Our conception of gene regulation specificity has undergone profound changes over the last 20 years. Previously, regulators were considered to control few genes, recognized with exquisite specificity by a 'lock and key' mechanism. However, recently genome-wide exploration of regulator binding site occupancy (whether on DNA or RNA targets) revealed extensive lists of molecular targets for every studied regulator. Such poor biochemical specificity suggested that each regulator controls many genes, collectively contributing to biological phenotypes. Here, I propose a third model, whereby regulators' biological specificity is only partially due to 'lock and key' biochemistry. Rather, regulators affect many genes at the microscopic scale, but biological consequences for most interactions are attenuated at the mesoscopic scale: only a few regulatory events propagate from microscopic to macroscopic scale; others are made inconsequential by homeostatic mechanisms. This model is well supported by the microRNA literature, and data suggest that it extends to other regulators. It reconciles contradicting observations from biochemistry and comparative genomics on one hand and in vivo genetics on the other hand, but this conceptual unification is obscured by common misconceptions and counter-intuitive modes of graphical display. Profound understanding of gene regulation requires conceptual clarification, and better suited statistical analyses and graphical representation.
{"title":"A new perspective on microRNA-guided gene regulation specificity, and its potential generalization to transcription factors and RNA-binding proteins.","authors":"Hervé Seitz","doi":"10.1093/nar/gkae694","DOIUrl":"10.1093/nar/gkae694","url":null,"abstract":"<p><p>Our conception of gene regulation specificity has undergone profound changes over the last 20 years. Previously, regulators were considered to control few genes, recognized with exquisite specificity by a 'lock and key' mechanism. However, recently genome-wide exploration of regulator binding site occupancy (whether on DNA or RNA targets) revealed extensive lists of molecular targets for every studied regulator. Such poor biochemical specificity suggested that each regulator controls many genes, collectively contributing to biological phenotypes. Here, I propose a third model, whereby regulators' biological specificity is only partially due to 'lock and key' biochemistry. Rather, regulators affect many genes at the microscopic scale, but biological consequences for most interactions are attenuated at the mesoscopic scale: only a few regulatory events propagate from microscopic to macroscopic scale; others are made inconsequential by homeostatic mechanisms. This model is well supported by the microRNA literature, and data suggest that it extends to other regulators. It reconciles contradicting observations from biochemistry and comparative genomics on one hand and in vivo genetics on the other hand, but this conceptual unification is obscured by common misconceptions and counter-intuitive modes of graphical display. Profound understanding of gene regulation requires conceptual clarification, and better suited statistical analyses and graphical representation.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141988482","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Daai Zhang, Dengwei Jue, Neil Smith, Chengcheng Zhong, E Jean Finnegan, Robert de Feyter, Ming-Bo Wang, Ian Greaves
Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides. The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript. 22nt siRNAs are under-represented due to competition between DCL2 and DCL4, while only a small number of 22nt miRNAs exist. Here we produce abundant 22nt siRNAs and other siRNA size classes using long hairpin RNA (hpRNA) transgenes. By introducing asymmetric bulges into the antisense strand of hpRNA, we shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs. The asymmetric hpRNAs effectively silenced a β-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes showed increased amounts of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes. This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. The 22nt asymmetric hpRNA constructs enhanced virus resistance in plants compared to the traditional hpRNA constructs.
