The SETD6 (SET domain-containing protein 6) protein lysine methyltransferase regulates various cellular processes including cancer initiation and progression. It monomethylates the transcription factor E2F1 (E2F transcription factor 1) and several other important proteins, but the functional consequences of many SETD6 mediated methylation events are unknown. In this study, the role of SETD6 mediated K117 monomethylation of E2F1 was investigated in prostate cancer cells. In chromatin binding and gene expression experiments, we identified distinct sets of genes that are bound and upregulated by methylated and unmethylated E2F1 indicating that E2F1 methylation by SETD6 directly modulates its chromatin interaction. In agreement with these findings, cellular data showed that E2F1 methylation affects oncogenic phenotypes. Mechanistically, we demonstrate with biochemical, cellular, and genomic assays that SETD6-mediated K117 methylation directly regulates the interaction of E2F1 and BRD4 by preventing K117 acetylation. Our data suggest that K117 methylation/acetylation represents a switch controlling bromodomain binding to E2F1 by which SETD6 methylation regulates different cellular effects of E2F1. Similar mechanisms may apply to the regulation of other transcription factors by SETD6.
{"title":"E2F1 K117 methylation by SETD6 disrupts BRD4-E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells.","authors":"Gizem Tugce Ulu,Margarita Kublanovsky,Raz Shalev,Tzofit Elbaz Biton,Michal Feldman,Sophia Murr,Jens Brockmeyer,Franziska Dorscht,Sara Weirich,Dan Levy,Albert Jeltsch","doi":"10.1093/nar/gkaf1513","DOIUrl":"https://doi.org/10.1093/nar/gkaf1513","url":null,"abstract":"The SETD6 (SET domain-containing protein 6) protein lysine methyltransferase regulates various cellular processes including cancer initiation and progression. It monomethylates the transcription factor E2F1 (E2F transcription factor 1) and several other important proteins, but the functional consequences of many SETD6 mediated methylation events are unknown. In this study, the role of SETD6 mediated K117 monomethylation of E2F1 was investigated in prostate cancer cells. In chromatin binding and gene expression experiments, we identified distinct sets of genes that are bound and upregulated by methylated and unmethylated E2F1 indicating that E2F1 methylation by SETD6 directly modulates its chromatin interaction. In agreement with these findings, cellular data showed that E2F1 methylation affects oncogenic phenotypes. Mechanistically, we demonstrate with biochemical, cellular, and genomic assays that SETD6-mediated K117 methylation directly regulates the interaction of E2F1 and BRD4 by preventing K117 acetylation. Our data suggest that K117 methylation/acetylation represents a switch controlling bromodomain binding to E2F1 by which SETD6 methylation regulates different cellular effects of E2F1. Similar mechanisms may apply to the regulation of other transcription factors by SETD6.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"177 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145986515","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}
{"title":"Correction to 'MetaflowX: a scalable and resource-efficient workflow for multi-strategy metagenomic analysis'.","authors":"","doi":"10.1093/nar/gkag015","DOIUrl":"https://doi.org/10.1093/nar/gkag015","url":null,"abstract":"","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"56 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145971940","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}
Alexander Geidies,Marius Nieke,Nils Witte,Benjamin J McLean,Maria Evangelopoulou,Cha San Koh,Stefanie Kuschel,Frauke Stölting,Matias D Zurbriggen,Hannes M Beyer
Crafting synthetic in vitro tissues with mammalian cells faces a shortage of methods to define spatial features. Optogenetic tissue engineering can provide the desired spatial and temporal control but requires stable genomic engineering to support long-term cultivation and high response resolution. Here, we developed BlueGENEs, a set of optimized optogenetic gene switches. BlueGENEs support rapid, stable cell line generation, including precision engineering into the human AAVS1 safe harbor locus. By combining a designer endonuclease and a phage integrase, the approach overcomes gene-disruptive effects of random gene delivery and enables reproducible cell line development. BlueGENEs comprise an optogenetic blue light-responsive gene switch, a synthetic response promoter, and selection strategies serving broad use scenarios. We generated various human cell lines for optical control of apoptotic cell fate, 3D tissue formation, and signals promoting cytoskeletal remodeling. Our results demonstrate the integration of optogenetic cells with bioprinting technologies, illustrating the potential of BlueGENEs in advancing the synthesis of de novo or patient-derived in vitro model systems.
