Lidiya Lisitskaya, Aleksandra Kuznetsova, Ivan Petushkov, Maya Alieva, Yuliya Zaitseva, Anna Olina, Ekaterina Kropocheva, Maria Lavrova, Anton Musabirov, Nikita Kuznetsov, Aleksei Agapov, Daria Gelfenbein, Andrey Kulbachinskiy
Argonautes are an evolutionarily conserved family of proteins that use guide oligonucleotides for specific recognition of nucleic acid targets. While eukaryotic Argonautes share a conserved structure and act in RNA interference, prokaryotic Argonautes (pAgos) display remarkable structural and functional variations, including various guide and target specificities. Most studied catalytically active pAgos use 5′-phosphorylated DNA guides to recognize and cleave DNA targets. Here, we describe a new group of pAgos from mesophilic bacteria that use RNA guides to cleave DNA targets and are active at physiological temperatures. In contrast to most characterized pAgo nucleases, these proteins can utilize guides of varying lengths containing either a 5′-phosphate or a 5′-hydroxyl group with similar efficiencies. Measurements of the kinetics of target DNA binding show that the annealing of the guide–target duplex in pAgo occurs in an ordered way and that pAgo increases the rate of guide–target interaction in the seed region. We show that the analyzed pAgos can produce alternative DNA products depending on the structure of guide RNA and the cleavage conditions. The results demonstrate that RNA-guided pAgo nucleases are more widespread than previously anticipated and that these pAgos have a relaxed specificity for target DNA cleavage.
{"title":"Specificity of DNA targeting by RNA-guided pAgo nucleases","authors":"Lidiya Lisitskaya, Aleksandra Kuznetsova, Ivan Petushkov, Maya Alieva, Yuliya Zaitseva, Anna Olina, Ekaterina Kropocheva, Maria Lavrova, Anton Musabirov, Nikita Kuznetsov, Aleksei Agapov, Daria Gelfenbein, Andrey Kulbachinskiy","doi":"10.1093/nar/gkag077","DOIUrl":"https://doi.org/10.1093/nar/gkag077","url":null,"abstract":"Argonautes are an evolutionarily conserved family of proteins that use guide oligonucleotides for specific recognition of nucleic acid targets. While eukaryotic Argonautes share a conserved structure and act in RNA interference, prokaryotic Argonautes (pAgos) display remarkable structural and functional variations, including various guide and target specificities. Most studied catalytically active pAgos use 5′-phosphorylated DNA guides to recognize and cleave DNA targets. Here, we describe a new group of pAgos from mesophilic bacteria that use RNA guides to cleave DNA targets and are active at physiological temperatures. In contrast to most characterized pAgo nucleases, these proteins can utilize guides of varying lengths containing either a 5′-phosphate or a 5′-hydroxyl group with similar efficiencies. Measurements of the kinetics of target DNA binding show that the annealing of the guide–target duplex in pAgo occurs in an ordered way and that pAgo increases the rate of guide–target interaction in the seed region. We show that the analyzed pAgos can produce alternative DNA products depending on the structure of guide RNA and the cleavage conditions. The results demonstrate that RNA-guided pAgo nucleases are more widespread than previously anticipated and that these pAgos have a relaxed specificity for target DNA cleavage.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"248 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146101699","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}
Horizontal gene transfer is a major driver of bacterial evolution and the global dissemination of antibiotic resistance genes (ARGs). Conjugative plasmids play a crucial role in ARG spread across hosts within their host range, yet the genetic and functional determinants shaping plasmid host range remain poorly understood. Here, we systematically analyzed the gene content of conjugative/mobilizable plasmids derived from Enterobacterales from public databases and found that two distinct survival strategies were enriched in different host-range groups: a “stealth” strategy, which actively represses its own transcription by employing a global regulator hns, was particularly enriched in broad-host-range plasmids, whereas a “manipulative” strategy, which promotes its establishment by manipulating host machineries including SOS response and defense systems, was more common in narrow-host-range plasmids. Plasmids employing either strategy constituted the majority of conjugative plasmids analyzed, and accumulated significantly more ARGs than plasmids with neither strategy. Our data further suggested that stealth plasmids facilitate the acquisition of emerging ARGs, while manipulative plasmids amplify the copy number of established ARGs. This “stealth-first” model successfully recapitulated historical ARG dissemination patterns. These findings provide critical insights into the relationship between plasmid survival strategies and host range, advancing our understanding of the global patterns underlying plasmid-mediated ARG transmission.