{"title":"Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence.","authors":"Daai Zhang, Dengwei Jue, Neil Smith, Chengcheng Zhong, E Jean Finnegan, Robert de Feyter, Ming-Bo Wang, Ian Greaves","doi":"10.1093/nar/gkae573","DOIUrl":"10.1093/nar/gkae573","url":null,"abstract":"<p><p>Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides. The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript. 22nt siRNAs are under-represented due to competition between DCL2 and DCL4, while only a small number of 22nt miRNAs exist. Here we produce abundant 22nt siRNAs and other siRNA size classes using long hairpin RNA (hpRNA) transgenes. By introducing asymmetric bulges into the antisense strand of hpRNA, we shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs. The asymmetric hpRNAs effectively silenced a β-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes showed increased amounts of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes. This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. The 22nt asymmetric hpRNA constructs enhanced virus resistance in plants compared to the traditional hpRNA constructs.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141534906","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Camila Gonzalo-Hansen, Barbara Steurer, Roel C Janssens, Di Zhou, Marjolein van Sluis, Hannes Lans, Jurgen A Marteijn
DNA damage severely impedes gene transcription by RNA polymerase II (Pol II), causing cellular dysfunction. Transcription-Coupled Nucleotide Excision Repair (TC-NER) specifically removes such transcription-blocking damage. TC-NER initiation relies on the CSB, CSA and UVSSA proteins; loss of any results in complete TC-NER deficiency. Strikingly, UVSSA deficiency results in UV-Sensitive Syndrome (UVSS), with mild cutaneous symptoms, while loss of CSA or CSB activity results in the severe Cockayne Syndrome (CS), characterized by neurodegeneration and premature aging. Thus far the underlying mechanism for these contrasting phenotypes remains unclear. Live-cell imaging approaches reveal that in TC-NER proficient cells, lesion-stalled Pol II is swiftly resolved, while in CSA and CSB knockout (KO) cells, elongating Pol II remains damage-bound, likely obstructing other DNA transacting processes and shielding the damage from alternative repair pathways. In contrast, in UVSSA KO cells, Pol II is cleared from the damage via VCP-mediated proteasomal degradation which is fully dependent on the CRL4CSA ubiquitin ligase activity. This Pol II degradation might provide access for alternative repair mechanisms, such as GG-NER, to remove the damage. Collectively, our data indicate that the inability to clear lesion-stalled Pol II from the chromatin, rather than TC-NER deficiency, causes the severe phenotypes observed in CS.
DNA 损伤会严重阻碍 RNA 聚合酶 II(Pol II)的基因转录,造成细胞功能障碍。转录偶联核苷酸切除修复(TC-NER)可专门清除此类转录阻碍损伤。TC-NER 的启动依赖于 CSB、CSA 和 UVSSA 蛋白;任何一种蛋白的缺失都会导致 TC-NER 的完全缺失。令人震惊的是,UVSSA 缺乏会导致轻微皮肤症状的紫外线敏感综合征(UVSS),而 CSA 或 CSB 活性丧失则会导致严重的科克恩综合征(CS),其特征是神经变性和过早衰老。迄今为止,这些截然不同的表型的内在机制仍不清楚。活细胞成像方法显示,在 TC-NER 熟练掌握的细胞中,病变停滞的 Pol II 会被迅速清除,而在 CSA 和 CSB 基因敲除(KO)细胞中,伸长的 Pol II 仍与损伤绑定,很可能会阻碍其他 DNA 转录过程,并使损伤免受替代修复途径的影响。相反,在 UVSSA KO 细胞中,Pol II 通过 VCP 介导的蛋白酶体降解从损伤中清除,而这种降解完全依赖于 CRL4CSA 泛素连接酶的活性。Pol II 的降解可能为 GG-NER 等替代修复机制提供了清除损伤的途径。总之,我们的数据表明,不能从染色质中清除病变停滞的 Pol II,而不是 TC-NER 缺乏,是 CS 中观察到的严重表型的原因。
{"title":"Differential processing of RNA polymerase II at DNA damage correlates with transcription-coupled repair syndrome severity.","authors":"Camila Gonzalo-Hansen, Barbara Steurer, Roel C Janssens, Di Zhou, Marjolein van Sluis, Hannes Lans, Jurgen A Marteijn","doi":"10.1093/nar/gkae618","DOIUrl":"10.1093/nar/gkae618","url":null,"abstract":"<p><p>DNA damage severely impedes gene transcription by RNA polymerase II (Pol II), causing cellular dysfunction. Transcription-Coupled Nucleotide Excision Repair (TC-NER) specifically removes such transcription-blocking damage. TC-NER initiation relies on the CSB, CSA and UVSSA proteins; loss of any results in complete TC-NER deficiency. Strikingly, UVSSA deficiency results in UV-Sensitive Syndrome (UVSS), with mild cutaneous symptoms, while loss of CSA or CSB activity results in the severe Cockayne Syndrome (CS), characterized by neurodegeneration and premature aging. Thus far the underlying mechanism for these contrasting phenotypes remains unclear. Live-cell imaging approaches reveal that in TC-NER proficient cells, lesion-stalled Pol II is swiftly resolved, while in CSA and CSB knockout (KO) cells, elongating Pol II remains damage-bound, likely obstructing other DNA transacting processes and shielding the damage from alternative repair pathways. In contrast, in UVSSA KO cells, Pol II is cleared from the damage via VCP-mediated proteasomal degradation which is fully dependent on the CRL4CSA ubiquitin ligase activity. This Pol II degradation might provide access for alternative repair mechanisms, such as GG-NER, to remove the damage. Collectively, our data indicate that the inability to clear lesion-stalled Pol II from the chromatin, rather than TC-NER deficiency, causes the severe phenotypes observed in CS.