Pub Date : 2024-07-15DOI: 10.1038/s41594-024-01358-8
Pedro Beltrao
The idea of a scientific discovery is often linked to the eureka moment of a lone scientist, which then transforms our thinking. However, scientific discoveries are never made by individuals in isolation. They build on the work of countless researchers, and often require interdisciplinary and collaborative teams of researchers.
{"title":"The power of scientific collaborations and the future of structural biology","authors":"Pedro Beltrao","doi":"10.1038/s41594-024-01358-8","DOIUrl":"10.1038/s41594-024-01358-8","url":null,"abstract":"The idea of a scientific discovery is often linked to the eureka moment of a lone scientist, which then transforms our thinking. However, scientific discoveries are never made by individuals in isolation. They build on the work of countless researchers, and often require interdisciplinary and collaborative teams of researchers.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 9","pages":"1309-1310"},"PeriodicalIF":12.5,"publicationDate":"2024-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141618205","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-11DOI: 10.1038/s41594-024-01356-w
Elena Lavdovskaia, Elisa Hanitsch, Andreas Linden, Martin Pašen, Venkatapathi Challa, Yehor Horokhovskyi, Hanna P. Roetschke, Franziska Nadler, Luisa Welp, Emely Steube, Marleen Heinrichs, Mandy Mong-Quyen Mai, Henning Urlaub, Juliane Liepe, Ricarda Richter-Dennerlein
Mitochondria contain dedicated ribosomes (mitoribosomes), which synthesize the mitochondrial-encoded core components of the oxidative phosphorylation complexes. The RNA and protein components of mitoribosomes are encoded on two different genomes (mitochondrial and nuclear) and are assembled into functional complexes with the help of dedicated factors inside the organelle. Defects in mitoribosome biogenesis are associated with severe human diseases, yet the molecular pathway of mitoribosome assembly remains poorly understood. Here, we applied a multidisciplinary approach combining biochemical isolation and analysis of native mitoribosomal assembly complexes with quantitative mass spectrometry and mathematical modeling to reconstitute the entire assembly pathway of the human mitoribosome. We show that, in contrast to its bacterial and cytosolic counterparts, human mitoribosome biogenesis involves the formation of ribosomal protein-only modules, which then assemble on the appropriate ribosomal RNA moiety in a coordinated fashion. The presence of excess protein-only modules primed for assembly rationalizes how mitochondria cope with the challenge of forming a protein-rich ribonucleoprotein complex of dual genetic origin. This study provides a comprehensive roadmap of mitoribosome biogenesis, from very early to late maturation steps, and highlights the evolutionary divergence from its bacterial ancestor. Lavdovskaia, Hanitsch, Linden et al. provide a comprehensive roadmap of mitoribosome biogenesis and establish that mitochondria use a unique pathway for the assembly of their translation machinery.
{"title":"A roadmap for ribosome assembly in human mitochondria","authors":"Elena Lavdovskaia, Elisa Hanitsch, Andreas Linden, Martin Pašen, Venkatapathi Challa, Yehor Horokhovskyi, Hanna P. Roetschke, Franziska Nadler, Luisa Welp, Emely Steube, Marleen Heinrichs, Mandy Mong-Quyen Mai, Henning Urlaub, Juliane Liepe, Ricarda Richter-Dennerlein","doi":"10.1038/s41594-024-01356-w","DOIUrl":"10.1038/s41594-024-01356-w","url":null,"abstract":"Mitochondria contain dedicated ribosomes (mitoribosomes), which synthesize the mitochondrial-encoded core components of the oxidative phosphorylation complexes. The RNA and protein components of mitoribosomes are encoded on two different genomes (mitochondrial and nuclear) and are assembled into functional complexes with the help of dedicated factors inside the organelle. Defects in mitoribosome biogenesis are associated with severe human diseases, yet the molecular pathway of mitoribosome assembly remains poorly understood. Here, we applied a multidisciplinary approach combining biochemical isolation and analysis of native mitoribosomal assembly complexes with quantitative mass spectrometry and mathematical modeling to reconstitute the entire assembly pathway of the human mitoribosome. We show that, in contrast to its bacterial and cytosolic counterparts, human mitoribosome biogenesis involves the formation of ribosomal protein-only modules, which then assemble on the appropriate ribosomal RNA moiety in a coordinated fashion. The presence of excess protein-only modules primed for assembly rationalizes how mitochondria cope with the challenge of forming a protein-rich ribonucleoprotein complex of dual genetic origin. This study provides a comprehensive roadmap of mitoribosome biogenesis, from very early to late maturation steps, and highlights the evolutionary divergence from its bacterial ancestor. Lavdovskaia, Hanitsch, Linden et al. provide a comprehensive roadmap of mitoribosome biogenesis and establish that mitochondria use a unique pathway for the assembly of their translation machinery.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 12","pages":"1898-1908"},"PeriodicalIF":12.5,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41594-024-01356-w.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141584474","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-10DOI: 10.1038/s41594-024-01355-x