{"title":"Host range and antibiotic resistance dissemination are shaped by distinct survival strategies of conjugative plasmids","authors":"Ryuichi Ono, Naoki Konno, Yuki Nishimura, Chikara Furusawa","doi":"10.1093/nar/gkaf1479","DOIUrl":"https://doi.org/10.1093/nar/gkaf1479","url":null,"abstract":"Horizontal gene transfer is a major driver of bacterial evolution and the global dissemination of antibiotic resistance genes (ARGs). Conjugative plasmids play a crucial role in ARG spread across hosts within their host range, yet the genetic and functional determinants shaping plasmid host range remain poorly understood. Here, we systematically analyzed the gene content of conjugative/mobilizable plasmids derived from Enterobacterales from public databases and found that two distinct survival strategies were enriched in different host-range groups: a “stealth” strategy, which actively represses its own transcription by employing a global regulator hns, was particularly enriched in broad-host-range plasmids, whereas a “manipulative” strategy, which promotes its establishment by manipulating host machineries including SOS response and defense systems, was more common in narrow-host-range plasmids. Plasmids employing either strategy constituted the majority of conjugative plasmids analyzed, and accumulated significantly more ARGs than plasmids with neither strategy. Our data further suggested that stealth plasmids facilitate the acquisition of emerging ARGs, while manipulative plasmids amplify the copy number of established ARGs. This “stealth-first” model successfully recapitulated historical ARG dissemination patterns. These findings provide critical insights into the relationship between plasmid survival strategies and host range, advancing our understanding of the global patterns underlying plasmid-mediated ARG transmission.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"142 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146033191","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}
Ibraheem Alshareedah, Sushil Pangeni, Paul A Dewan, Masayoshi Honda, Ting-Wei Liao, Maria Spies, Taekjip Ha
Human RAD52 is a prime target for synthetic lethality approaches to treat cancers with deficiency in homologous recombination. Among multiple cellular roles, RAD52's functions in homologous recombination repair and stalled replication fork protection appear to substitute for those of the tumor suppressor protein BRCA2. However, the mechanistic details of how RAD52 substitutes for BRCA2 functions are only beginning to emerge. RAD52 forms an oligomeric ring enveloped by ∼200-residue-long disordered regions, forming a highly multivalent and branched protein complex that promotes supramolecular assembly. Here, we demonstrate that RAD52 undergoes homotypic phase separation, forming condensates that recruit key homologous recombination factors, including single-stranded DNA (ssDNA), replication protein A (RPA), and the RAD51 recombinase. Furthermore, we show that RAD52 phase separation is regulated by its interaction partners such as ssDNA and RPA. Through fluorescence microscopy, we observe that RAD52 promotes the formation of RAD51-ssDNA fibrillar structures. To resolve the fine architecture of these fibrils, we employed single-molecule super-resolution imaging via DNA-PAINT and atomic force microscopy, revealing that RAD51 fibrils comprise bundles of individual RAD51 nucleoprotein filaments. Additionally, we show that RAD52 induces end-to-end tethering of RAD51 nucleoprotein filaments. Collectively, these findings highlight distinctive macromolecular organizational features of RAD52 that may underpin its diverse cellular functions.