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141634140","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ashley L Cooney, Laura Marquez Loza, Kenan Najdawi, Christian M Brommel, Paul B McCray, Patrick L Sinn
A fundamental challenge for cystic fibrosis (CF) gene therapy is ensuring sufficient transduction of airway epithelia to achieve therapeutic correction. Hypertonic saline (HTS) is frequently administered to people with CF to enhance mucus clearance. HTS transiently disrupts epithelial cell tight junctions, but its ability to improve gene transfer has not been investigated. Here, we asked if increasing the concentration of NaCl enhances the transduction efficiency of three gene therapy vectors: adenovirus, AAV, and lentiviral vectors. Vectors formulated with 3-7% NaCl exhibited markedly increased transduction for all three platforms, leading to anion channel correction in primary cultures of human CF epithelial cells and enhanced gene transfer in mouse and pig airways in vivo. The mechanism of transduction enhancement involved tonicity but not osmolarity or pH. Formulating vectors with a high ionic strength solution is a simple strategy to greatly enhance efficacy and immediately improve preclinical or clinical applications.
{"title":"High ionic strength vector formulations enhance gene transfer to airway epithelia.","authors":"Ashley L Cooney, Laura Marquez Loza, Kenan Najdawi, Christian M Brommel, Paul B McCray, Patrick L Sinn","doi":"10.1093/nar/gkae640","DOIUrl":"10.1093/nar/gkae640","url":null,"abstract":"<p><p>A fundamental challenge for cystic fibrosis (CF) gene therapy is ensuring sufficient transduction of airway epithelia to achieve therapeutic correction. Hypertonic saline (HTS) is frequently administered to people with CF to enhance mucus clearance. HTS transiently disrupts epithelial cell tight junctions, but its ability to improve gene transfer has not been investigated. Here, we asked if increasing the concentration of NaCl enhances the transduction efficiency of three gene therapy vectors: adenovirus, AAV, and lentiviral vectors. Vectors formulated with 3-7% NaCl exhibited markedly increased transduction for all three platforms, leading to anion channel correction in primary cultures of human CF epithelial cells and enhanced gene transfer in mouse and pig airways in vivo. The mechanism of transduction enhancement involved tonicity but not osmolarity or pH. Formulating vectors with a high ionic strength solution is a simple strategy to greatly enhance efficacy and immediately improve preclinical or clinical applications.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141792988","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ian L Morgan, Shannon J McKie, Rachel Kim, Yeonee Seol, Jing Xu, Gabor M Harami, Anthony Maxwell, Keir C Neuman
Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability and a number of clinically important anticancer and antibacterial drugs, e.g. quinolones. We developed a simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type II topo cleavage complexes on DNA in vitro. Using SHAN-seq, we detected Escherichia coli gyrase and topoisomerase IV cleavage complexes at hundreds of sites on supercoiled pBR322 DNA, approximately one site every ten bp, with frequencies that varied by two-to-three orders of magnitude. These sites included previously identified sites and 20-50-fold more new sites. We show that the location and frequency of cleavage complexes at these sites are enzyme-specific and vary substantially in the presence of the quinolone, ciprofloxacin, but not with DNA supercoil chirality, i.e. negative versus positive supercoiling. SHAN-seq's exquisite sensitivity provides an unprecedented single-nucleotide resolution view of the distribution of gyrase and topoisomerase IV cleavage complexes on DNA. Moreover, the discovery that these enzymes can cleave DNA at orders of magnitude more sites than the relatively few previously known sites resolves the apparent paradox of how these enzymes resolve topological problems throughout the genome.