Thomas E. Wales, Aleksandra Pajak, Alžběta Roeselová, Santosh Shivakumaraswamy, Steven Howell, Svend Kjær, F. Ulrich Hartl, John R. Engen, David Balchin
Protein folding in vivo begins during synthesis on the ribosome and is modulated by molecular chaperones that engage the nascent polypeptide. How these features of protein biogenesis influence the maturation pathway of nascent proteins is incompletely understood. Here, we use hydrogen–deuterium exchange mass spectrometry to define, at peptide resolution, the cotranslational chaperone-assisted folding pathway of Escherichia coli dihydrofolate reductase. The nascent polypeptide folds along an unanticipated pathway through structured intermediates not populated during refolding from denaturant. Association with the ribosome allows these intermediates to form, as otherwise destabilizing carboxy-terminal sequences remain confined in the ribosome exit tunnel. Trigger factor binds partially folded states without disrupting their structure, and the nascent chain is poised to complete folding immediately upon emergence of the C terminus from the exit tunnel. By mapping interactions between the nascent chain and ribosomal proteins, we trace the path of the emerging polypeptide during synthesis. Our work reveals new mechanisms by which cellular factors shape the conformational search for the native state. The authors follow the folding dynamics of a nascent protein trapped during its synthesis, showing how the ribosome and a molecular chaperone shape the pathway of protein folding.
体内蛋白质的折叠始于核糖体上的合成过程，并受到与新生多肽结合的分子伴侣的调节。人们对蛋白质生物发生的这些特征如何影响新生蛋白质的成熟途径尚不完全清楚。在这里，我们利用氢氘交换质谱法，以肽段分辨率确定了大肠杆菌二氢叶酸还原酶的共翻译伴侣辅助折叠途径。新生多肽沿着一条意料之外的途径折叠，经过的结构中间体在从变性剂重新折叠过程中没有出现。与核糖体的结合使这些中间体得以形成，否则破坏稳定的羧基末端序列将被限制在核糖体出口隧道中。触发因子与部分折叠状态结合而不会破坏它们的结构，新生链准备好在 C 端从出口隧道出现后立即完成折叠。通过绘制新生链与核糖体蛋白之间的相互作用图，我们追踪了合成过程中新生多肽的路径。我们的研究揭示了细胞因素影响原生态构象搜索的新机制。
{"title":"Resolving chaperone-assisted protein folding on the ribosome at the peptide level","authors":"Thomas E. Wales, Aleksandra Pajak, Alžběta Roeselová, Santosh Shivakumaraswamy, Steven Howell, Svend Kjær, F. Ulrich Hartl, John R. Engen, David Balchin","doi":"10.1038/s41594-024-01355-x","DOIUrl":"10.1038/s41594-024-01355-x","url":null,"abstract":"Protein folding in vivo begins during synthesis on the ribosome and is modulated by molecular chaperones that engage the nascent polypeptide. How these features of protein biogenesis influence the maturation pathway of nascent proteins is incompletely understood. Here, we use hydrogen–deuterium exchange mass spectrometry to define, at peptide resolution, the cotranslational chaperone-assisted folding pathway of Escherichia coli dihydrofolate reductase. The nascent polypeptide folds along an unanticipated pathway through structured intermediates not populated during refolding from denaturant. Association with the ribosome allows these intermediates to form, as otherwise destabilizing carboxy-terminal sequences remain confined in the ribosome exit tunnel. Trigger factor binds partially folded states without disrupting their structure, and the nascent chain is poised to complete folding immediately upon emergence of the C terminus from the exit tunnel. By mapping interactions between the nascent chain and ribosomal proteins, we trace the path of the emerging polypeptide during synthesis. Our work reveals new mechanisms by which cellular factors shape the conformational search for the native state. The authors follow the folding dynamics of a nascent protein trapped during its synthesis, showing how the ribosome and a molecular chaperone shape the pathway of protein folding.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 12","pages":"1888-1897"},"PeriodicalIF":12.5,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41594-024-01355-x.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141577168","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-08DOI: 10.1038/s41594-024-01350-2
Rohit Roy, Hashim M. Al-Hashimi
AlphaFold 3 represents a breakthrough in predicting the 3D structures of complexes directly from their sequences, offering insights into biomolecular interactions. Extending predictions to molecular behavior and function requires a shift from viewing biomolecules as static 3D structures to dynamic conformational ensembles.