{"title":"Optogenetic BlueGENEs engineered into a human safe harbor locus.","authors":"Alexander Geidies,Marius Nieke,Nils Witte,Benjamin J McLean,Maria Evangelopoulou,Cha San Koh,Stefanie Kuschel,Frauke Stölting,Matias D Zurbriggen,Hannes M Beyer","doi":"10.1093/nar/gkaf1461","DOIUrl":"https://doi.org/10.1093/nar/gkaf1461","url":null,"abstract":"Crafting synthetic in vitro tissues with mammalian cells faces a shortage of methods to define spatial features. Optogenetic tissue engineering can provide the desired spatial and temporal control but requires stable genomic engineering to support long-term cultivation and high response resolution. Here, we developed BlueGENEs, a set of optimized optogenetic gene switches. BlueGENEs support rapid, stable cell line generation, including precision engineering into the human AAVS1 safe harbor locus. By combining a designer endonuclease and a phage integrase, the approach overcomes gene-disruptive effects of random gene delivery and enables reproducible cell line development. BlueGENEs comprise an optogenetic blue light-responsive gene switch, a synthetic response promoter, and selection strategies serving broad use scenarios. We generated various human cell lines for optical control of apoptotic cell fate, 3D tissue formation, and signals promoting cytoskeletal remodeling. Our results demonstrate the integration of optogenetic cells with bioprinting technologies, illustrating the potential of BlueGENEs in advancing the synthesis of de novo or patient-derived in vitro model systems.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"267 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145971947","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}
Tong Wu,Youhang Li,Yuqin Zhao,Elodie Bournique,Pedro Ortega,Minghua Nie,Yiqing Wang,Hailong Wang,Rémi Buisson,Ian D Hickson,Michael N Boddy,Xiaohua Wu
R-loops play essential physiological roles but also pose a significant threat to genome stability, particularly during replication, by exacerbating transcription-replication conflicts (TRCs). In this study, we have uncovered a critical role of the SMC5/6 complex in resolving TRCs to preserve fork integrity. We identified the SMC5/6 complex as a synthetic lethal partner of senataxin (SETX), an RNA/DNA helicase critical for removing R-loops that arise during replication. We demonstrated that in SETX-deficient cells, the SMC5/6 complex is recruited to TRCs in response to the buildup of DNA supercoiling and facilitates the recruitment of the BLM/TOP3A/RMI1/RMI2 complex (BTRR). Once recruited, BTRR acts to resolve the TRCs in a manner dependent on the catalytic activity of TOP3A. BTRR is also required for FANCM accumulation at TRCs, which activates the FANCD2 pathway to resolve TRCs. These studies underscore the role of SMC5/6 in sensing TRCs and define the SMC5/6-BTRR-FANCM-FANCD2 axis as an important player in mitigating TRC-induced genome instability. Our findings also provide therapeutic opportunities for targeting this axis for effective treatment of SETX-deficient tumors.
{"title":"The SMC5/SMC6 complex is critical for resolving R-loop-induced transcription-replication conflicts.","authors":"Tong Wu,Youhang Li,Yuqin Zhao,Elodie Bournique,Pedro Ortega,Minghua Nie,Yiqing Wang,Hailong Wang,Rémi Buisson,Ian D Hickson,Michael N Boddy,Xiaohua Wu","doi":"10.1093/nar/gkaf1537","DOIUrl":"https://doi.org/10.1093/nar/gkaf1537","url":null,"abstract":"R-loops play essential physiological roles but also pose a significant threat to genome stability, particularly during replication, by exacerbating transcription-replication conflicts (TRCs). In this study, we have uncovered a critical role of the SMC5/6 complex in resolving TRCs to preserve fork integrity. We identified the SMC5/6 complex as a synthetic lethal partner of senataxin (SETX), an RNA/DNA helicase critical for removing R-loops that arise during replication. We demonstrated that in SETX-deficient cells, the SMC5/6 complex is recruited to TRCs in response to the buildup of DNA supercoiling and facilitates the recruitment of the BLM/TOP3A/RMI1/RMI2 complex (BTRR). Once recruited, BTRR acts to resolve the TRCs in a manner dependent on the catalytic activity of TOP3A. BTRR is also required for FANCM accumulation at TRCs, which activates the FANCD2 pathway to resolve TRCs. These studies underscore the role of SMC5/6 in sensing TRCs and define the SMC5/6-BTRR-FANCM-FANCD2 axis as an important player in mitigating TRC-induced genome instability. Our findings also provide therapeutic opportunities for targeting this axis for effective treatment of SETX-deficient tumors.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"29 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145971958","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}
Joseph S Romanowski, Kevin M Myles, Zach N Adelman