II 型拓扑异构酶(topos)是一类无处不在的重要酶,可在 DNA 上形成瞬时的酶结合双链断裂,称为裂解复合物。这些裂解复合物在 DNA 上的位置和频率对细胞功能、基因组稳定性和一些临床上重要的抗癌和抗菌药物(如喹诺酮类药物)非常重要。我们开发了一种简单的高精度末端测序(SHAN-seq)方法,可以灵敏地绘制体外 DNA 上的 II 型拓扑裂解复合物。利用 SHAN-seq,我们在超卷曲 pBR322 DNA 上的数百个位点检测到了大肠杆菌回旋酶和拓扑异构酶 IV 的裂解复合物,大约每十个 bp 就有一个位点,频率相差两到三个数量级。这些位点包括以前发现的位点和多出 20-50 倍的新位点。我们的研究表明,这些位点上的裂解复合物的位置和频率具有酶的特异性,在喹诺酮类药物环丙沙星存在的情况下变化很大,但与 DNA 的超螺旋手性(即负超螺旋与正超螺旋)无关。SHAN-seq的高灵敏度为回旋酶和拓扑异构酶IV裂解复合物在DNA上的分布提供了前所未有的单核苷酸分辨率视图。此外,发现这些酶能裂解 DNA 的位点比以前已知的相对较少的位点要多得多,这就解决了这些酶如何解决整个基因组拓扑问题的明显悖论。
{"title":"Highly sensitive mapping of in vitro type II topoisomerase DNA cleavage sites with SHAN-seq.","authors":"Ian L Morgan, Shannon J McKie, Rachel Kim, Yeonee Seol, Jing Xu, Gabor M Harami, Anthony Maxwell, Keir C Neuman","doi":"10.1093/nar/gkae638","DOIUrl":"10.1093/nar/gkae638","url":null,"abstract":"<p><p>Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stability and a number of clinically important anticancer and antibacterial drugs, e.g. quinolones. We developed a simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type II topo cleavage complexes on DNA in vitro. Using SHAN-seq, we detected Escherichia coli gyrase and topoisomerase IV cleavage complexes at hundreds of sites on supercoiled pBR322 DNA, approximately one site every ten bp, with frequencies that varied by two-to-three orders of magnitude. These sites included previously identified sites and 20-50-fold more new sites. We show that the location and frequency of cleavage complexes at these sites are enzyme-specific and vary substantially in the presence of the quinolone, ciprofloxacin, but not with DNA supercoil chirality, i.e. negative versus positive supercoiling. SHAN-seq's exquisite sensitivity provides an unprecedented single-nucleotide resolution view of the distribution of gyrase and topoisomerase IV cleavage complexes on DNA. Moreover, the discovery that these enzymes can cleave DNA at orders of magnitude more sites than the relatively few previously known sites resolves the apparent paradox of how these enzymes resolve topological problems throughout the genome.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141897930","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The combination of morphogenetic and transcription factors together with the synergic aid of noncoding RNAs and their cognate RNA binding proteins contribute to shape motor neurons (MN) identity. Here, we extend the noncoding perspective of human MN, by detailing the molecular and biological activity of CyCoNP (as Cytoplasmic Coordinator of Neural Progenitors) a highly expressed and MN-enriched human lncRNA. Through in silico prediction, in vivo RNA purification and loss of function experiments followed by RNA-sequencing, we found that CyCoNP sustains a specific neuron differentiation program, required for the physiology of both neuroblastoma cells and hiPSC-derived MN, which mainly involves miR-4492 and NCAM1 mRNA. We propose a novel lncRNA-mediated 'dual mode' of action, in which CyCoNP acts in trans as a classical RNA sponge by sequestering miR-4492 from its pro-neuronal targets, including NCAM1 mRNA, and at the same time it plays an additional role in cis by interacting with NCAM1 mRNA and regulating the availability and localization of the miR-4492 in its proximity. These data highlight novel insights into the noncoding RNA-mediated control of human neuron physiology and point out the importance of lncRNA-mediated interactions for the spatial distribution of regulatory molecules.