{"title":"The human RAD52 complex undergoes phase separation and facilitates bundling and end-to-end tethering of RAD51 presynaptic filaments.","authors":"Ibraheem Alshareedah, Sushil Pangeni, Paul A Dewan, Masayoshi Honda, Ting-Wei Liao, Maria Spies, Taekjip Ha","doi":"10.1093/nar/gkag043","DOIUrl":"10.1093/nar/gkag043","url":null,"abstract":"<p><p>Human RAD52 is a prime target for synthetic lethality approaches to treat cancers with deficiency in homologous recombination. Among multiple cellular roles, RAD52's functions in homologous recombination repair and stalled replication fork protection appear to substitute for those of the tumor suppressor protein BRCA2. However, the mechanistic details of how RAD52 substitutes for BRCA2 functions are only beginning to emerge. RAD52 forms an oligomeric ring enveloped by ∼200-residue-long disordered regions, forming a highly multivalent and branched protein complex that promotes supramolecular assembly. Here, we demonstrate that RAD52 undergoes homotypic phase separation, forming condensates that recruit key homologous recombination factors, including single-stranded DNA (ssDNA), replication protein A (RPA), and the RAD51 recombinase. Furthermore, we show that RAD52 phase separation is regulated by its interaction partners such as ssDNA and RPA. Through fluorescence microscopy, we observe that RAD52 promotes the formation of RAD51-ssDNA fibrillar structures. To resolve the fine architecture of these fibrils, we employed single-molecule super-resolution imaging via DNA-PAINT and atomic force microscopy, revealing that RAD51 fibrils comprise bundles of individual RAD51 nucleoprotein filaments. Additionally, we show that RAD52 induces end-to-end tethering of RAD51 nucleoprotein filaments. Collectively, these findings highlight distinctive macromolecular organizational features of RAD52 that may underpin its diverse cellular functions.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"54 3","pages":""},"PeriodicalIF":13.1,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12848948/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146065419","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Zi Gao,Yiewoon Chong,Xiaomei He,Jun Yuan,Yinsheng Wang
Guanine quadruplexes (G4s) are unique secondary structures of nucleic acids with functions in many biological processes. Understanding the functions of DNA G4s requires knowledge about their recognition by cellular proteins. Here, we developed a method involving photoclick chemistry and LC-MS/MS-based quantitative proteomics for uncovering G4-binding proteins (G4BPs). By incorporating a photoactivatable ortho-nitrobenzylamine moiety into G4 DNA probes and employing UVA irradiation along with stringent washing, we identified 99 proteins enriched with G4 structures derived from the human telomere. By employing fluorescence anisotropy, fluorescence resonance energy transfer, immunofluorescence microscopy, and ChIP-seq analyses, we demonstrated the abilities of one of these proteins, HELLS, in binding and resolving G4 structures in vitro and in chromatin. We also found that HELLS-G4 interaction modulates gene expression. Together, we developed a photoclick chemistry-based approach for identifying G4BPs. The approach allowed for the identification of a number of new G4BPs, and we also documented novel functions of one of these proteins, i.e. HELLS, in recognizing and unwinding G4 structures in vitro and in cells.
{"title":"Photoclick chemistry led to the identification of HELLS as a helicase for DNA G-quadruplexes.","authors":"Zi Gao,Yiewoon Chong,Xiaomei He,Jun Yuan,Yinsheng Wang","doi":"10.1093/nar/gkag034","DOIUrl":"https://doi.org/10.1093/nar/gkag034","url":null,"abstract":"Guanine quadruplexes (G4s) are unique secondary structures of nucleic acids with functions in many biological processes. Understanding the functions of DNA G4s requires knowledge about their recognition by cellular proteins. Here, we developed a method involving photoclick chemistry and LC-MS/MS-based quantitative proteomics for uncovering G4-binding proteins (G4BPs). By incorporating a photoactivatable ortho-nitrobenzylamine moiety into G4 DNA probes and employing UVA irradiation along with stringent washing, we identified 99 proteins enriched with G4 structures derived from the human telomere. By employing fluorescence anisotropy, fluorescence resonance energy transfer, immunofluorescence microscopy, and ChIP-seq analyses, we demonstrated the abilities of one of these proteins, HELLS, in binding and resolving G4 structures in vitro and in chromatin. We also found that HELLS-G4 interaction modulates gene expression. Together, we developed a photoclick chemistry-based approach for identifying G4BPs. The approach allowed for the identification of a number of new G4BPs, and we also documented novel functions of one of these proteins, i.e. HELLS, in recognizing and unwinding G4 structures in vitro and in cells.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"382 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146015324","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}
Emily A Warman,David Forrest,Anne M Stringer,Joseph T Wade,David C Grainger
In prokaryotes, bidirectional promoters are pseudo-symmetrical DNA sequences that stimulate divergent transcription. Ubiquitous, and far more likely to drive messenger RNA production than directional promoters, nothing is known about their control. For example, symmetry allows bidirectional promoters to engage RNA polymerase in two possible orientations. As one binding event prevents the other, there is potential for regulation at this step. Here, we show that basal transcription, from all five tested bidirectional promoters, is too low for RNA polymerase competition. Hence, synthesis of one RNA does not impact the divergent pair. Conversely, if transcription in one direction is substantially activated, divergent RNA production can be repressed. Often, this results from RNA polymerase competition alone. Unexpectedly, this also impacts population-level gene expression noise. Specifically, if transcription is constrained, by RNA polymerase interference, cell-to-cell variation is reduced. We anticipate that our findings will help to establish rules for understanding bidirectional promoters, which have hardly been studied, in many bacteria.