Programmable site-specific nucleases have revolutionized the field of genetics, and in the field of mosquito vector control, gene editing by these tools has inspired a new wave of population control approaches that aim to prevent disease transmission. Little is known of how DNA repair is prioritized in mosquitoes, which diverged from the nearest model system (Drosophila) by >200 million years, despite site-specific gene editing now being commonplace. Here, we report a scalable, high-throughput platform for studying DNA double-stranded DNA break (DSB) repair in mosquitoes by delivering CRISPR/Cas9, I-SceI, or other nucleases to Aedes aegypti embryos, capable of measuring single-strand annealing (SSA), non-homologous end joining, and microhomology-mediated end-joining (MMEJ) repair outcomes. We find CRISPR/Cas9 can induce deletions of up to 8.6 kb through SSA repair and is tolerant of resection distances of 3.5 kb. Indel events were insensitive to lig4 knockouts, and across 20 synthetic guide RNAs (sgRNAs) representing 5 locations in 2 transgenic strains were almost exclusively attributed to MMEJ repair, establishing MMEJ as the dominant form of repair in A. aegypti at CRISPR/Cas9 DSBs. This information is critical to our understanding of how DNA repair shapes processes required for genetic control strategies involving gene drive action/resistance as well as transgene stability.
{"title":"Microhomology-mediated end joining is the predominant form of DNA repair in the mosquito Aedes aegypti with implications for gene editing, gene drive, and transgene removal","authors":"Joseph S Romanowski, Kevin M Myles, Zach N Adelman","doi":"10.1093/nar/gkaf1532","DOIUrl":"https://doi.org/10.1093/nar/gkaf1532","url":null,"abstract":"Programmable site-specific nucleases have revolutionized the field of genetics, and in the field of mosquito vector control, gene editing by these tools has inspired a new wave of population control approaches that aim to prevent disease transmission. Little is known of how DNA repair is prioritized in mosquitoes, which diverged from the nearest model system (Drosophila) by >200 million years, despite site-specific gene editing now being commonplace. Here, we report a scalable, high-throughput platform for studying DNA double-stranded DNA break (DSB) repair in mosquitoes by delivering CRISPR/Cas9, I-SceI, or other nucleases to Aedes aegypti embryos, capable of measuring single-strand annealing (SSA), non-homologous end joining, and microhomology-mediated end-joining (MMEJ) repair outcomes. We find CRISPR/Cas9 can induce deletions of up to 8.6 kb through SSA repair and is tolerant of resection distances of 3.5 kb. Indel events were insensitive to lig4 knockouts, and across 20 synthetic guide RNAs (sgRNAs) representing 5 locations in 2 transgenic strains were almost exclusively attributed to MMEJ repair, establishing MMEJ as the dominant form of repair in A. aegypti at CRISPR/Cas9 DSBs. This information is critical to our understanding of how DNA repair shapes processes required for genetic control strategies involving gene drive action/resistance as well as transgene stability.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"29 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145986435","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 rapid proliferation of trajectory inference methods for single-cell RNA-seq data has allowed researchers to investigate complex biological processes by examining underlying gene expression dynamics. After estimating a latent cell ordering, statistical models are used to identify genes exhibiting changes in expression significantly associated with progression through the trajectory. While a few techniques for trajectory differential expression exist, most rely on generalized additive models to account for the inherent nonlinearity of gene expression dynamics. As such, the results can be difficult to interpret, and biological conclusions rely on subjective visual inspections. To address this challenge, we propose single-cell linear adaptive negative-binomial expression (scLANE) testing, which is built around an interpretable generalized linear model and handles nonlinearity with basis splines chosen empirically for each gene. In addition, extensions to estimating equations and mixed models allow for reliable trajectory testing under complex experimental designs. After validating the accuracy of scLANE under several simulation scenarios, we applied it to a set of diverse biological datasets and demonstrated its ability to provide novel biological information when used downstream of both pseudotime and RNA velocity estimation methods. scLANE is freely available as an R package through Bioconductor at https://bioconductor.org/packages/scLANE/, and is also accessible via a web server leveraging high-performance computing resources at https://sclane.rc.ufl.edu/.
