Pub Date : 2024-08-07DOI: 10.1038/s41594-024-01370-y
Johann J. Roske, Joseph T. P. Yeeles
During chromosome replication, the nascent leading strand is synthesized by DNA polymerase epsilon (Pol ε), which associates with the sliding clamp processivity factor proliferating cell nuclear antigen (PCNA) to form a processive holoenzyme. For high-fidelity DNA synthesis, Pol ε relies on nucleotide selectivity and its proofreading ability to detect and excise a misincorporated nucleotide. Here, we present cryo-electron microscopy (cryo-EM) structures of human Pol ε in complex with PCNA, DNA and an incoming nucleotide, revealing how Pol ε associates with PCNA through its PCNA-interacting peptide box and additional unique features of its catalytic domain. Furthermore, by solving a series of cryo-EM structures of Pol ε at a mismatch-containing DNA, we elucidate how Pol ε senses and edits a misincorporated nucleotide. Our structures delineate steps along an intramolecular switching mechanism between polymerase and exonuclease activities, providing the basis for a proofreading mechanism in B-family replicative polymerases.
在染色体复制过程中,新生前导链由 DNA 聚合酶ε(Pol ε)合成,它与滑动钳加工因子增殖细胞核抗原(PCNA)结合形成一个加工全酶。Pol ε依靠核苷酸选择性及其校对能力来检测和切除错误结合的核苷酸,从而实现高保真的DNA合成。在这里,我们展示了人Pol ε与PCNA、DNA和输入核苷酸复合物的冷冻电子显微镜(cryo-EM)结构,揭示了Pol ε如何通过其PCNA-interacting肽盒与PCNA结合,以及其催化结构域的其他独特特征。此外,通过解决 Pol ε 在含错配 DNA 上的一系列低温电子显微镜结构,我们阐明了 Pol ε 如何感知和编辑误入的核苷酸。我们的结构描述了聚合酶和外切酶活性之间分子内切换机制的步骤,为 B-家族复制聚合酶的校对机制提供了基础。
{"title":"Structural basis for processive daughter-strand synthesis and proofreading by the human leading-strand DNA polymerase Pol ε","authors":"Johann J. Roske, Joseph T. P. Yeeles","doi":"10.1038/s41594-024-01370-y","DOIUrl":"https://doi.org/10.1038/s41594-024-01370-y","url":null,"abstract":"<p>During chromosome replication, the nascent leading strand is synthesized by DNA polymerase epsilon (Pol ε), which associates with the sliding clamp processivity factor proliferating cell nuclear antigen (PCNA) to form a processive holoenzyme. For high-fidelity DNA synthesis, Pol ε relies on nucleotide selectivity and its proofreading ability to detect and excise a misincorporated nucleotide. Here, we present cryo-electron microscopy (cryo-EM) structures of human Pol ε in complex with PCNA, DNA and an incoming nucleotide, revealing how Pol ε associates with PCNA through its PCNA-interacting peptide box and additional unique features of its catalytic domain. Furthermore, by solving a series of cryo-EM structures of Pol ε at a mismatch-containing DNA, we elucidate how Pol ε senses and edits a misincorporated nucleotide. Our structures delineate steps along an intramolecular switching mechanism between polymerase and exonuclease activities, providing the basis for a proofreading mechanism in B-family replicative polymerases.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141899782","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-26DOI: 10.1038/s41594-024-01366-8
Jasleen Gill
CRISPR–Cas enzymes have enabled us to manipulate the genetic code with unparalleled precision and efficiency. Here I explore the structural and biochemical intricacies that govern the functionality of CRISPR–Cas technologies, emphasizing the need for a nuanced mechanistic understanding to overcome current limitations and pave the way for safer and more effective genome-editing applications in medicine and research.
CRISPR–Cas enzymes have emerged as a molecular scalpel for scientists and physicians, who are now able to target and manipulate our genetic code efficiently and precisely. Over the past ten years, scientists have leveraged the ability of these enzymes to target specific genomic regions, beginning with cytosine and adenine base editors, and followed by prime and click editing technologies1 that expanded editing to transversion mutations, insertions and deletions. CRISPR-based technologies have made detecting and treating disease, drug and genetic screening, and creating genetically modified crops more accessible than ever before.