{"title":"Bidirectional promoters in Escherichia coli: regulatory rules and implications for gene expression noise.","authors":"Emily A Warman,David Forrest,Anne M Stringer,Joseph T Wade,David C Grainger","doi":"10.1093/nar/gkag028","DOIUrl":"https://doi.org/10.1093/nar/gkag028","url":null,"abstract":"In prokaryotes, bidirectional promoters are pseudo-symmetrical DNA sequences that stimulate divergent transcription. Ubiquitous, and far more likely to drive messenger RNA production than directional promoters, nothing is known about their control. For example, symmetry allows bidirectional promoters to engage RNA polymerase in two possible orientations. As one binding event prevents the other, there is potential for regulation at this step. Here, we show that basal transcription, from all five tested bidirectional promoters, is too low for RNA polymerase competition. Hence, synthesis of one RNA does not impact the divergent pair. Conversely, if transcription in one direction is substantially activated, divergent RNA production can be repressed. Often, this results from RNA polymerase competition alone. Unexpectedly, this also impacts population-level gene expression noise. Specifically, if transcription is constrained, by RNA polymerase interference, cell-to-cell variation is reduced. We anticipate that our findings will help to establish rules for understanding bidirectional promoters, which have hardly been studied, in many bacteria.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"31 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146015327","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}
Eukaryotic ribosome synthesis is a highly complex, multistep process that is best characterized in the yeast Saccharomyces cerevisiae. It is orchestrated by >200 ribosome assembly factors and 75 small nucleolar ribonucleoproteins (snoRNPs), which guide site-specific chemical modifications of precursor rRNA (pre-rRNA). While canonical box C/D snoRNPs guide 2'-O-methylation, the atypical box C/D snoRNPs snR4 and snR45 guide acetylation of 18S rRNA residues C1280 and C1773, respectively, catalyzed by the acetyltransferase Kre33. Here, we identify and characterize Ynl050c/Sni445 as a novel ribosome assembly factor and previously unrecognized auxiliary component of the snR4 and snR45 box C/D snoRNPs. Sni445 associates with snR4 and snR45 in their free form and is required for their stable incorporation into 90S pre-ribosomes. Genetic interactions link Sni445 and the snR4 and snR45 snoRNAs to ribosomal proteins Rps20 (uS10) and Rps14 (uS11), which are positioned near the respective acetylation sites in the 40S subunit. Moreover, Sni445 physically interacts with Kre33 within the 90S pre-ribosome, and its absence abolishes acetylation of C1280 and C1773. Our findings suggest that Sni445 facilitates the recruitment of snR4 and snR45 snoRNPs to 90S particles and might promote their interaction with Kre33, thereby enabling the site-specific acetylation of 18S rRNA by Kre33.
