Pub Date : 2024-04-29DOI: 10.1038/s41594-024-01301-x
Ivan B. Lomakin, Minh Ho, Christopher G. Bunick
The human cytoskeleton consists of three major classes of filaments: microfilaments, microtubules and intermediate filaments. Here, we summarize recent progress in deciphering the structure and function of intermediate filaments and their implications for human disease.
{"title":"Unveiling the secrets of vimentin filament architecture relevant to human disease","authors":"Ivan B. Lomakin, Minh Ho, Christopher G. Bunick","doi":"10.1038/s41594-024-01301-x","DOIUrl":"10.1038/s41594-024-01301-x","url":null,"abstract":"The human cytoskeleton consists of three major classes of filaments: microfilaments, microtubules and intermediate filaments. Here, we summarize recent progress in deciphering the structure and function of intermediate filaments and their implications for human disease.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 6","pages":"849-851"},"PeriodicalIF":16.8,"publicationDate":"2024-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140808424","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 : 2024-04-29DOI: 10.1038/s41594-024-01284-9
Zhang Feng, Omar E. Alvarenga, Alessio Accardi
Activation of Ca2+-dependent TMEM16 scramblases induces phosphatidylserine externalization, a key step in multiple signaling processes. Current models suggest that the TMEM16s scramble lipids by deforming the membrane near a hydrophilic groove and that Ca2+ dependence arises from the different association of lipids with an open or closed groove. However, the molecular rearrangements underlying groove opening and how lipids reorganize outside the closed groove remain unknown. Here we directly visualize how lipids associate at the closed groove of Ca2+-bound fungal nhTMEM16 in nanodiscs using cryo-EM. Functional experiments pinpoint lipid–protein interaction sites critical for closed groove scrambling. Structural and functional analyses suggest groove opening entails the sequential appearance of two π-helical turns in the groove-lining TM6 helix and identify critical rearrangements. Finally, we show that the choice of scaffold protein and lipids affects the conformations of nhTMEM16 and their distribution, highlighting a key role of these factors in cryo-EM structure determination. The authors used cryo-EM to visualize the arrangement of lipids at the closed groove of a TMEM16 scramblase and to reveal that both the structures and distributions of the protein’s conformations depend on the lipid composition and nanodisc scaffold.
{"title":"Structural basis of closed groove scrambling by a TMEM16 protein","authors":"Zhang Feng, Omar E. Alvarenga, Alessio Accardi","doi":"10.1038/s41594-024-01284-9","DOIUrl":"10.1038/s41594-024-01284-9","url":null,"abstract":"Activation of Ca2+-dependent TMEM16 scramblases induces phosphatidylserine externalization, a key step in multiple signaling processes. Current models suggest that the TMEM16s scramble lipids by deforming the membrane near a hydrophilic groove and that Ca2+ dependence arises from the different association of lipids with an open or closed groove. However, the molecular rearrangements underlying groove opening and how lipids reorganize outside the closed groove remain unknown. Here we directly visualize how lipids associate at the closed groove of Ca2+-bound fungal nhTMEM16 in nanodiscs using cryo-EM. Functional experiments pinpoint lipid–protein interaction sites critical for closed groove scrambling. Structural and functional analyses suggest groove opening entails the sequential appearance of two π-helical turns in the groove-lining TM6 helix and identify critical rearrangements. Finally, we show that the choice of scaffold protein and lipids affects the conformations of nhTMEM16 and their distribution, highlighting a key role of these factors in cryo-EM structure determination. The authors used cryo-EM to visualize the arrangement of lipids at the closed groove of a TMEM16 scramblase and to reveal that both the structures and distributions of the protein’s conformations depend on the lipid composition and nanodisc scaffold.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 10","pages":"1468-1481"},"PeriodicalIF":12.5,"publicationDate":"2024-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140808343","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 : 2024-04-26DOI: 10.1038/s41594-024-01291-w
Alex Chialastri, Saumya Sarkar, Elizabeth E. Schauer, Shyl Lamba, Siddharth S. Dey
Inheritance of 5-methylcytosine from one cell generation to the next by DNA methyltransferase 1 (DNMT1) plays a key role in regulating cellular identity. While recent work has shown that the activity of DNMT1 is imprecise, it remains unclear how the fidelity of DNMT1 is tuned in different genomic and cell state contexts. Here we describe Dyad-seq, a method to quantify the genome-wide methylation status of cytosines at the resolution of individual CpG dinucleotides to find that the fidelity of DNMT1-mediated maintenance methylation is related to the local density of DNA methylation and the landscape of histone modifications. To gain deeper insights into methylation/demethylation turnover dynamics, we first extended Dyad-seq to quantify all combinations of 5-methylcytosine and 5-hydroxymethylcytosine at individual CpG dyads. Next, to understand how cell state transitions impact maintenance methylation, we scaled the method down to jointly profile genome-wide methylation levels, maintenance methylation fidelity and the transcriptome from single cells (scDyad&T-seq). Using scDyad&T-seq, we demonstrate that, while distinct cell states can substantially impact the activity of the maintenance methylation machinery, locally there exists an intrinsic relationship between DNA methylation density, histone modifications and DNMT1-mediated maintenance methylation fidelity that is independent of cell state. Here the authors develop a method to quantify all combinations of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine at individual CpG dyads, including in single cells, to identify the relationship between the local 5mC density, histone marks and maintenance methylation fidelity.