{"title":"AlphaFold3 takes a step toward decoding molecular behavior and biological computation","authors":"Rohit Roy, Hashim M. Al-Hashimi","doi":"10.1038/s41594-024-01350-2","DOIUrl":"10.1038/s41594-024-01350-2","url":null,"abstract":"AlphaFold 3 represents a breakthrough in predicting the 3D structures of complexes directly from their sequences, offering insights into biomolecular interactions. Extending predictions to molecular behavior and function requires a shift from viewing biomolecules as static 3D structures to dynamic conformational ensembles.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 7","pages":"997-1000"},"PeriodicalIF":12.5,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141556979","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-08DOI: 10.1038/s41594-024-01354-y
Sven M. Lange, Matthew R. McFarland, Frederic Lamoliatte, Thomas Carroll, Logesvaran Krshnan, Anna Pérez-Ràfols, Dominika Kwasna, Linnan Shen, Iona Wallace, Isobel Cole, Lee A. Armstrong, Axel Knebel, Clare Johnson, Virginia De Cesare, Yogesh Kulathu
Branched ubiquitin (Ub) chains constitute a sizable fraction of Ub polymers in human cells. Despite their abundance, our understanding of branched Ub function in cell signaling has been stunted by the absence of accessible methods and tools. Here we identify cellular branched-chain-specific binding proteins and devise approaches to probe K48–K63-branched Ub function. We establish a method to monitor cleavage of linkages within complex Ub chains and unveil ATXN3 and MINDY as debranching enzymes. We engineer a K48–K63 branch-specific nanobody and reveal the molecular basis of its specificity in crystal structures of nanobody-branched Ub chain complexes. Using this nanobody, we detect increased K48–K63-Ub branching following valosin-containing protein (VCP)/p97 inhibition and after DNA damage. Together with our discovery that multiple VCP/p97-associated proteins bind to or debranch K48–K63-linked Ub, these results suggest a function for K48–K63-branched chains in VCP/p97-related processes. Here the authors assemble a toolkit to probe K48–K63-branched ubiquitin chain function. By identifying specific binders and deubiquitinases and engineering a specific nanobody, they reveal the importance of these chains in p97-dependent processes.