{"title":"Interpretable trajectory inference with single-cell linear adaptive negative-binomial expression (scLANE) testing.","authors":"Jack R Leary, Xiaoru Dong, Rhonda Bacher","doi":"10.1093/nar/gkaf1494","DOIUrl":"10.1093/nar/gkaf1494","url":null,"abstract":"<p><p>The rapid proliferation of trajectory inference methods for single-cell RNA-seq data has allowed researchers to investigate complex biological processes by examining underlying gene expression dynamics. After estimating a latent cell ordering, statistical models are used to identify genes exhibiting changes in expression significantly associated with progression through the trajectory. While a few techniques for trajectory differential expression exist, most rely on generalized additive models to account for the inherent nonlinearity of gene expression dynamics. As such, the results can be difficult to interpret, and biological conclusions rely on subjective visual inspections. To address this challenge, we propose single-cell linear adaptive negative-binomial expression (scLANE) testing, which is built around an interpretable generalized linear model and handles nonlinearity with basis splines chosen empirically for each gene. In addition, extensions to estimating equations and mixed models allow for reliable trajectory testing under complex experimental designs. After validating the accuracy of scLANE under several simulation scenarios, we applied it to a set of diverse biological datasets and demonstrated its ability to provide novel biological information when used downstream of both pseudotime and RNA velocity estimation methods. scLANE is freely available as an R package through Bioconductor at https://bioconductor.org/packages/scLANE/, and is also accessible via a web server leveraging high-performance computing resources at https://sclane.rc.ufl.edu/.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"54 2","pages":""},"PeriodicalIF":13.1,"publicationDate":"2026-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12802912/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145984974","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}
The co-evolution of immune and metabolic systems has endowed immune signaling pathways with distinct control of cellular metabolism. Innate immune transcription factors, such as nuclear factor κB (NF-κB), have thus emerged as key regulators of adaptive metabolic responses to changes in diet and nutrition. Utilizing chromatin accessibility genomics, we found that Drosophila NF-κB (Relish) can restrain nutrient-dependent metabolic transcriptional programs that control cellular catabolism of energy substrates, divergent from the protein’s canonical role as a transcriptional activator. NF-κB/Relish restricts chromatin accessibility through modulating histone acetylation at metabolic target gene loci, which restrains metabolic gene transcription and blocks excessive activation of nutrient-dependent metabolic programs. Targeted genetic screening revealed that histone deacetylase 6 interacts with NF-κB/Relish at NF-κB DNA regulatory motifs to limit chromatin accessibility and repress metabolic transcriptional programs. These results highlight that innate immune transcription factors can epigenetically restrain cellular catabolism to fine-tune nutrient-dependent metabolic adaptation.
{"title":"NF-κB restrains nutrient-dependent transcription programs through chromatin modulation in Drosophila","authors":"Xiangshuo Kong, Conghui Li, Jason Karpac","doi":"10.1093/nar/gkaf1530","DOIUrl":"https://doi.org/10.1093/nar/gkaf1530","url":null,"abstract":"The co-evolution of immune and metabolic systems has endowed immune signaling pathways with distinct control of cellular metabolism. Innate immune transcription factors, such as nuclear factor κB (NF-κB), have thus emerged as key regulators of adaptive metabolic responses to changes in diet and nutrition. Utilizing chromatin accessibility genomics, we found that Drosophila NF-κB (Relish) can restrain nutrient-dependent metabolic transcriptional programs that control cellular catabolism of energy substrates, divergent from the protein’s canonical role as a transcriptional activator. NF-κB/Relish restricts chromatin accessibility through modulating histone acetylation at metabolic target gene loci, which restrains metabolic gene transcription and blocks excessive activation of nutrient-dependent metabolic programs. Targeted genetic screening revealed that histone deacetylase 6 interacts with NF-κB/Relish at NF-κB DNA regulatory motifs to limit chromatin accessibility and repress metabolic transcriptional programs. These results highlight that innate immune transcription factors can epigenetically restrain cellular catabolism to fine-tune nutrient-dependent metabolic adaptation.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"18 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145972021","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}