DNA 甲基转移酶 1（DNMT1）将 5-甲基胞嘧啶从一代细胞遗传到下一代细胞，在调节细胞特性方面起着关键作用。虽然最近的研究表明 DNMT1 的活性并不精确，但仍不清楚 DNMT1 的保真度在不同的基因组和细胞状态下是如何调整的。在这里，我们描述了 Dyad-seq，这是一种以单个 CpG 二核苷酸为分辨率量化全基因组胞嘧啶甲基化状态的方法，发现 DNMT1 介导的维持甲基化的保真度与 DNA 甲基化的局部密度和组蛋白修饰景观有关。为了更深入地了解甲基化/去甲基化的周转动态，我们首先扩展了 Dyad-seq 来量化单个 CpG 二聚体上 5-甲基胞嘧啶和 5-羟甲基胞嘧啶的所有组合。接下来，为了了解细胞状态转变如何影响甲基化的维持，我们缩小了该方法的规模，以联合剖析全基因组甲基化水平、甲基化维持保真度和来自单细胞的转录组（scDyad&T-seq）。利用 scDyad&T-seq，我们证明，虽然不同的细胞状态会对甲基化维持机制的活性产生重大影响，但在 DNA 甲基化密度、组蛋白修饰和 DNMT1 介导的甲基化维持保真度之间存在着与细胞状态无关的内在联系。
{"title":"Combinatorial quantification of 5mC and 5hmC at individual CpG dyads and the transcriptome in single cells reveals modulators of DNA methylation maintenance fidelity","authors":"Alex Chialastri, Saumya Sarkar, Elizabeth E. Schauer, Shyl Lamba, Siddharth S. Dey","doi":"10.1038/s41594-024-01291-w","DOIUrl":"10.1038/s41594-024-01291-w","url":null,"abstract":"Inheritance of 5-methylcytosine from one cell generation to the next by DNA methyltransferase 1 (DNMT1) plays a key role in regulating cellular identity. While recent work has shown that the activity of DNMT1 is imprecise, it remains unclear how the fidelity of DNMT1 is tuned in different genomic and cell state contexts. Here we describe Dyad-seq, a method to quantify the genome-wide methylation status of cytosines at the resolution of individual CpG dinucleotides to find that the fidelity of DNMT1-mediated maintenance methylation is related to the local density of DNA methylation and the landscape of histone modifications. To gain deeper insights into methylation/demethylation turnover dynamics, we first extended Dyad-seq to quantify all combinations of 5-methylcytosine and 5-hydroxymethylcytosine at individual CpG dyads. Next, to understand how cell state transitions impact maintenance methylation, we scaled the method down to jointly profile genome-wide methylation levels, maintenance methylation fidelity and the transcriptome from single cells (scDyad&T-seq). Using scDyad&T-seq, we demonstrate that, while distinct cell states can substantially impact the activity of the maintenance methylation machinery, locally there exists an intrinsic relationship between DNA methylation density, histone modifications and DNMT1-mediated maintenance methylation fidelity that is independent of cell state. Here the authors develop a method to quantify all combinations of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine at individual CpG dyads, including in single cells, to identify the relationship between the local 5mC density, histone marks and maintenance methylation fidelity.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 8","pages":"1296-1308"},"PeriodicalIF":12.5,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140648652","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 : 2024-04-26DOI: 10.1038/s41594-024-01295-6
Shashank Khare, Miryam I. Villalba, Juan C. Canul-Tec, Arantza Balsebre Cajiao, Anand Kumar, Marija Backovic, Felix A. Rey, Els Pardon, Jan Steyaert, Camilo Perez, Nicolas Reyes
Human syncytin-1 and suppressyn are cellular proteins of retroviral origin involved in cell–cell fusion events to establish the maternal–fetal interface in the placenta. In cell culture, they restrict infections from members of the largest interference group of vertebrate retroviruses, and are regarded as host immunity factors expressed during development. At the core of the syncytin-1 and suppressyn functions are poorly understood mechanisms to recognize a common cellular receptor, the membrane transporter ASCT2. Here, we present cryo-electron microscopy structures of human ASCT2 in complexes with the receptor-binding domains of syncytin-1 and suppressyn. Despite their evolutionary divergence, the two placental proteins occupy similar positions in ASCT2, and are stabilized by the formation of a hybrid β-sheet or ‘clamp’ with the receptor. Structural predictions of the receptor-binding domains of extant retroviruses indicate overlapping binding interfaces and clamping sites with ASCT2, revealing a competition mechanism between the placental proteins and the retroviruses. Our work uncovers a common ASCT2 recognition mechanism by a large group of endogenous and disease-causing retroviruses, and provides high-resolution views on how placental human proteins exert morphological and immunological functions. The structures of the retrovirus-derived human syncytin-1 and suppressyn in complexes with their shared receptor reveal an ancient cellular recognition mechanism that underlies key morphological and immunological functions in placenta.