{"title":"VCP/p97-associated proteins are binders and debranching enzymes of K48–K63-branched ubiquitin chains","authors":"Sven M. Lange, Matthew R. McFarland, Frederic Lamoliatte, Thomas Carroll, Logesvaran Krshnan, Anna Pérez-Ràfols, Dominika Kwasna, Linnan Shen, Iona Wallace, Isobel Cole, Lee A. Armstrong, Axel Knebel, Clare Johnson, Virginia De Cesare, Yogesh Kulathu","doi":"10.1038/s41594-024-01354-y","DOIUrl":"10.1038/s41594-024-01354-y","url":null,"abstract":"Branched ubiquitin (Ub) chains constitute a sizable fraction of Ub polymers in human cells. Despite their abundance, our understanding of branched Ub function in cell signaling has been stunted by the absence of accessible methods and tools. Here we identify cellular branched-chain-specific binding proteins and devise approaches to probe K48–K63-branched Ub function. We establish a method to monitor cleavage of linkages within complex Ub chains and unveil ATXN3 and MINDY as debranching enzymes. We engineer a K48–K63 branch-specific nanobody and reveal the molecular basis of its specificity in crystal structures of nanobody-branched Ub chain complexes. Using this nanobody, we detect increased K48–K63-Ub branching following valosin-containing protein (VCP)/p97 inhibition and after DNA damage. Together with our discovery that multiple VCP/p97-associated proteins bind to or debranch K48–K63-linked Ub, these results suggest a function for K48–K63-branched chains in VCP/p97-related processes. Here the authors assemble a toolkit to probe K48–K63-branched ubiquitin chain function. By identifying specific binders and deubiquitinases and engineering a specific nanobody, they reveal the importance of these chains in p97-dependent processes.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 12","pages":"1872-1887"},"PeriodicalIF":12.5,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41594-024-01354-y.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141556737","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-08DOI: 10.1038/s41594-024-01347-x
Agnel Sfeir
Curiosity-driven and fundamental discovery science must be justified in its importance to human health and translational potential for practical applications and cures. However, many groundbreaking discoveries occur through the freedom to ask fundamental questions — the how and why — without knowing where they lead. Presented here is an example of a clinical target that emerged from a seemingly simple question in chromosome biology.
{"title":"Obscure DNA sequences unveil a new cancer target","authors":"Agnel Sfeir","doi":"10.1038/s41594-024-01347-x","DOIUrl":"10.1038/s41594-024-01347-x","url":null,"abstract":"Curiosity-driven and fundamental discovery science must be justified in its importance to human health and translational potential for practical applications and cures. However, many groundbreaking discoveries occur through the freedom to ask fundamental questions — the how and why — without knowing where they lead. Presented here is an example of a clinical target that emerged from a seemingly simple question in chromosome biology.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 9","pages":"1311-1312"},"PeriodicalIF":12.5,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141556978","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-02DOI: 10.1038/s41594-024-01340-4
Ilias Skeparnias, Charles Bou-Nader, Dimitrios G. Anastasakis, Lixin Fan, Yun-Xing Wang, Markus Hafner, Jinwei Zhang
The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) long noncoding RNA (lncRNA) has key roles in regulating transcription, splicing, tumorigenesis, etc. Its maturation and stabilization require precise processing by RNase P, which simultaneously initiates the biogenesis of a 3′ cytoplasmic MALAT1-associated small cytoplasmic RNA (mascRNA). mascRNA was proposed to fold into a transfer RNA (tRNA)-like secondary structure but lacks eight conserved linking residues required by the canonical tRNA fold. Here we report crystal structures of human mascRNA before and after processing, which reveal an ultracompact, quasi-tRNA-like structure. Despite lacking all linker residues, mascRNA faithfully recreates the characteristic ‘elbow’ feature of tRNAs to recruit RNase P and ElaC homolog protein 2 (ELAC2) for processing, which exhibit distinct substrate specificities. Rotation and repositioning of the D-stem and anticodon regions preclude mascRNA from aminoacylation, avoiding interference with translation. Therefore, a class of metazoan lncRNA loci uses a previously unrecognized, unusually streamlined quasi-tRNA architecture to recruit select tRNA-processing enzymes while excluding others to drive bespoke RNA biogenesis, processing and maturation. The authors uncover a Père David’s deer-like design for long noncoding RNAs such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), which partially mimics the transfer RNA (tRNA) structure to recruit select tRNA processing enzymes for maturation and to create novel regulatory RNAs such as the MALAT1-associated small cytoplasmic RNA.