{"title":"Dissecting the mechanism of CRISPR–Cas technologies to design efficient biotechnologies","authors":"Jasleen Gill","doi":"10.1038/s41594-024-01366-8","DOIUrl":"https://doi.org/10.1038/s41594-024-01366-8","url":null,"abstract":"<p>CRISPR–Cas enzymes have enabled us to manipulate the genetic code with unparalleled precision and efficiency. Here I explore the structural and biochemical intricacies that govern the functionality of CRISPR–Cas technologies, emphasizing the need for a nuanced mechanistic understanding to overcome current limitations and pave the way for safer and more effective genome-editing applications in medicine and research.</p><p>CRISPR–Cas enzymes have emerged as a molecular scalpel for scientists and physicians, who are now able to target and manipulate our genetic code efficiently and precisely. Over the past ten years, scientists have leveraged the ability of these enzymes to target specific genomic regions, beginning with cytosine and adenine base editors, and followed by prime and click editing technologies<sup>1</sup> that expanded editing to transversion mutations, insertions and deletions. CRISPR-based technologies have made detecting and treating disease, drug and genetic screening, and creating genetically modified crops more accessible than ever before.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141764281","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-26DOI: 10.1038/s41594-024-01367-7
Sichen Pan, Karin Gries, Benjamin D. Engel, Michael Schroda, Christoph A. Haselwandter, Simon Scheuring
The biogenesis and maintenance of thylakoid membranes require vesicle-inducing protein in plastids 1 (VIPP1). VIPP1 is a member of the endosomal sorting complex required for transport-III (ESCRT-III) superfamily, whose members form diverse filament-based supramolecular structures that facilitate membrane deformation and fission. VIPP1 cryo-electron microscopy (EM) structures in solution revealed helical rods and baskets of stacked rings, with amphipathic membrane-binding domains in the lumen. However, how VIPP1 interacts with membranes remains largely unknown. Here, using high-speed atomic force microscopy (HS-AFM), we show that VIPP1 assembles into right-handed chiral spirals and regular polygons on supported lipid bilayers via ESCRT-III-like filament assembly and dynamics. VIPP1 filaments grow clockwise into spirals through polymerization at a ring-shaped central polymerization hub, and into polygons through clockwise polymerization at the sector peripheries. Interestingly, VIPP1 initially forms Archimedean spirals, which upon maturation transform into logarithmic spirals through lateral annealing of strands to the outermore low-curvature spiral turns.
{"title":"The cyanobacterial protein VIPP1 forms ESCRT-III-like structures on lipid bilayers","authors":"Sichen Pan, Karin Gries, Benjamin D. Engel, Michael Schroda, Christoph A. Haselwandter, Simon Scheuring","doi":"10.1038/s41594-024-01367-7","DOIUrl":"https://doi.org/10.1038/s41594-024-01367-7","url":null,"abstract":"<p>The biogenesis and maintenance of thylakoid membranes require vesicle-inducing protein in plastids 1 (VIPP1). VIPP1 is a member of the endosomal sorting complex required for transport-III (ESCRT-III) superfamily, whose members form diverse filament-based supramolecular structures that facilitate membrane deformation and fission. VIPP1 cryo-electron microscopy (EM) structures in solution revealed helical rods and baskets of stacked rings, with amphipathic membrane-binding domains in the lumen. However, how VIPP1 interacts with membranes remains largely unknown. Here, using high-speed atomic force microscopy (HS-AFM), we show that VIPP1 assembles into right-handed chiral spirals and regular polygons on supported lipid bilayers via ESCRT-III-like filament assembly and dynamics. VIPP1 filaments grow clockwise into spirals through polymerization at a ring-shaped central polymerization hub, and into polygons through clockwise polymerization at the sector peripheries. Interestingly, VIPP1 initially forms Archimedean spirals, which upon maturation transform into logarithmic spirals through lateral annealing of strands to the outermore low-curvature spiral turns.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141764278","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-24DOI: 10.1038/s41594-024-01353-z
Michael C. Lanz, Shuyuan Zhang, Matthew P. Swaffer, Inbal Ziv, Luisa Hernández Götz, Jacob Kim, Frank McCarthy, Daniel F. Jarosz, Joshua E. Elias, Jan M. Skotheim
Cell size is tightly controlled in healthy tissues and single-celled organisms, but it remains unclear how cell size influences physiology. Increasing cell size was recently shown to remodel the proteomes of cultured human cells, demonstrating that large and small cells of the same type can be compositionally different. In the present study, we utilize the natural heterogeneity of hepatocyte ploidy and yeast genetics to establish that the ploidy-to-cell size ratio is a highly conserved determinant of proteome composition. In both mammalian and yeast cells, genome dilution by cell growth elicits a starvation-like phenotype, suggesting that growth in large cells is restricted by genome concentration in a manner that mimics a limiting nutrient. Moreover, genome dilution explains some proteomic changes ascribed to yeast aging. Overall, our data indicate that genome concentration drives changes in cell composition independently of external environmental cues.