{"title":"Receptor-recognition and antiviral mechanisms of retrovirus-derived human proteins","authors":"Shashank Khare, Miryam I. Villalba, Juan C. Canul-Tec, Arantza Balsebre Cajiao, Anand Kumar, Marija Backovic, Felix A. Rey, Els Pardon, Jan Steyaert, Camilo Perez, Nicolas Reyes","doi":"10.1038/s41594-024-01295-6","DOIUrl":"10.1038/s41594-024-01295-6","url":null,"abstract":"Human syncytin-1 and suppressyn are cellular proteins of retroviral origin involved in cell–cell fusion events to establish the maternal–fetal interface in the placenta. In cell culture, they restrict infections from members of the largest interference group of vertebrate retroviruses, and are regarded as host immunity factors expressed during development. At the core of the syncytin-1 and suppressyn functions are poorly understood mechanisms to recognize a common cellular receptor, the membrane transporter ASCT2. Here, we present cryo-electron microscopy structures of human ASCT2 in complexes with the receptor-binding domains of syncytin-1 and suppressyn. Despite their evolutionary divergence, the two placental proteins occupy similar positions in ASCT2, and are stabilized by the formation of a hybrid β-sheet or ‘clamp’ with the receptor. Structural predictions of the receptor-binding domains of extant retroviruses indicate overlapping binding interfaces and clamping sites with ASCT2, revealing a competition mechanism between the placental proteins and the retroviruses. Our work uncovers a common ASCT2 recognition mechanism by a large group of endogenous and disease-causing retroviruses, and provides high-resolution views on how placental human proteins exert morphological and immunological functions. The structures of the retrovirus-derived human syncytin-1 and suppressyn in complexes with their shared receptor reveal an ancient cellular recognition mechanism that underlies key morphological and immunological functions in placenta.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 9","pages":"1368-1376"},"PeriodicalIF":12.5,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140648820","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 : 2024-04-25DOI: 10.1038/s41594-024-01276-9
Jiangtao Zhang, Chunhua Zhan, Junping Fan, Dian Wu, Ruixue Zhang, Di Wu, Xinyao Chen, Ying Lu, Ming Li, Min Lin, Jianke Gong, Daohua Jiang
RNA uptake by cells is critical for RNA-mediated gene interference (RNAi) and RNA-based therapeutics. In Caenorhabditis elegans, RNAi is systemic as a result of SID-1-mediated double-stranded RNA (dsRNA) across cells. Despite the functional importance, the underlying mechanisms of dsRNA internalization by SID-1 remain elusive. Here we describe cryogenic electron microscopy structures of SID-1, SID-1–dsRNA complex and human SID-1 homologs SIDT1 and SIDT2, elucidating the structural basis of dsRNA recognition and import by SID-1. The homodimeric SID-1 homologs share conserved architecture, but only SID-1 possesses the molecular determinants within its extracellular domains for distinguishing dsRNA from single-stranded RNA and DNA. We show that the removal of the long intracellular loop between transmembrane helix 1 and 2 attenuates dsRNA uptake and systemic RNAi in vivo, suggesting a possible endocytic mechanism of SID-1-mediated dsRNA internalization. Our study provides mechanistic insights into dsRNA internalization by SID-1, which may facilitate the development of dsRNA applications based on SID-1. In C. elegans, systemic RNAi is initiated by SID-1-mediated dsRNA internalization. Here the authors present cryo-EM structures of SID-1 homologs and the SID-1–dsRNA complex, elucidating the structural basis for dsRNA recognition and uptake by SID-1.
