Kuo-Chieh Liao, Majid Eshaghi, Zebin Hong, Tzuen Yih Saw, Jian An Jovi Lim, Jian Han, Jong Ghut Ashley Aw, Kiat Yee Tan, Aixin Yap, Xiang Gao, Youzhi Anthony Cheng, Su Ying Lim, You Zhi Nicholas Cheang, Wilfried A A Saron, Abhay P S Rathore, Li Zhang, Bhuvaneshwari Shunmuganathan, Rashi Gupta, Siang Ling Isabelle Tan, Xinlei Qian, Kiren Purushotorman, Nagavidya Subramaniam, Leah A Vardy, Paul A Macary, Ashley John, Yi Yan Yang, Sylvie Alonso, Haiwei Song, Roland G Huber, Yue Wan
Circular RNAs are an increasingly important class of RNA molecules that can be engineered as RNA vaccines and therapeutics. Here, we screened eight different group I introns for their ability to circularize and delineated different features that are important for their function. First, we identified the Scytalidium dimidiatum group I intron as causing minimal innate immune activation inside cells, underscoring its potential to serve as an effective RNA vaccine without triggering unwanted reactogenicity. Additionally, mechanistic RNA structure analysis was used to identify the P9 domain as important for circularization, showing that swapping sequences can restore pairing to improve the circularization of poor circularizers. We also determined the diversity of sequence requirements for the exon 1 and exon 2 (E1 and E2) domains of different group I introns and engineered a S1 tag within the domains for positive purification of circular RNAs. In addition, this flexibility in E1 and E2 enables substitution with less immunostimulatory sequences to enhance protein production. Our work deepens the understanding of the properties of group I introns, expands the panel of introns that can be used, and improves the manufacturing process to generate circular RNAs for vaccines and therapeutics.
{"title":"Characterization of group I introns in generating circular RNAs as vaccines","authors":"Kuo-Chieh Liao, Majid Eshaghi, Zebin Hong, Tzuen Yih Saw, Jian An Jovi Lim, Jian Han, Jong Ghut Ashley Aw, Kiat Yee Tan, Aixin Yap, Xiang Gao, Youzhi Anthony Cheng, Su Ying Lim, You Zhi Nicholas Cheang, Wilfried A A Saron, Abhay P S Rathore, Li Zhang, Bhuvaneshwari Shunmuganathan, Rashi Gupta, Siang Ling Isabelle Tan, Xinlei Qian, Kiren Purushotorman, Nagavidya Subramaniam, Leah A Vardy, Paul A Macary, Ashley John, Yi Yan Yang, Sylvie Alonso, Haiwei Song, Roland G Huber, Yue Wan","doi":"10.1093/nar/gkaf089","DOIUrl":"https://doi.org/10.1093/nar/gkaf089","url":null,"abstract":"Circular RNAs are an increasingly important class of RNA molecules that can be engineered as RNA vaccines and therapeutics. Here, we screened eight different group I introns for their ability to circularize and delineated different features that are important for their function. First, we identified the Scytalidium dimidiatum group I intron as causing minimal innate immune activation inside cells, underscoring its potential to serve as an effective RNA vaccine without triggering unwanted reactogenicity. Additionally, mechanistic RNA structure analysis was used to identify the P9 domain as important for circularization, showing that swapping sequences can restore pairing to improve the circularization of poor circularizers. We also determined the diversity of sequence requirements for the exon 1 and exon 2 (E1 and E2) domains of different group I introns and engineered a S1 tag within the domains for positive purification of circular RNAs. In addition, this flexibility in E1 and E2 enables substitution with less immunostimulatory sequences to enhance protein production. Our work deepens the understanding of the properties of group I introns, expands the panel of introns that can be used, and improves the manufacturing process to generate circular RNAs for vaccines and therapeutics.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"10 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143546398","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 RNA-binding protein ProQ interacts with many transcripts in the bacterial cell. ProQ binding is associated with increased messenger RNA (mRNA) levels, but a mechanistic explanation for this effect has been lacking. In Salmonella Typhimurium, ProQ affects key traits associated with infection, including motility and intracellular survival. However, the direct links between ProQ activity and these phenotypes are not well understood. Here, we demonstrate that ProQ promotes biofilm formation, another virulence-associated phenotype. This effect is strictly dependent on sigma factor RpoS. ProQ increases both RpoS protein and rpoS mRNA levels, but neither affects rpoS transcription nor translation. The rpoS mRNA is a ProQ target, and expression of the rpoS 3′UTR alone is strongly dependent on ProQ. RpoS expression becomes independent of ProQ in strains lacking poly(A) polymerase I (PAPI), indicating that ProQ protects against 3′ end-dependent decay. Indeed, purified ProQ inhibits PAPI-mediated polyadenylation at RNA 3′ ends. Finally, PAPI is required for ProQ’s effect on expression of genes involved in biofilm, motility, osmotic stress, and virulence, indicating that inhibition of polyadenylation is a general function of ProQ.