{"title":"Structural basis of MALAT1 RNA maturation and mascRNA biogenesis","authors":"Ilias Skeparnias, Charles Bou-Nader, Dimitrios G. Anastasakis, Lixin Fan, Yun-Xing Wang, Markus Hafner, Jinwei Zhang","doi":"10.1038/s41594-024-01340-4","DOIUrl":"10.1038/s41594-024-01340-4","url":null,"abstract":"The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) long noncoding RNA (lncRNA) has key roles in regulating transcription, splicing, tumorigenesis, etc. Its maturation and stabilization require precise processing by RNase P, which simultaneously initiates the biogenesis of a 3′ cytoplasmic MALAT1-associated small cytoplasmic RNA (mascRNA). mascRNA was proposed to fold into a transfer RNA (tRNA)-like secondary structure but lacks eight conserved linking residues required by the canonical tRNA fold. Here we report crystal structures of human mascRNA before and after processing, which reveal an ultracompact, quasi-tRNA-like structure. Despite lacking all linker residues, mascRNA faithfully recreates the characteristic ‘elbow’ feature of tRNAs to recruit RNase P and ElaC homolog protein 2 (ELAC2) for processing, which exhibit distinct substrate specificities. Rotation and repositioning of the D-stem and anticodon regions preclude mascRNA from aminoacylation, avoiding interference with translation. Therefore, a class of metazoan lncRNA loci uses a previously unrecognized, unusually streamlined quasi-tRNA architecture to recruit select tRNA-processing enzymes while excluding others to drive bespoke RNA biogenesis, processing and maturation. The authors uncover a Père David’s deer-like design for long noncoding RNAs such as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), which partially mimics the transfer RNA (tRNA) structure to recruit select tRNA processing enzymes for maturation and to create novel regulatory RNAs such as the MALAT1-associated small cytoplasmic RNA.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 11","pages":"1655-1668"},"PeriodicalIF":12.5,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141489614","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-02DOI: 10.1038/s41594-024-01345-z
Léa Mammri, Paul T. Conduit
Microtubules within cells often have 13 protofilaments but are nucleated by multi-protein y-TuRCs complexes that display 14 γ-tubulin molecules. High-resolution cryo-EM structures of γ-TuRCs after nucleation show that these γ-TuRCs ‘close’ during nucleation to display only 13 γ-tubulin molecules for protofilament assembly.
{"title":"Cryo-EM structures of γ-TuRC reveal molecular insights into microtubule nucleation","authors":"Léa Mammri, Paul T. Conduit","doi":"10.1038/s41594-024-01345-z","DOIUrl":"10.1038/s41594-024-01345-z","url":null,"abstract":"Microtubules within cells often have 13 protofilaments but are nucleated by multi-protein y-TuRCs complexes that display 14 γ-tubulin molecules. High-resolution cryo-EM structures of γ-TuRCs after nucleation show that these γ-TuRCs ‘close’ during nucleation to display only 13 γ-tubulin molecules for protofilament assembly.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 7","pages":"1004-1006"},"PeriodicalIF":12.5,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141489617","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-02DOI: 10.1038/s41594-024-01344-0
Marina C. Nocente, Anida Mesihovic Karamitsos, Emilie Drouineau, Manon Soleil, Waad Albawardi, Cécile Dulary, Florence Ribierre, Hélène Picaud, Olivier Alibert, Joël Acker, Marie Kervella, Jean-Christophe Aude, Nick Gilbert, Françoise Ochsenbein, Sophie Chantalat, Matthieu Gérard
The canonical BRG/BRM-associated factor (cBAF) complex is essential for chromatin opening at enhancers in mammalian cells. However, the nature of the open chromatin remains unclear. Here, we show that, in addition to producing histone-free DNA, cBAF generates stable hemisome-like subnucleosomal particles containing the four core histones associated with 50–80 bp of DNA. Our genome-wide analysis indicates that cBAF makes these particles by targeting and splitting fragile nucleosomes. In mouse embryonic stem cells, these subnucleosomes become an in vivo binding substrate for the master transcription factor OCT4 independently of the presence of OCT4 DNA motifs. At enhancers, the OCT4–subnucleosome interaction increases OCT4 occupancy and amplifies the genomic interval bound by OCT4 by up to one order of magnitude compared to the region occupied on histone-free DNA. We propose that cBAF-dependent subnucleosomes orchestrate a molecular mechanism that projects OCT4 function in chromatin opening beyond its DNA motifs. Here, the authors show that the canonical BRG/BRM-associated factor (SWI/SNF) chromatin remodeler generates subnucleosomes containing 50–80 bp of DNA associated with the four core histones. These hemisome-like particles interact with OCT4 to expand its binding domain at enhancers.