{"title":"Structural insights into double-stranded RNA recognition and transport by SID-1","authors":"Jiangtao Zhang, Chunhua Zhan, Junping Fan, Dian Wu, Ruixue Zhang, Di Wu, Xinyao Chen, Ying Lu, Ming Li, Min Lin, Jianke Gong, Daohua Jiang","doi":"10.1038/s41594-024-01276-9","DOIUrl":"10.1038/s41594-024-01276-9","url":null,"abstract":"RNA uptake by cells is critical for RNA-mediated gene interference (RNAi) and RNA-based therapeutics. In Caenorhabditis elegans, RNAi is systemic as a result of SID-1-mediated double-stranded RNA (dsRNA) across cells. Despite the functional importance, the underlying mechanisms of dsRNA internalization by SID-1 remain elusive. Here we describe cryogenic electron microscopy structures of SID-1, SID-1–dsRNA complex and human SID-1 homologs SIDT1 and SIDT2, elucidating the structural basis of dsRNA recognition and import by SID-1. The homodimeric SID-1 homologs share conserved architecture, but only SID-1 possesses the molecular determinants within its extracellular domains for distinguishing dsRNA from single-stranded RNA and DNA. We show that the removal of the long intracellular loop between transmembrane helix 1 and 2 attenuates dsRNA uptake and systemic RNAi in vivo, suggesting a possible endocytic mechanism of SID-1-mediated dsRNA internalization. Our study provides mechanistic insights into dsRNA internalization by SID-1, which may facilitate the development of dsRNA applications based on SID-1. In C. elegans, systemic RNAi is initiated by SID-1-mediated dsRNA internalization. Here the authors present cryo-EM structures of SID-1 homologs and the SID-1–dsRNA complex, elucidating the structural basis for dsRNA recognition and uptake by SID-1.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 7","pages":"1095-1104"},"PeriodicalIF":12.5,"publicationDate":"2024-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140642165","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 : 2024-04-25DOI: 10.1038/s41594-024-01290-x
Petra Vizjak, Dieter Kamp, Nicola Hepp, Alessandro Scacchetti, Mariano Gonzalez Pisfil, Joseph Bartho, Mario Halic, Peter B. Becker, Michaela Smolle, Johannes Stigler, Felix Mueller-Planitz
How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. Here we investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI’s mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI’s diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a ‘monkey-bar’ model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner, qualitatively agree with our data. We speculate that monkey-bar mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies. Chromatin condensation does not impede nucleosome sliding by ISWI remodelers. Notably, ATP energy is used not only for remodeling but also for enzyme mobility and to prevent solidification of chromatin. A ‘monkey-bar’ model rationalizes the findings.