Jennie L Hibma, Lia M Munson, Joshua D Jones, Taylor M Nye, Kristin S Koutmou, Lyle A Simmons
Ribosomal RNA (rRNA) methylation is conserved across biology, yet the effect of rRNA methylation on ribosome function is poorly understood. In this work, we identify a biological function for the rRNA 2′-O-methylcytidine methyltransferase TlyA, conserved between Bacillus subtilis and Mycobacterium tuberculosis (Mtb). The tlyA deletion in B. subtilis confers a cold sensitive phenotype and resistance to aminoglycoside and cyclic polypeptide antibiotics. We show that ∆tlyA cells have ribosome assembly defects characterized by accumulation of the 50S subunit. Using a genetic approach, we tested the importance of potential catalytic residues and S-adenosyl-l-methionine (SAM) cofactor binding sites identified based on sequence alignments with other rRNA methyltransferases. We show that B. subtilis TlyA uses the common rRNA methyltransferase catalytic triad KDK and SAM binding motif GxSxG. This differs from TlyA from Mtb, which requires an additional tetrapeptide linker. Together our work demonstrates that B. subtilis tlyA is critical for ribosome assembly and we identify key residues for TlyA function in vivo. Since Escherichia coli lacks TlyA or a functional equivalent, our work highlights key differences in ribosome maturation between B. subtilis, Mtb, and more divergent Gram-negative bacteria providing new insight into rRNA maturation and antibiotic resistance mechanisms.
核糖体RNA （rRNA）甲基化在整个生物学中是保守的，但rRNA甲基化对核糖体功能的影响尚不清楚。在这项工作中，我们确定了在枯草芽孢杆菌和结核分枝杆菌（Mtb）之间保守的rRNA 2 ' - o -甲基胞苷甲基转移酶（TlyA）的生物学功能。枯草芽孢杆菌的tlyA缺失赋予其冷敏感表型和对氨基糖苷类和环多肽类抗生素的抗性。我们发现，∆tlyA细胞具有以50S亚基积累为特征的核糖体组装缺陷。利用遗传学方法，我们测试了潜在催化残基和s -腺苷-l-蛋氨酸（SAM）辅因子结合位点的重要性，这些位点是根据与其他rRNA甲基转移酶的序列比对确定的。我们发现枯草芽孢杆菌TlyA使用共同的rRNA甲基转移酶催化三元组KDK和SAM结合基序gxxsxg。这与TlyA和Mtb不同，后者需要额外的四肽连接器。总之，我们的工作表明枯草芽孢杆菌tlyA对核糖体组装至关重要，我们在体内鉴定了tlyA功能的关键残基。由于大肠杆菌缺乏TlyA或功能等同物，我们的工作强调了枯草芽孢杆菌，Mtb和更多不同的革兰氏阴性菌之间核糖体成熟的关键差异，为rRNA成熟和抗生素耐药机制提供了新的见解。
{"title":"TlyA is a 23S and 16S 2′-O-methylcytidine methyltransferase important for ribosome assembly in Bacillus subtilis","authors":"Jennie L Hibma, Lia M Munson, Joshua D Jones, Taylor M Nye, Kristin S Koutmou, Lyle A Simmons","doi":"10.1093/nar/gkaf1531","DOIUrl":"https://doi.org/10.1093/nar/gkaf1531","url":null,"abstract":"Ribosomal RNA (rRNA) methylation is conserved across biology, yet the effect of rRNA methylation on ribosome function is poorly understood. In this work, we identify a biological function for the rRNA 2′-O-methylcytidine methyltransferase TlyA, conserved between Bacillus subtilis and Mycobacterium tuberculosis (Mtb). The tlyA deletion in B. subtilis confers a cold sensitive phenotype and resistance to aminoglycoside and cyclic polypeptide antibiotics. We show that ∆tlyA cells have ribosome assembly defects characterized by accumulation of the 50S subunit. Using a genetic approach, we tested the importance of potential catalytic residues and S-adenosyl-l-methionine (SAM) cofactor binding sites identified based on sequence alignments with other rRNA methyltransferases. We show that B. subtilis TlyA uses the common rRNA methyltransferase catalytic triad KDK and SAM binding motif GxSxG. This differs from TlyA from Mtb, which requires an additional tetrapeptide linker. Together our work demonstrates that B. subtilis tlyA is critical for ribosome assembly and we identify key residues for TlyA function in vivo. Since Escherichia coli lacks TlyA or a functional equivalent, our work highlights key differences in ribosome maturation between B. subtilis, Mtb, and more divergent Gram-negative bacteria providing new insight into rRNA maturation and antibiotic resistance mechanisms.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"57 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145972024","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}