{"title":"ProQ prevents mRNA degradation through inhibition of poly(A) polymerase","authors":"Sofia Bergman, Christopher Birk, Erik Holmqvist","doi":"10.1093/nar/gkaf103","DOIUrl":"https://doi.org/10.1093/nar/gkaf103","url":null,"abstract":"The RNA-binding protein ProQ interacts with many transcripts in the bacterial cell. ProQ binding is associated with increased messenger RNA (mRNA) levels, but a mechanistic explanation for this effect has been lacking. In Salmonella Typhimurium, ProQ affects key traits associated with infection, including motility and intracellular survival. However, the direct links between ProQ activity and these phenotypes are not well understood. Here, we demonstrate that ProQ promotes biofilm formation, another virulence-associated phenotype. This effect is strictly dependent on sigma factor RpoS. ProQ increases both RpoS protein and rpoS mRNA levels, but neither affects rpoS transcription nor translation. The rpoS mRNA is a ProQ target, and expression of the rpoS 3′UTR alone is strongly dependent on ProQ. RpoS expression becomes independent of ProQ in strains lacking poly(A) polymerase I (PAPI), indicating that ProQ protects against 3′ end-dependent decay. Indeed, purified ProQ inhibits PAPI-mediated polyadenylation at RNA 3′ ends. Finally, PAPI is required for ProQ’s effect on expression of genes involved in biofilm, motility, osmotic stress, and virulence, indicating that inhibition of polyadenylation is a general function of ProQ.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"26 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143546400","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}
Martyna Węglewska, Joanna Gracz-Bernaciak, Sophia Bałdysz, Grzegorz Nowicki, Jakub Barylski
Group I introns are self-splicing ribozymes that can be found in eukaryotes, prokaryotes, and quite often in their viruses. The distribution, structure, and splicing of group I introns in genes of some phage taxa like the Tevenvirinae or Twortwirinae was extensively studied. On the other hand, the prevalence of intervening sequences in most other clades of bacterial viruses remains mostly unexplored. In this paper, we describe group I autocatalytic introns in genes of phages from the Bastillevirinae subfamily. This taxon belongs to the Herelleviridae family and consists of 15 genera and 37 species, including viruses with strong antimicrobial potential. A bioinformatic search for intron-related RNA structures revealed the presence of 45 intervening sequences within 37 genes that belong to four gene families. Eight of the nine genes selected for experimental validation were spliced—four only in an infected bacteria but additional four self-spliced in vitro. Interestingly, one of the studied genes undergoes alternative splicing. To sum up, our findings expand the knowledge on the distribution and diversity of group I introns and shed new light on this neglected aspect of phage transcriptomics. Additionally, in the course of our study, we demonstrated the effectiveness of nanopore sequencing in elucidating prokaryotic splicing mechanisms.