{"title":"Genome dilution by cell growth drives starvation-like proteome remodeling in mammalian and yeast cells","authors":"Michael C. Lanz, Shuyuan Zhang, Matthew P. Swaffer, Inbal Ziv, Luisa Hernández Götz, Jacob Kim, Frank McCarthy, Daniel F. Jarosz, Joshua E. Elias, Jan M. Skotheim","doi":"10.1038/s41594-024-01353-z","DOIUrl":"https://doi.org/10.1038/s41594-024-01353-z","url":null,"abstract":"<p>Cell size is tightly controlled in healthy tissues and single-celled organisms, but it remains unclear how cell size influences physiology. Increasing cell size was recently shown to remodel the proteomes of cultured human cells, demonstrating that large and small cells of the same type can be compositionally different. In the present study, we utilize the natural heterogeneity of hepatocyte ploidy and yeast genetics to establish that the ploidy-to-cell size ratio is a highly conserved determinant of proteome composition. In both mammalian and yeast cells, genome dilution by cell growth elicits a starvation-like phenotype, suggesting that growth in large cells is restricted by genome concentration in a manner that mimics a limiting nutrient. Moreover, genome dilution explains some proteomic changes ascribed to yeast aging. Overall, our data indicate that genome concentration drives changes in cell composition independently of external environmental cues.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141755099","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-22DOI: 10.1038/s41594-024-01348-w
Victor R. A. Dubach, Pablo San Segundo-Acosta, Bonnie J. Murphy
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) have a major role in the physiology of eukaryotic cells by mediating reactive oxygen species production. Evolutionarily distant proteins with the NOX catalytic core have been found in bacteria, including Streptococcus pneumoniae NOX (SpNOX), which is proposed as a model for studying NOXs because of its high activity and stability in detergent micelles. We present here cryo-electron microscopy structures of substrate-free and nicotinamide adenine dinucleotide (NADH)-bound SpNOX and of NADPH-bound wild-type and F397A SpNOX under turnover conditions. These high-resolution structures provide insights into the electron-transfer pathway and reveal a hydride-transfer mechanism regulated by the displacement of F397. We conducted structure-guided mutagenesis and biochemical analyses that explain the absence of substrate specificity toward NADPH and suggest the mechanism behind constitutive activity. Our study presents the structural basis underlying SpNOX enzymatic activity and sheds light on its potential in vivo function.
{"title":"Structural and mechanistic insights into Streptococcus pneumoniae NADPH oxidase","authors":"Victor R. A. Dubach, Pablo San Segundo-Acosta, Bonnie J. Murphy","doi":"10.1038/s41594-024-01348-w","DOIUrl":"https://doi.org/10.1038/s41594-024-01348-w","url":null,"abstract":"<p>Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) have a major role in the physiology of eukaryotic cells by mediating reactive oxygen species production. Evolutionarily distant proteins with the NOX catalytic core have been found in bacteria, including <i>Streptococcus pneumoniae</i> NOX (SpNOX), which is proposed as a model for studying NOXs because of its high activity and stability in detergent micelles. We present here cryo-electron microscopy structures of substrate-free and nicotinamide adenine dinucleotide (NADH)-bound SpNOX and of NADPH-bound wild-type and F397A SpNOX under turnover conditions. These high-resolution structures provide insights into the electron-transfer pathway and reveal a hydride-transfer mechanism regulated by the displacement of F397. We conducted structure-guided mutagenesis and biochemical analyses that explain the absence of substrate specificity toward NADPH and suggest the mechanism behind constitutive activity. Our study presents the structural basis underlying SpNOX enzymatic activity and sheds light on its potential in vivo function.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141737018","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","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.
{"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":"https://doi.org/10.1038/s41594-024-01356-w","url":null,"abstract":"<p>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.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141584474","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","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.
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":"https://doi.org/10.1038/s41594-024-01355-x","url":null,"abstract":"<p>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 <i>Escherichia coli</i> 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.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141577168","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","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.
{"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":"https://doi.org/10.1038/s41594-024-01354-y","url":null,"abstract":"<p>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.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141556737","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","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.
{"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":"https://doi.org/10.1038/s41594-024-01340-4","url":null,"abstract":"<p>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 <i>ElaC</i> 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.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"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":0,"RegionCategory":"","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.
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":"https://doi.org/10.1038/s41594-024-01344-0","url":null,"abstract":"<p>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.</p>","PeriodicalId":18822,"journal":{"name":"Nature structural & molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"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":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}