{"title":"CyCoNP lncRNA establishes cis and trans RNA-RNA interactions to supervise neuron physiology.","authors":"Fabio Desideri, Alessandro Grazzi, Michela Lisi, Adriano Setti, Tiziana Santini, Alessio Colantoni, Gabriele Proietti, Andrea Carvelli, Gian Gaetano Tartaglia, Monica Ballarino, Irene Bozzoni","doi":"10.1093/nar/gkae590","DOIUrl":"10.1093/nar/gkae590","url":null,"abstract":"<p><p>The combination of morphogenetic and transcription factors together with the synergic aid of noncoding RNAs and their cognate RNA binding proteins contribute to shape motor neurons (MN) identity. Here, we extend the noncoding perspective of human MN, by detailing the molecular and biological activity of CyCoNP (as Cytoplasmic Coordinator of Neural Progenitors) a highly expressed and MN-enriched human lncRNA. Through in silico prediction, in vivo RNA purification and loss of function experiments followed by RNA-sequencing, we found that CyCoNP sustains a specific neuron differentiation program, required for the physiology of both neuroblastoma cells and hiPSC-derived MN, which mainly involves miR-4492 and NCAM1 mRNA. We propose a novel lncRNA-mediated 'dual mode' of action, in which CyCoNP acts in trans as a classical RNA sponge by sequestering miR-4492 from its pro-neuronal targets, including NCAM1 mRNA, and at the same time it plays an additional role in cis by interacting with NCAM1 mRNA and regulating the availability and localization of the miR-4492 in its proximity. These data highlight novel insights into the noncoding RNA-mediated control of human neuron physiology and point out the importance of lncRNA-mediated interactions for the spatial distribution of regulatory molecules.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141580361","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The ribosome small subunit (SSU) is assembled by the SSU processome which contains approximately 70 non-ribosomal protein factors. Whilst the biochemical mechanisms of the SSU processome in 18S rRNA processing and maturation have been extensively studied, how SSU processome components enter the nucleolus has yet to be systematically investigated. Here, in examining the nucleolar localization of 50 human SSU processome components, we found that UTP3, together with another 24 proteins, enter the nucleolus autonomously. For the remaining 25 proteins we found that UTP3/SAS10 assists the nucleolar localization of five proteins (MPP10, UTP25, EMG1 and the two UTP-B components UTP12 and UTP13), likely through its interaction with nuclear importin α. This 'ferrying' function of UTP3 was then confirmed as conserved in the zebrafish. We also found that knockdown of human UTP3 impairs cleavage at the A0-site while loss-of-function of either utp3/sas10 or utp13/tbl3 in zebrafish causes the accumulation of aberrantly processed 5'ETS products, which highlights the crucial role of UTP3 in mediating 5'ETS processing. Mechanistically, we found that UTP3 facilitates the degradation of processed 5'ETS by recruiting the RNA exosome component EXOSC10 to the nucleolus. These findings lay the groundwork for studying the mechanism of cytoplasm-to-nucleolus trafficking of SSU processome components.
{"title":"A UTP3-dependent nucleolar translocation pathway facilitates pre-rRNA 5'ETS processing.","authors":"Jiayang Bao, Baochun Su, Zheyan Chen, Zhaoxiang Sun, Jinrong Peng, Shuyi Zhao","doi":"10.1093/nar/gkae631","DOIUrl":"10.1093/nar/gkae631","url":null,"abstract":"<p><p>The ribosome small subunit (SSU) is assembled by the SSU processome which contains approximately 70 non-ribosomal protein factors. Whilst the biochemical mechanisms of the SSU processome in 18S rRNA processing and maturation have been extensively studied, how SSU processome components enter the nucleolus has yet to be systematically investigated. Here, in examining the nucleolar localization of 50 human SSU processome components, we found that UTP3, together with another 24 proteins, enter the nucleolus autonomously. For the remaining 25 proteins we found that UTP3/SAS10 assists the nucleolar localization of five proteins (MPP10, UTP25, EMG1 and the two UTP-B components UTP12 and UTP13), likely through its interaction with nuclear importin α. This 'ferrying' function of UTP3 was then confirmed as conserved in the zebrafish. We also found that knockdown of human UTP3 impairs cleavage at the A0-site while loss-of-function of either utp3/sas10 or utp13/tbl3 in zebrafish causes the accumulation of aberrantly processed 5'ETS products, which highlights the crucial role of UTP3 in mediating 5'ETS processing. Mechanistically, we found that UTP3 facilitates the degradation of processed 5'ETS by recruiting the RNA exosome component EXOSC10 to the nucleolus. These findings lay the groundwork for studying the mechanism of cytoplasm-to-nucleolus trafficking of SSU processome components.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141734698","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Eugenia S Bardossy, Sebastiano Volpe, Yasutsugu Suzuki, Fernando Merwaiss, Santiago Faraj, Mónica Montes, Maria-Carla Saleh, Diego E Alvarez, Claudia V Filomatori
Chikungunya virus (CHIKV) is a rapidly spreading re-emergent virus transmitted from mosquitoes to humans. The emergence of epidemic variants has been associated with changes in the viral genome, such as the duplication of repeated sequences in the 3' untranslated region (UTR). Indeed, blocks of repeated sequences seemingly favor RNA recombination, providing the virus with a unique ability to continuously change the 3'UTR architecture during host switching. In this work, we provide experimental data on the molecular mechanism of RNA recombination and describe specific sequence and structural elements in the viral 3'UTR that favor template switching of the viral RNA-dependent RNA polymerase on the 3'UTR. Furthermore, we found that a 3'UTR deletion mutant that exhibits markedly delayed replication in mosquito cells and impaired transmission in vivo, recombines in reference laboratory strains of mosquitoes. Altogether, our data provide novel experimental evidence indicating that RNA recombination can act as a nucleic acid repair mechanism to add repeated sequences that are associated to high viral fitness in mosquito during chikungunya virus replication.