{"title":"Sni445 recruits box C/D snoRNPs snR4 and snR45 to guide ribosomal RNA acetylation by Kre33.","authors":"Jutta Hafner,Ingrid Zierler,Hussein Hamze,Sébastien Favre,Matthias Thoms,Natalia Kunowska,Sarah Rimser,Benjamin Albert,Tomas Caetano,Marion Aguirrebengoa,Roland Beckmann,Ulrich Stelzl,Dieter Kressler,Anthony K Henras,Brigitte Pertschy","doi":"10.1093/nar/gkag030","DOIUrl":"https://doi.org/10.1093/nar/gkag030","url":null,"abstract":"Eukaryotic ribosome synthesis is a highly complex, multistep process that is best characterized in the yeast Saccharomyces cerevisiae. It is orchestrated by >200 ribosome assembly factors and 75 small nucleolar ribonucleoproteins (snoRNPs), which guide site-specific chemical modifications of precursor rRNA (pre-rRNA). While canonical box C/D snoRNPs guide 2'-O-methylation, the atypical box C/D snoRNPs snR4 and snR45 guide acetylation of 18S rRNA residues C1280 and C1773, respectively, catalyzed by the acetyltransferase Kre33. Here, we identify and characterize Ynl050c/Sni445 as a novel ribosome assembly factor and previously unrecognized auxiliary component of the snR4 and snR45 box C/D snoRNPs. Sni445 associates with snR4 and snR45 in their free form and is required for their stable incorporation into 90S pre-ribosomes. Genetic interactions link Sni445 and the snR4 and snR45 snoRNAs to ribosomal proteins Rps20 (uS10) and Rps14 (uS11), which are positioned near the respective acetylation sites in the 40S subunit. Moreover, Sni445 physically interacts with Kre33 within the 90S pre-ribosome, and its absence abolishes acetylation of C1280 and C1773. Our findings suggest that Sni445 facilitates the recruitment of snR4 and snR45 snoRNPs to 90S particles and might promote their interaction with Kre33, thereby enabling the site-specific acetylation of 18S rRNA by Kre33.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"42 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146056906","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}
RNA G-quadruplexes (rG4s), formed through guanine self-recognition into stacked tetrads, serve as critical regulators of gene expression, yet their comprehensive mapping and dynamic regulation in physiological contexts remain technically challenging. Here, we develop Ultra-low-input rG4-seq (ULI-rG4-seq), enabling precise rG4 detection enabling precise rG4 detection with ∼140 bp resolution in samples as small as 100 oocytes, and reveal notable enrichment of rG4s near crucial regulatory regions, particularly transcription start sites and end sites. This technological advance, combined with Trim-away or oocyte-specific knockout of DHX36 (also known as G4R1 or RHAU), an rG4-specific helicase, reveals acute and chronic loss of DHX36 leads to opposing effects on rG4 levels. This observation extends beyond the traditional view of helicases as unwinding enzymes and suggests sophisticated cellular mechanisms maintaining RNA structural homeostasis. Through integrated analysis of rG4 landscapes and DHX36-binding profiles, we demonstrate coordination between cytoplasmic rG4 regulation and nuclear gene expression, revealing how RNA structure dynamics orchestrate RNA stability and translation, thereby influencing transcriptional elongation, genome stability, and alternative splicing. Finally, we show that deletion of DHX36 resulted in decreased oocyte quality, premature ovarian failure and complete female infertility due to transcriptional defects and genome instability related to R-loop accumulation. These technological and conceptual advances not only deepen our understanding of RNA-based regulation but also open new therapeutic possibilities for diseases involving RNA structure.