染色质酶如何在凝聚的染色质中工作，以及它们如何保持染色质内部的扩散流动性，这些问题仍有待探索。在这里，我们利用果蝇的 ISWI 重塑 ATP 酶研究了这些难题，该酶可使核小体沿 DNA 滑动。染色质纤维的折叠并不影响体外滑动。染色质凝聚体内外的催化率也相当。除了 ATP 水解可能发生的情况外，ISWI 会交联，从而使凝集物变硬。ISWI 在凝聚体中的流动性也需要活性水解。因此，ATP水解产生的能量可促进ISWI在染色质中的扩散，并阻止ISWI交联染色质。在 "猴条 "模型的分子动力学模拟中，ISWI先抓住相邻的核小体，然后以依赖于ATP水解的方式从一个核小体中撤出，再重新与另一个核小体结合。我们推测，"猴条 "机制可能与其他染色质因子共享，重塑因子突变导致的染色质动力学变化可能导致病变。
{"title":"ISWI catalyzes nucleosome sliding in condensed nucleosome arrays","authors":"Petra Vizjak, Dieter Kamp, Nicola Hepp, Alessandro Scacchetti, Mariano Gonzalez Pisfil, Joseph Bartho, Mario Halic, Peter B. Becker, Michaela Smolle, Johannes Stigler, Felix Mueller-Planitz","doi":"10.1038/s41594-024-01290-x","DOIUrl":"10.1038/s41594-024-01290-x","url":null,"abstract":"How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. Here we investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI’s mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI’s diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a ‘monkey-bar’ model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner, qualitatively agree with our data. We speculate that monkey-bar mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies. Chromatin condensation does not impede nucleosome sliding by ISWI remodelers. Notably, ATP energy is used not only for remodeling but also for enzyme mobility and to prevent solidification of chromatin. A ‘monkey-bar’ model rationalizes the findings.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 9","pages":"1331-1340"},"PeriodicalIF":12.5,"publicationDate":"2024-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140642453","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 : 2024-04-25DOI: 10.1038/s41594-024-01293-8
Suyang Zhang
Spliceosome biogenesis and recycling remains a largely unexplored area. Two papers now reveal how protein chaperones remodel the 20S U5 snRNP, leading to formation of the U4/U6.U5 tri-snRNP.
{"title":"Illuminating the role of chaperones in spliceosome biogenesis and recycling","authors":"Suyang Zhang","doi":"10.1038/s41594-024-01293-8","DOIUrl":"10.1038/s41594-024-01293-8","url":null,"abstract":"Spliceosome biogenesis and recycling remains a largely unexplored area. Two papers now reveal how protein chaperones remodel the 20S U5 snRNP, leading to formation of the U4/U6.U5 tri-snRNP.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 5","pages":"735-736"},"PeriodicalIF":16.8,"publicationDate":"2024-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140642273","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}
The PIWI-interacting RNA (piRNA) pathway is an adaptive defense system wherein piRNAs guide PIWI family Argonaute proteins to recognize and silence ever-evolving selfish genetic elements and ensure genome integrity. Driven by this intensive host–pathogen arms race, the piRNA pathway and its targeted transposons have coevolved rapidly in a species-specific manner, but how the piRNA pathway adapts specifically to target silencing in mammals remains elusive. Here, we show that mouse MILI and human HILI piRNA-induced silencing complexes (piRISCs) bind and cleave targets more efficiently than their invertebrate counterparts from the sponge Ephydatia fluviatilis. The inherent functional differences comport with structural features identified by cryo-EM studies of piRISCs. In the absence of target, MILI and HILI piRISCs adopt a wider nucleic-acid-binding channel and display an extended prearranged piRNA seed as compared with EfPiwi piRISC, consistent with their ability to capture targets more efficiently than EfPiwi piRISC. In the presence of target, the seed gate—which enforces seed–target fidelity in microRNA RISC—adopts a relaxed state in mammalian piRISC, revealing how MILI and HILI tolerate seed–target mismatches to broaden the target spectrum. A vertebrate-specific lysine distorts the piRNA seed, shifting the trajectory of the piRNA–target duplex out of the central cleft and toward the PAZ lobe. Functional analyses reveal that this lysine promotes target binding and cleavage. Our study therefore provides a molecular basis for the piRNA targeting mechanism in mice and humans, and suggests that mammalian piRNA machinery can achieve broad target silencing using a limited supply of piRNA species. This study provides structural and biochemical insight into how mammalian PIWI proteins use a limited supply of piRNAs to silence a vast array of ever-evolving transposons in the germline.