{"title":"Molecular basis of RNA recombination in the 3'UTR of chikungunya virus genome.","authors":"Eugenia S Bardossy, Sebastiano Volpe, Yasutsugu Suzuki, Fernando Merwaiss, Santiago Faraj, Mónica Montes, Maria-Carla Saleh, Diego E Alvarez, Claudia V Filomatori","doi":"10.1093/nar/gkae650","DOIUrl":"10.1093/nar/gkae650","url":null,"abstract":"<p><p>Chikungunya virus (CHIKV) is a rapidly spreading re-emergent virus transmitted from mosquitoes to humans. The emergence of epidemic variants has been associated with changes in the viral genome, such as the duplication of repeated sequences in the 3' untranslated region (UTR). Indeed, blocks of repeated sequences seemingly favor RNA recombination, providing the virus with a unique ability to continuously change the 3'UTR architecture during host switching. In this work, we provide experimental data on the molecular mechanism of RNA recombination and describe specific sequence and structural elements in the viral 3'UTR that favor template switching of the viral RNA-dependent RNA polymerase on the 3'UTR. Furthermore, we found that a 3'UTR deletion mutant that exhibits markedly delayed replication in mosquito cells and impaired transmission in vivo, recombines in reference laboratory strains of mosquitoes. Altogether, our data provide novel experimental evidence indicating that RNA recombination can act as a nucleic acid repair mechanism to add repeated sequences that are associated to high viral fitness in mosquito during chikungunya virus replication.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141760096","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Xiao Li, Ivette Maria Menendez Perdomo, Victoria Rodrigues Alves Barbosa, Catherine Diao, Maja Tarailo-Graovac
FANCJ/BRIP1, initially identified as DOG-1 (Deletions Of G-rich DNA) in Caenorhabditis elegans, plays a critical role in genome integrity by facilitating DNA interstrand cross-link repair and resolving G-quadruplex structures. Its function is tightly linked to a conserved [4Fe-4S] cluster-binding motif, mutations of which contribute to Fanconi anemia and various cancers. This study investigates the critical role of the iron-sulfur (Fe-S) cluster in DOG-1 and its relationship with the cytosolic iron-sulfur protein assembly targeting complex (CTC). We found that a DOG-1 mutant, expected to be defective in Fe-S cluster binding, is primarily localized in the cytoplasm, leading to heightened DNA damage sensitivity and G-rich DNA deletions. We further discovered that the deletion of mms-19, a nonessential CTC component, also resulted in DOG-1 sequestered in cytoplasm and increased DNA damage sensitivity. Additionally, we identified that CIAO-1 and CIAO-2B are vital for DOG-1's stability and repair functions but unlike MMS-19 have essential roles in C. elegans. These findings confirm the CTC and Fe-S cluster as key elements in regulating DOG-1, crucial for genome integrity. Additionally, this study advances our understanding of the CTC's role in Fe-S protein regulation and development in C. elegans, offering a model to study its impact on multicellular organism development.