RNA g -四分体（rG4s）通过鸟嘌呤自我识别形成堆叠四分体，是基因表达的关键调控因子，但其在生理背景下的全面定位和动态调控仍然是技术上的挑战。在这里，我们开发了超低输入rG4-seq (ULI-rG4-seq)，能够精确检测rG4，能够在小到100个卵母细胞的样品中以~ 140 bp的分辨率精确检测rG4，并揭示了关键调控区域附近rG4s的显著富集，特别是转录起始位点和结束位点。这一技术进步，结合Trim-away或卵母细胞特异性敲除DHX36（也称为G4R1或RHAU），一种rG4特异性解旋酶，揭示了DHX36的急性和慢性损失对rG4水平的相反影响。这一观察结果超越了解旋酶作为解绕酶的传统观点，并表明维持RNA结构稳态的复杂细胞机制。通过对rG4景观和dhx36结合谱的综合分析，我们证明了细胞质rG4调控与核基因表达之间的协调，揭示了RNA结构动力学如何协调RNA的稳定性和翻译，从而影响转录延伸、基因组稳定性和选择性剪接。最后，我们发现DHX36的缺失导致卵母细胞质量下降，卵巢早衰和完全女性不育，这是由于与r环积累相关的转录缺陷和基因组不稳定。这些技术和概念上的进步不仅加深了我们对RNA调控的理解，也为涉及RNA结构的疾病开辟了新的治疗可能性。
{"title":"Ultra-low-input rG4-seq reveals the RNA G-quadruplex regulome in gene expression and genome integrity.","authors":"Tie-Gang Meng,Wei Yue,Chao Li,Di Gao,Zongchang Du,Li-Tao Yi,Shu-Chen Liu,Ke Xu,You-Hui Lu,Xue-Shan Ma,Jia-Ni Guo,Chang-Yin Zhou,Zhenyu Ju,Shi-Ming Luo,Ruibao Su,Xiang-Hong Ou,Qing-Yuan Sun","doi":"10.1093/nar/gkag040","DOIUrl":"https://doi.org/10.1093/nar/gkag040","url":null,"abstract":"RNA G-quadruplexes (rG4s), formed through guanine self-recognition into stacked tetrads, serve as critical regulators of gene expression, yet their comprehensive mapping and dynamic regulation in physiological contexts remain technically challenging. Here, we develop Ultra-low-input rG4-seq (ULI-rG4-seq), enabling precise rG4 detection enabling precise rG4 detection with ∼140 bp resolution in samples as small as 100 oocytes, and reveal notable enrichment of rG4s near crucial regulatory regions, particularly transcription start sites and end sites. This technological advance, combined with Trim-away or oocyte-specific knockout of DHX36 (also known as G4R1 or RHAU), an rG4-specific helicase, reveals acute and chronic loss of DHX36 leads to opposing effects on rG4 levels. This observation extends beyond the traditional view of helicases as unwinding enzymes and suggests sophisticated cellular mechanisms maintaining RNA structural homeostasis. Through integrated analysis of rG4 landscapes and DHX36-binding profiles, we demonstrate coordination between cytoplasmic rG4 regulation and nuclear gene expression, revealing how RNA structure dynamics orchestrate RNA stability and translation, thereby influencing transcriptional elongation, genome stability, and alternative splicing. Finally, we show that deletion of DHX36 resulted in decreased oocyte quality, premature ovarian failure and complete female infertility due to transcriptional defects and genome instability related to R-loop accumulation. These technological and conceptual advances not only deepen our understanding of RNA-based regulation but also open new therapeutic possibilities for diseases involving RNA structure.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"102 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146056755","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}
Conjugative plasmids are key drivers of bacterial adaptation, enabling the horizontal transfer of accessory genes within and across diverse microbial populations, yet annotating them remains challenging due to their highly mosaic genetic architectures and inconsistent gene naming conventions that complicate functional predictions and comparative analyses. To address this, we developed PlasAnn, a database designed specifically for genes encoded on natural plasmids, paired with a dedicated annotation pipeline (available via Bioconda or through the URL https://plasann.rochester.edu/). The curated database provides highly accurate, plasmid-type-specific gene names with standardized functional annotations, enabling direct comparison across plasmids without manual curation or specialized expertise, while the integrated annotation tool incorporates other common plasmid features for a fast, one-stop solution that outperforms broad prokaryotic genome annotation pipelines in both accuracy and efficiency. We demonstrate PlasAnn's utility by showing that plasmid accessory genes from different groups often share conserved repertoires, suggesting dynamic, modular networks of interconnected genes, and by revealing that plasmid-encoded transposable elements frequently carry genes related to bacterial adaptation beyond antibiotic resistance, including metabolism, virulence, and stress responses, emphasizing their broader contributions to fitness and adaptability. These insights, not captured by current field-standard tools, highlight how PlasAnn improves plasmid annotation and advances our understanding of plasmid biology, microbial ecology, and evolution.