{"title":"Self-splicing introns in genes of Bastillevirinae bacteriophages","authors":"Martyna Węglewska, Joanna Gracz-Bernaciak, Sophia Bałdysz, Grzegorz Nowicki, Jakub Barylski","doi":"10.1093/nar/gkaf121","DOIUrl":"https://doi.org/10.1093/nar/gkaf121","url":null,"abstract":"Group I introns are self-splicing ribozymes that can be found in eukaryotes, prokaryotes, and quite often in their viruses. The distribution, structure, and splicing of group I introns in genes of some phage taxa like the Tevenvirinae or Twortwirinae was extensively studied. On the other hand, the prevalence of intervening sequences in most other clades of bacterial viruses remains mostly unexplored. In this paper, we describe group I autocatalytic introns in genes of phages from the Bastillevirinae subfamily. This taxon belongs to the Herelleviridae family and consists of 15 genera and 37 species, including viruses with strong antimicrobial potential. A bioinformatic search for intron-related RNA structures revealed the presence of 45 intervening sequences within 37 genes that belong to four gene families. Eight of the nine genes selected for experimental validation were spliced—four only in an infected bacteria but additional four self-spliced in vitro. Interestingly, one of the studied genes undergoes alternative splicing. To sum up, our findings expand the knowledge on the distribution and diversity of group I introns and shed new light on this neglected aspect of phage transcriptomics. Additionally, in the course of our study, we demonstrated the effectiveness of nanopore sequencing in elucidating prokaryotic splicing mechanisms.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"1 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143546399","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}
Parental stress can be encoded into altered epigenetic information to influence their offspring. Concurrently, it is vital for the preservation of a parent's epigenetic information, despite environmental challenges, to ensure accurate inheritance by the next generation. Nevertheless, the complexities of this process and the specific molecular mechanisms involved are not yet fully understood. Here we report that Argonaute CSR-1A potentiates the recovery of histone H3 lysine 9 trimethylation (H3K9me3) in spermatocyte to secure the developmental competence of male offspring. CSR-1A employs its repetitive RG motif to engage with putative histone 3 lysine 9 (H3K9) methyltransferases SET-25 and -32, and helps to restore repressive H3K9me3 chromatin marks following heat-stress, protecting the late development of somatic cells in the progeny. Finally, among the genes regulated by CSR-1A, we identified dim-1, at which decreased H3K9me3 persists in the progeny, and RNAi of dim-1 mitigates the somatic defects associated with csr-1a loss under stress. Thus, CSR-1A coordinates a paternal epigenetic program that shields development from the influences of the paternal environment. We speculate that, driven by both natural environmental stressors and the unique characteristics of spermatogenic chromatin, the emergence of multiple RG motif-featured and spermatogenesis-specific CSR-1A and small RNA serves as a protective strategy to safeguard against variability in the orchestration of inherited developmental programs from the paternal lineage.
{"title":"Argonaute CSR-1A promotes H3K9me3 maintenance to protect somatic development in offspring","authors":"Di Rao, Dengfeng Li, Lili Li, Junchao Xue, Shikui Tu, En-Zhi Shen","doi":"10.1093/nar/gkaf127","DOIUrl":"https://doi.org/10.1093/nar/gkaf127","url":null,"abstract":"Parental stress can be encoded into altered epigenetic information to influence their offspring. Concurrently, it is vital for the preservation of a parent's epigenetic information, despite environmental challenges, to ensure accurate inheritance by the next generation. Nevertheless, the complexities of this process and the specific molecular mechanisms involved are not yet fully understood. Here we report that Argonaute CSR-1A potentiates the recovery of histone H3 lysine 9 trimethylation (H3K9me3) in spermatocyte to secure the developmental competence of male offspring. CSR-1A employs its repetitive RG motif to engage with putative histone 3 lysine 9 (H3K9) methyltransferases SET-25 and -32, and helps to restore repressive H3K9me3 chromatin marks following heat-stress, protecting the late development of somatic cells in the progeny. Finally, among the genes regulated by CSR-1A, we identified dim-1, at which decreased H3K9me3 persists in the progeny, and RNAi of dim-1 mitigates the somatic defects associated with csr-1a loss under stress. Thus, CSR-1A coordinates a paternal epigenetic program that shields development from the influences of the paternal environment. We speculate that, driven by both natural environmental stressors and the unique characteristics of spermatogenic chromatin, the emergence of multiple RG motif-featured and spermatogenesis-specific CSR-1A and small RNA serves as a protective strategy to safeguard against variability in the orchestration of inherited developmental programs from the paternal lineage.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"229 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143546406","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}