{"title":"The critical role of the iron-sulfur cluster and CTC components in DOG-1/BRIP1 function in Caenorhabditis elegans.","authors":"Xiao Li, Ivette Maria Menendez Perdomo, Victoria Rodrigues Alves Barbosa, Catherine Diao, Maja Tarailo-Graovac","doi":"10.1093/nar/gkae617","DOIUrl":"10.1093/nar/gkae617","url":null,"abstract":"<p><p>FANCJ/BRIP1, initially identified as DOG-1 (Deletions Of G-rich DNA) in Caenorhabditis elegans, plays a critical role in genome integrity by facilitating DNA interstrand cross-link repair and resolving G-quadruplex structures. Its function is tightly linked to a conserved [4Fe-4S] cluster-binding motif, mutations of which contribute to Fanconi anemia and various cancers. This study investigates the critical role of the iron-sulfur (Fe-S) cluster in DOG-1 and its relationship with the cytosolic iron-sulfur protein assembly targeting complex (CTC). We found that a DOG-1 mutant, expected to be defective in Fe-S cluster binding, is primarily localized in the cytoplasm, leading to heightened DNA damage sensitivity and G-rich DNA deletions. We further discovered that the deletion of mms-19, a nonessential CTC component, also resulted in DOG-1 sequestered in cytoplasm and increased DNA damage sensitivity. Additionally, we identified that CIAO-1 and CIAO-2B are vital for DOG-1's stability and repair functions but unlike MMS-19 have essential roles in C. elegans. These findings confirm the CTC and Fe-S cluster as key elements in regulating DOG-1, crucial for genome integrity. Additionally, this study advances our understanding of the CTC's role in Fe-S protein regulation and development in C. elegans, offering a model to study its impact on multicellular organism development.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141620529","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Guy Assa, Nechama Kalter, Michael Rosenberg, Avigail Beck, Oshry Markovich, Tanya Gontmakher, Ayal Hendel, Zohar Yakhini
Off-target effects present a significant impediment to the safe and efficient use of CRISPR-Cas genome editing. Since off-target activity is influenced by the genomic sequence, the presence of sequence variants leads to varying on- and off-target profiles among different alleles or individuals. However, a reliable tool that quantifies genome editing activity in an allelic context is not available. Here, we introduce CRISPECTOR2.0, an extended version of our previously published software tool CRISPECTOR, with an allele-specific editing activity quantification option. CRISPECTOR2.0 enables reference-free, allele-aware, precise quantification of on- and off-target activity, by using de novo sample-specific single nucleotide variant (SNV) detection and statistical-based allele-calling algorithms. We demonstrate CRISPECTOR2.0 efficacy in analyzing samples containing multiple alleles and quantifying allele-specific editing activity, using data from diverse cell types, including primary human cells, plants, and an original extensive human cell line database. We identified instances where an SNV induced changes in the protospacer adjacent motif sequence, resulting in allele-specific editing. Intriguingly, differential allelic editing was also observed in regions carrying distal SNVs, hinting at the involvement of additional epigenetic factors. Our findings highlight the importance of allele-specific editing measurement as a milestone in the adaptation of efficient, accurate, and safe personalized genome editing.
{"title":"Quantifying allele-specific CRISPR editing activity with CRISPECTOR2.0.","authors":"Guy Assa, Nechama Kalter, Michael Rosenberg, Avigail Beck, Oshry Markovich, Tanya Gontmakher, Ayal Hendel, Zohar Yakhini","doi":"10.1093/nar/gkae651","DOIUrl":"10.1093/nar/gkae651","url":null,"abstract":"<p><p>Off-target effects present a significant impediment to the safe and efficient use of CRISPR-Cas genome editing. Since off-target activity is influenced by the genomic sequence, the presence of sequence variants leads to varying on- and off-target profiles among different alleles or individuals. However, a reliable tool that quantifies genome editing activity in an allelic context is not available. Here, we introduce CRISPECTOR2.0, an extended version of our previously published software tool CRISPECTOR, with an allele-specific editing activity quantification option. CRISPECTOR2.0 enables reference-free, allele-aware, precise quantification of on- and off-target activity, by using de novo sample-specific single nucleotide variant (SNV) detection and statistical-based allele-calling algorithms. We demonstrate CRISPECTOR2.0 efficacy in analyzing samples containing multiple alleles and quantifying allele-specific editing activity, using data from diverse cell types, including primary human cells, plants, and an original extensive human cell line database. We identified instances where an SNV induced changes in the protospacer adjacent motif sequence, resulting in allele-specific editing. Intriguingly, differential allelic editing was also observed in regions carrying distal SNVs, hinting at the involvement of additional epigenetic factors. Our findings highlight the importance of allele-specific editing measurement as a milestone in the adaptation of efficient, accurate, and safe personalized genome editing.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":16.6,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141792989","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}