{"title":"PlasAnn: a curated plasmid-specific database and annotation pipeline for standardized gene and function analysis.","authors":"Habibul Islam,Abhishek Sharma,Jordan Blair,Allison J Lopatkin","doi":"10.1093/nar/gkaf1507","DOIUrl":"https://doi.org/10.1093/nar/gkaf1507","url":null,"abstract":"Conjugative plasmids are key drivers of bacterial adaptation, enabling the horizontal transfer of accessory genes within and across diverse microbial populations, yet annotating them remains challenging due to their highly mosaic genetic architectures and inconsistent gene naming conventions that complicate functional predictions and comparative analyses. To address this, we developed PlasAnn, a database designed specifically for genes encoded on natural plasmids, paired with a dedicated annotation pipeline (available via Bioconda or through the URL https://plasann.rochester.edu/). The curated database provides highly accurate, plasmid-type-specific gene names with standardized functional annotations, enabling direct comparison across plasmids without manual curation or specialized expertise, while the integrated annotation tool incorporates other common plasmid features for a fast, one-stop solution that outperforms broad prokaryotic genome annotation pipelines in both accuracy and efficiency. We demonstrate PlasAnn's utility by showing that plasmid accessory genes from different groups often share conserved repertoires, suggesting dynamic, modular networks of interconnected genes, and by revealing that plasmid-encoded transposable elements frequently carry genes related to bacterial adaptation beyond antibiotic resistance, including metabolism, virulence, and stress responses, emphasizing their broader contributions to fitness and adaptability. These insights, not captured by current field-standard tools, highlight how PlasAnn improves plasmid annotation and advances our understanding of plasmid biology, microbial ecology, and evolution.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"77 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146056758","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}
Zhongwen Cao,Xingyuan Chen,Yuxiang Sun,Yinsheng Wang
While METTL1 is a well-established m7G writer protein, its protein-protein interaction network remains largely unexplored. To map the METTL1 interactome in HEK293T cells, we employed APEX2-mediated proximity labeling coupled with LC-MS/MS analysis. This approach allowed for the identification of 60 and 18 unique proteins significantly enriched in the METTL1 proximity proteome compared to enhanced green fluorescent protein (EGFP) and nuclear localization signal (NLS) controls, respectively. Among these proteins, we found exportin-5 (XPO5), a nuclear export factor critical for pre-miRNA transport. We validated the METTL1-XPO5 interaction by co-immunoprecipitation and western blot analysis. Strikingly, genetic ablation of METTL1 caused XPO5 to redistribute to the cytosol, which in turn accelerated pre-miRNA export and enhanced miRNA maturation. This function of METTL1 was independent of its canonical m7G methyltransferase activity. Mechanistically, we found that METTL1 facilitates ERK-mediated phosphorylation of XPO5, thereby promoting its nuclear retention. Accordingly, constitutive activation of ERK was sufficient to restore nuclear XPO5 localization in METTL1-deficient cells. In summary, our study uncovers a non-canonical role for METTL1 in regulating the subcellular distribution of XPO5 and pre-miRNA export, revealing a novel mechanism of miRNA maturation that extends METTL1's function beyond m7G methylation.