{"title":"Mammalian PIWI–piRNA–target complexes reveal features for broad and efficient target silencing","authors":"Zhiqing Li, Zhenzhen Li, Yuqi Zhang, Lunni Zhou, Qikui Xu, Lili Li, Lin Zeng, Junchao Xue, Huilin Niu, Jing Zhong, Qilu Yu, Dengfeng Li, Miao Gui, Yongping Huang, Shikui Tu, Zhao Zhang, Chun-Qing Song, Jianping Wu, En-Zhi Shen","doi":"10.1038/s41594-024-01287-6","DOIUrl":"10.1038/s41594-024-01287-6","url":null,"abstract":"The PIWI-interacting RNA (piRNA) pathway is an adaptive defense system wherein piRNAs guide PIWI family Argonaute proteins to recognize and silence ever-evolving selfish genetic elements and ensure genome integrity. Driven by this intensive host–pathogen arms race, the piRNA pathway and its targeted transposons have coevolved rapidly in a species-specific manner, but how the piRNA pathway adapts specifically to target silencing in mammals remains elusive. Here, we show that mouse MILI and human HILI piRNA-induced silencing complexes (piRISCs) bind and cleave targets more efficiently than their invertebrate counterparts from the sponge Ephydatia fluviatilis. The inherent functional differences comport with structural features identified by cryo-EM studies of piRISCs. In the absence of target, MILI and HILI piRISCs adopt a wider nucleic-acid-binding channel and display an extended prearranged piRNA seed as compared with EfPiwi piRISC, consistent with their ability to capture targets more efficiently than EfPiwi piRISC. In the presence of target, the seed gate—which enforces seed–target fidelity in microRNA RISC—adopts a relaxed state in mammalian piRISC, revealing how MILI and HILI tolerate seed–target mismatches to broaden the target spectrum. A vertebrate-specific lysine distorts the piRNA seed, shifting the trajectory of the piRNA–target duplex out of the central cleft and toward the PAZ lobe. Functional analyses reveal that this lysine promotes target binding and cleavage. Our study therefore provides a molecular basis for the piRNA targeting mechanism in mice and humans, and suggests that mammalian piRNA machinery can achieve broad target silencing using a limited supply of piRNA species. This study provides structural and biochemical insight into how mammalian PIWI proteins use a limited supply of piRNAs to silence a vast array of ever-evolving transposons in the germline.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 8","pages":"1222-1231"},"PeriodicalIF":12.5,"publicationDate":"2024-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140639963","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}
The heterogeneity of CARM1 controls first cell fate bias during early mouse development. However, how this heterogeneity is established is unknown. Here, we show that Carm1 mRNA is of a variety of specific exon-skipping splicing (ESS) isoforms in mouse two-cell to four-cell embryos that contribute to CARM1 heterogeneity. Disruption of paraspeckles promotes the ESS of Carm1 precursor mRNAs (pre-mRNAs). LincGET, but not Neat1, is required for paraspeckle assembly and inhibits the ESS of Carm1 pre-mRNAs in mouse two-cell to four-cell embryos. We further find that LincGET recruits paraspeckles to the Carm1 gene locus through HNRNPU. Interestingly, PCBP1 binds the Carm1 pre-mRNAs and promotes its ESS in the absence of LincGET. Finally, we find that the ESS seen in mouse two-cell to four-cell embryos decreases CARM1 protein levels and leads to trophectoderm fate bias. Our findings demonstrate that alternative splicing of CARM1 has an important role in first cell fate determination. The heterogeneity of CARM1 for the first cell fate bias in mice arises from exon-skipping splicing of Carm1 pre-mRNAs, which is regulated by the competition between LincGET-paraspeckles and splicing speckles for binding to the Carm1 locus.
{"title":"Alternative splicing of CARM1 regulated by LincGET-guided paraspeckles biases the first cell fate in mammalian early embryos","authors":"Jiaqiang Wang, Yiwei Zhang, Jiaze Gao, Guihai Feng, Chao Liu, Xueke Li, Pengcheng Li, Zhonghua Liu, Falong Lu, Leyun Wang, Wei Li, Qi Zhou, Yusheng Liu","doi":"10.1038/s41594-024-01292-9","DOIUrl":"10.1038/s41594-024-01292-9","url":null,"abstract":"The heterogeneity of CARM1 controls first cell fate bias during early mouse development. However, how this heterogeneity is established is unknown. Here, we show that Carm1 mRNA is of a variety of specific exon-skipping splicing (ESS) isoforms in mouse two-cell to four-cell embryos that contribute to CARM1 heterogeneity. Disruption of paraspeckles promotes the ESS of Carm1 precursor mRNAs (pre-mRNAs). LincGET, but not Neat1, is required for paraspeckle assembly and inhibits the ESS of Carm1 pre-mRNAs in mouse two-cell to four-cell embryos. We further find that LincGET recruits paraspeckles to the Carm1 gene locus through HNRNPU. Interestingly, PCBP1 binds the Carm1 pre-mRNAs and promotes its ESS in the absence of LincGET. Finally, we find that the ESS seen in mouse two-cell to four-cell embryos decreases CARM1 protein levels and leads to trophectoderm fate bias. Our findings demonstrate that alternative splicing of CARM1 has an important role in first cell fate determination. The heterogeneity of CARM1 for the first cell fate bias in mice arises from exon-skipping splicing of Carm1 pre-mRNAs, which is regulated by the competition between LincGET-paraspeckles and splicing speckles for binding to the Carm1 locus.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 9","pages":"1341-1354"},"PeriodicalIF":12.5,"publicationDate":"2024-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41594-024-01292-9.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140639898","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 : 2024-04-22DOI: 10.1038/s41594-024-01272-z
