genome stability, R-loop formation and resolution is strictly controlled by RNAse H and helicases. In a screening for RNA helicases, previously described as RNA-DNA hybrid interactors, that control genome integrity, we identified for the first time DDX37 and DDX50. Depletion of DDX37 and DDX50 promotes DNA damage, as demonstrated by H2AX phosphorylation and increased comet tail length. In addition, knock down of these RNA helicases decreases the DNA replication track length and leads to RPA focus formation, results that are indicative of replication stress. Downregulation of DDX37 and DDX50 triggers an increase in RNA-DNA hybrids, that can be reverted by the overexpression of RNase H1. Interestingly, inhibition of transcription prevented the increased RNA-DNA hybrid formation and DNA damage upon DDX37 or DDX50 depletion. Together these results demonstrate that DDX37 and DDX50 are important for resolving RNA-DNA hybrids appearing during transcription and thereby preventing DNA damage by replication stress.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 10","pages":"Article 169061"},"PeriodicalIF":4.7,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565541","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bacterial Regulatory Circuits are Linked and Extended by Small RNAs","authors":"Susan Gottesman","doi":"10.1016/j.jmb.2025.169059","DOIUrl":"10.1016/j.jmb.2025.169059","url":null,"abstract":"<div><div>I was lucky to start my research career as the molecular biology revolution was taking hold, providing a constantly increasing set of tools and questions to investigate. Starting from a fascination with bacteria and their ability to adapt to different conditions, I’ve investigated post-translational mechanisms and their role in the ability of <em>E. coli</em> to respond to stress. My research career has been primarily at the National Institutes of Health, where I run a group within the Laboratory of Molecular Biology, NCI and hold the title of NIH Distinguished Investigator. Our lab has been interested in both energy-dependent proteolysis, discussed very briefly here, and small regulatory RNAs (sRNAs). The major group of such sRNAs act by pairing with target mRNAs with the aid of the RNA chaperone Hfq, mediating both positive and negative regulation of translation and mRNA stability. Both in our own lab and in a continuing and highly productive collaboration with the laboratory of Gisela (Gigi) Storz, we have used global approaches to identify novel sRNAs, identified how many of them are regulated, both at the level of transcription and stability, and worked on understanding the role of these sRNAs in regulatory networks. Our continued work explores regulators of sRNA and Hfq function. Here, Gigi and I have split summaries of our findings, and hope that our two chapters will be read together.</div></div>","PeriodicalId":369,"journal":{"name":"Journal of Molecular Biology","volume":"437 11","pages":"Article 169059"},"PeriodicalIF":4.7,"publicationDate":"2025-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565537","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}