{"title":"METTL1 interacts with XPO5 to modulate pre-miRNA export.","authors":"Zhongwen Cao,Xingyuan Chen,Yuxiang Sun,Yinsheng Wang","doi":"10.1093/nar/gkag037","DOIUrl":"https://doi.org/10.1093/nar/gkag037","url":null,"abstract":"While METTL1 is a well-established m7G writer protein, its protein-protein interaction network remains largely unexplored. To map the METTL1 interactome in HEK293T cells, we employed APEX2-mediated proximity labeling coupled with LC-MS/MS analysis. This approach allowed for the identification of 60 and 18 unique proteins significantly enriched in the METTL1 proximity proteome compared to enhanced green fluorescent protein (EGFP) and nuclear localization signal (NLS) controls, respectively. Among these proteins, we found exportin-5 (XPO5), a nuclear export factor critical for pre-miRNA transport. We validated the METTL1-XPO5 interaction by co-immunoprecipitation and western blot analysis. Strikingly, genetic ablation of METTL1 caused XPO5 to redistribute to the cytosol, which in turn accelerated pre-miRNA export and enhanced miRNA maturation. This function of METTL1 was independent of its canonical m7G methyltransferase activity. Mechanistically, we found that METTL1 facilitates ERK-mediated phosphorylation of XPO5, thereby promoting its nuclear retention. Accordingly, constitutive activation of ERK was sufficient to restore nuclear XPO5 localization in METTL1-deficient cells. In summary, our study uncovers a non-canonical role for METTL1 in regulating the subcellular distribution of XPO5 and pre-miRNA export, revealing a novel mechanism of miRNA maturation that extends METTL1's function beyond m7G methylation.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"397 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146056761","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}
Dilek Guneri,Christopher J Morris,Yiliang Ding,Timothy D Craggs,Steven S Smith,Zoë A E Waller
The insulin-linked polymorphic region (ILPR) is a variable number tandem repeat located in the promoter of the human insulin gene. This G-rich sequence can fold into four-stranded G-quadruplex DNA structures, while its complementary C-rich strand forms i-motifs. The ILPR varies in repeat number and sequence composition, but the relationship between sequence diversity, DNA structure, and insulin gene regulation remains poorly understood. Although both G-quadruplexes and i-motifs have been implicated in transcriptional control, their relative contributions, particularly when formed on complementary strands of the same locus, are unclear. Here, we characterized the structure and stability of nine ILPR-based sequences using biophysical techniques and luciferase reporter assays. We demonstrate that transcriptional activation in response to high glucose occurs only when both G-quadruplex and i-motif structures can form. Other combinations of structures do not induce transcription. Moreover, promoter activity correlated positively with i-motif stability, but not with G-quadruplex stability. These results suggest a model in which G-quadruplexes may act as an initiation site, while i-motifs act as modulators of insulin gene expression. Our findings underscore the importance of treating G-quadruplexes and i-motifs as a dynamic, interdependent system in both the regulation of gene expression and also the potential of these structures as therapeutic targets.
{"title":"i-Motif, not G-quadruplex, stability regulates insulin expression.","authors":"Dilek Guneri,Christopher J Morris,Yiliang Ding,Timothy D Craggs,Steven S Smith,Zoë A E Waller","doi":"10.1093/nar/gkag041","DOIUrl":"https://doi.org/10.1093/nar/gkag041","url":null,"abstract":"The insulin-linked polymorphic region (ILPR) is a variable number tandem repeat located in the promoter of the human insulin gene. This G-rich sequence can fold into four-stranded G-quadruplex DNA structures, while its complementary C-rich strand forms i-motifs. The ILPR varies in repeat number and sequence composition, but the relationship between sequence diversity, DNA structure, and insulin gene regulation remains poorly understood. Although both G-quadruplexes and i-motifs have been implicated in transcriptional control, their relative contributions, particularly when formed on complementary strands of the same locus, are unclear. Here, we characterized the structure and stability of nine ILPR-based sequences using biophysical techniques and luciferase reporter assays. We demonstrate that transcriptional activation in response to high glucose occurs only when both G-quadruplex and i-motif structures can form. Other combinations of structures do not induce transcription. Moreover, promoter activity correlated positively with i-motif stability, but not with G-quadruplex stability. These results suggest a model in which G-quadruplexes may act as an initiation site, while i-motifs act as modulators of insulin gene expression. Our findings underscore the importance of treating G-quadruplexes and i-motifs as a dynamic, interdependent system in both the regulation of gene expression and also the potential of these structures as therapeutic targets.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"71 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146069954","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}
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