Jiao Yang, Jingyang Li, Langxi Miao, Xu Gao, Wenhao Sun, Shuo Linghu, Guiping Ren, Bangya Peng, Shunkai Chen, Zhongqi Liu, Bo Wang, Ao Dong, Duo Huang, Jinrong Yuan, Yunkun Dang, Fan Lai
A universal characteristic of eukaryotic transcription is that the promoter recruits RNA polymerase II (RNAPII) to produce both precursor mRNAs (pre-mRNAs) and short unstable promoter upstream transcripts (PROMPTs) toward the opposite direction. However, how the transcription machinery selects the correct direction to produce pre-mRNAs is largely unknown. Here, through multiple acute auxin-inducible degradation systems, we show that rapid depletion of an RNAPII-binding protein complex, Integrator, results in robust PROMPT accumulation throughout the genome. Interestingly, the accumulation of PROMPTs is compensated by the reduction of pre-mRNA transcripts in actively transcribed genes. Consistently, Integrator depletion alters the distribution of polymerase between the sense and antisense directions, which is marked by increased RNAPII-carboxy-terminal domain Tyr1 phosphorylation at PROMPT regions and a reduced Ser2 phosphorylation level at transcription start sites. Mechanistically, the endonuclease activity of Integrator is critical to suppress PROMPT production. Furthermore, our data indicate that the presence of U1 binding sites on nascent transcripts could counteract the cleavage activity of Integrator. In this process, the absence of robust U1 signal at most PROMPTs allows Integrator to suppress the antisense transcription and shift the transcriptional balance in favor of the sense direction. The authors investigate how the transcription machinery selects the correct direction to produce coding transcripts. Their results propose a universal mechanism by which Integrator licenses bidirectional transcription to determine the direction of eukaryotic pre-mRNA transcription.
{"title":"Transcription directionality is licensed by Integrator at active human promoters","authors":"Jiao Yang, Jingyang Li, Langxi Miao, Xu Gao, Wenhao Sun, Shuo Linghu, Guiping Ren, Bangya Peng, Shunkai Chen, Zhongqi Liu, Bo Wang, Ao Dong, Duo Huang, Jinrong Yuan, Yunkun Dang, Fan Lai","doi":"10.1038/s41594-024-01272-z","DOIUrl":"10.1038/s41594-024-01272-z","url":null,"abstract":"A universal characteristic of eukaryotic transcription is that the promoter recruits RNA polymerase II (RNAPII) to produce both precursor mRNAs (pre-mRNAs) and short unstable promoter upstream transcripts (PROMPTs) toward the opposite direction. However, how the transcription machinery selects the correct direction to produce pre-mRNAs is largely unknown. Here, through multiple acute auxin-inducible degradation systems, we show that rapid depletion of an RNAPII-binding protein complex, Integrator, results in robust PROMPT accumulation throughout the genome. Interestingly, the accumulation of PROMPTs is compensated by the reduction of pre-mRNA transcripts in actively transcribed genes. Consistently, Integrator depletion alters the distribution of polymerase between the sense and antisense directions, which is marked by increased RNAPII-carboxy-terminal domain Tyr1 phosphorylation at PROMPT regions and a reduced Ser2 phosphorylation level at transcription start sites. Mechanistically, the endonuclease activity of Integrator is critical to suppress PROMPT production. Furthermore, our data indicate that the presence of U1 binding sites on nascent transcripts could counteract the cleavage activity of Integrator. In this process, the absence of robust U1 signal at most PROMPTs allows Integrator to suppress the antisense transcription and shift the transcriptional balance in favor of the sense direction. The authors investigate how the transcription machinery selects the correct direction to produce coding transcripts. Their results propose a universal mechanism by which Integrator licenses bidirectional transcription to determine the direction of eukaryotic pre-mRNA transcription.","PeriodicalId":49141,"journal":{"name":"Nature Structural & Molecular Biology","volume":"31 8","pages":"1208-1221"},"PeriodicalIF":12.5,"publicationDate":"2024-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140632294","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}
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