典型的 BRG/BRM 相关因子（cBAF）复合物对哺乳动物细胞中增强子的染色质开放至关重要。然而，开放染色质的性质仍不清楚。在这里，我们发现除了产生无组蛋白的 DNA 外，cBAF 还能产生稳定的半球状亚核糖体颗粒，其中包含与 50-80 bp DNA 相关的四个核心组蛋白。我们的全基因组分析表明，cBAF通过靶向和分裂脆弱的核小体来产生这些颗粒。在小鼠胚胎干细胞中，这些亚核小体成为主转录因子OCT4的体内结合底物，与OCT4 DNA基序的存在无关。在增强子上，OCT4与亚核小体的相互作用增加了OCT4的占据率，与无组蛋白DNA占据的区域相比，OCT4结合的基因组间隔扩大了一个数量级。我们认为，依赖于cBAF的亚核小体协调了一种分子机制，将OCT4在染色质开放中的功能投射到其DNA基团之外。
{"title":"cBAF generates subnucleosomes that expand OCT4 binding and function beyond DNA motifs at enhancers","authors":"Marina C. Nocente, Anida Mesihovic Karamitsos, Emilie Drouineau, Manon Soleil, Waad Albawardi, Cécile Dulary, Florence Ribierre, Hélène Picaud, Olivier Alibert, Joël Acker, Marie Kervella, Jean-Christophe Aude, Nick Gilbert, Françoise Ochsenbein, Sophie Chantalat, Matthieu Gérard","doi":"10.1038/s41594-024-01344-0","DOIUrl":"10.1038/s41594-024-01344-0","url":null,"abstract":"The canonical BRG/BRM-associated factor (cBAF) complex is essential for chromatin opening at enhancers in mammalian cells. However, the nature of the open chromatin remains unclear. Here, we show that, in addition to producing histone-free DNA, cBAF generates stable hemisome-like subnucleosomal particles containing the four core histones associated with 50–80 bp of DNA. Our genome-wide analysis indicates that cBAF makes these particles by targeting and splitting fragile nucleosomes. In mouse embryonic stem cells, these subnucleosomes become an in vivo binding substrate for the master transcription factor OCT4 independently of the presence of OCT4 DNA motifs. At enhancers, the OCT4–subnucleosome interaction increases OCT4 occupancy and amplifies the genomic interval bound by OCT4 by up to one order of magnitude compared to the region occupied on histone-free DNA. We propose that cBAF-dependent subnucleosomes orchestrate a molecular mechanism that projects OCT4 function in chromatin opening beyond its DNA motifs. Here, the authors show that the canonical BRG/BRM-associated factor (SWI/SNF) chromatin remodeler generates subnucleosomes containing 50–80 bp of DNA associated with the four core histones. These hemisome-like particles interact with OCT4 to expand its binding domain at enhancers.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 11","pages":"1756-1768"},"PeriodicalIF":12.5,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141489595","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-01DOI: 10.1038/s41594-024-01336-0
Wenxin Hu, Amit Kumar, Syed Faraz Ahmed, Shijiao Qi, David K. G. Ma, Honglin Chen, Gurjeet J. Singh, Joshua M. L. Casan, Michelle Haber, Ilia Voskoboinik, Matthew R. McKay, Joseph A. Trapani, Paul G. Ekert, Mohamed Fareh
The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized. Here, we present single-base tiled screening and computational analyses that identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. We show that the de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient CRISPR RNAs (crRNAs). These validated design principles (integrated into an online tool, https://cas13target.azurewebsites.net/ ) can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer–target mutagenesis revealed that PspCas13b can tolerate only up to four mismatches and requires ~26-nucleotide base pairing with the target to activate its nuclease domains, highlighting its superior specificity compared to other RNA or DNA interference tools. On the basis of this targeting resolution, we predict an extremely low probability of PspCas13b having off-target effects on other cellular transcripts. Proteomic analysis validated this prediction and showed that, unlike other Cas13 orthologs, PspCas13b exhibits potent on-target activity and lacks collateral effects. This study uses single-base tiled screens, bioinformatics, comprehensive mutagenesis and proteomics to provide a high-resolution view of RNA silencing with PspCas13b. It reveals design principles for potent silencing without collateral effects.
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