CRISPR-Cas-based live-cell imaging has rapidly become a central technology for studying genome dynamics with high specificity and flexibility. By coupling nuclease-deactivated Cas (dCas) with programmable guide RNAs, genomic loci can be tracked in living cells, providing direct insights into nuclear organization and chromatin behavior. While repetitive regions such as telomeres and centromeres are readily visualized, labeling non-repetitive loci remains more challenging due to weak signals and high background. Recent advances, including multicolor labeling strategies, innovative amplification systems based on dCas9 and single-guide RNA (sgRNA) engineering, and integration with novel fluorescent reporters, have markedly expanded the applicability of CRISPR imaging across the genome. These developments have expanded the multiplexing capacity of CRISPR imaging, improved signal-to-background ratios, and even enabled the visualization of non-repetitive genomic loci. Nonetheless, key challenges remain, including cellular toxicity, replication stress, and genomic instability associated with prolonged CRISPR expression. In this review, we summarize recent advances in CRISPR live-cell imaging and highlight key design trade-offs and biological constraints.
{"title":"Illuminating the genome: emerging approaches in CRISPR-Cas live-cell imaging.","authors":"Zhiguang Xiao,Yujie Sun","doi":"10.1093/nar/gkaf1540","DOIUrl":"https://doi.org/10.1093/nar/gkaf1540","url":null,"abstract":"CRISPR-Cas-based live-cell imaging has rapidly become a central technology for studying genome dynamics with high specificity and flexibility. By coupling nuclease-deactivated Cas (dCas) with programmable guide RNAs, genomic loci can be tracked in living cells, providing direct insights into nuclear organization and chromatin behavior. While repetitive regions such as telomeres and centromeres are readily visualized, labeling non-repetitive loci remains more challenging due to weak signals and high background. Recent advances, including multicolor labeling strategies, innovative amplification systems based on dCas9 and single-guide RNA (sgRNA) engineering, and integration with novel fluorescent reporters, have markedly expanded the applicability of CRISPR imaging across the genome. These developments have expanded the multiplexing capacity of CRISPR imaging, improved signal-to-background ratios, and even enabled the visualization of non-repetitive genomic loci. Nonetheless, key challenges remain, including cellular toxicity, replication stress, and genomic instability associated with prolonged CRISPR expression. In this review, we summarize recent advances in CRISPR live-cell imaging and highlight key design trade-offs and biological constraints.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"18 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2026-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145971862","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}
Si-Young Choi,Haemin Park,Sung-Soo Kim,Hyungmin Kim,Sanghyo Park,Hyunsook Lee
We report that Aurora B kinase-mediated phosphorylation is essential for BubR1 acetylation at lysine 250 (K250), a modification required to preserve the mitotic checkpoint complex (MCC) and ensure accurate chromosome segregation. This Aurora B-BubR1 acetylation axis provides a mechanistic explanation for how kinetochore-microtubule attachment status is transduced to spindle assembly checkpoint (SAC) activity. Aurora B phosphorylates BubR1 at Serine 39 (and Ser16) in response to unattachment, and this phosphorylation is indispensable for subsequent K250 acetylation. Using a monoclonal anti-AcK250 antibody in structured illumination microscopy, we demonstrate that BubR1 acetylation sustains the fibrous corona, as shown by the crescent-shaped expansion of ZW10 and MAD2 surrounding kinetochores. Loss of either CENP-E or BubR1 acetylation abolishes fibrous corona, indicating that the interaction between acetylated BubR1 and CENP-E connects lateral attachment with the prevention of premature corona disassembly until proper end-on attachment is achieved. Disruption of Aurora B-mediated phosphorylation compromises K250 acetylation, fibrous corona maintenance, and MCC stability, whereas expression of a K250 acetylation-mimetic BubR1 rescues these defects in S16A/S39A phosphorylation-deficient mutants. Together, our findings establish a phosphorylation-acetylation cascade in BubR1 as a critical SAC signaling pathway and identify this axis as a promising therapeutic target in cancers driven by chromosomal instability.
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