Sophie I Jeanjean, Yimin Shen, Lise M Hardy, Antoine Daunay, Marc Delépine, Zuzana Gerber, Antonio Alberdi, Emmanuel Tubacher, Jean-François Deleuze, Alexandre How-Kit
Microsatellites are short tandem repeats (STRs) of a motif of 1–6 nucleotides that are ubiquitous in almost all genomes and widely used in many biomedical applications. However, despite the development of next-generation sequencing (NGS) over the past two decades with new technologies coming to the market, accurately sequencing and genotyping STRs, particularly homopolymers, remain very challenging today due to several technical limitations. This leads in many cases to erroneous allele calls and difficulty in correctly identifying the genuine allele distribution in a sample. Here, we assessed several second and third-generation sequencing approaches in their capability to correctly determine the length of microsatellites using plasmids containing A/T homopolymers, AC/TG or AT/TA dinucleotide STRs of variable length. Standard polymerase chain reaction (PCR)-free and PCR-containing, single Unique Molecular Indentifier (UMI) and dual UMI ‘duplex sequencing’ protocols were evaluated using Illumina short-read sequencing, and two PCR-free protocols using PacBio and Oxford Nanopore Technologies long-read sequencing. Several bioinformatics algorithms were developed to correctly identify microsatellite alleles from sequencing data, including four and two modes for generating standard and combined consensus alleles, respectively. We provided a detailed analysis and comparison of these approaches and made several recommendations for the accurate determination of microsatellite allele length.
{"title":"A detailed analysis of second and third-generation sequencing approaches for accurate length determination of short tandem repeats and homopolymers","authors":"Sophie I Jeanjean, Yimin Shen, Lise M Hardy, Antoine Daunay, Marc Delépine, Zuzana Gerber, Antonio Alberdi, Emmanuel Tubacher, Jean-François Deleuze, Alexandre How-Kit","doi":"10.1093/nar/gkaf131","DOIUrl":"https://doi.org/10.1093/nar/gkaf131","url":null,"abstract":"Microsatellites are short tandem repeats (STRs) of a motif of 1–6 nucleotides that are ubiquitous in almost all genomes and widely used in many biomedical applications. However, despite the development of next-generation sequencing (NGS) over the past two decades with new technologies coming to the market, accurately sequencing and genotyping STRs, particularly homopolymers, remain very challenging today due to several technical limitations. This leads in many cases to erroneous allele calls and difficulty in correctly identifying the genuine allele distribution in a sample. Here, we assessed several second and third-generation sequencing approaches in their capability to correctly determine the length of microsatellites using plasmids containing A/T homopolymers, AC/TG or AT/TA dinucleotide STRs of variable length. Standard polymerase chain reaction (PCR)-free and PCR-containing, single Unique Molecular Indentifier (UMI) and dual UMI ‘duplex sequencing’ protocols were evaluated using Illumina short-read sequencing, and two PCR-free protocols using PacBio and Oxford Nanopore Technologies long-read sequencing. Several bioinformatics algorithms were developed to correctly identify microsatellite alleles from sequencing data, including four and two modes for generating standard and combined consensus alleles, respectively. We provided a detailed analysis and comparison of these approaches and made several recommendations for the accurate determination of microsatellite allele length.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"29 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143546405","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}
Jianzhong Lin, Songwei Ni, Baiyuan Li, Yunxue Guo, Xinyu Gao, Yabo Liu, Lingxian Yi, Pengxia Wang, Ran Chen, Jianyun Yao, Thomas K Wood, Xiaoxue Wang
Conjugative plasmids, major vehicles for the spread of antibiotic resistance genes, often contain multiple toxin–antitoxin (TA) systems. However, the physiological functions of TA systems remain obscure. By studying two TA families commonly found on colistin-resistant IncI2 mcr-1-bearing plasmids, we discovered that the HicAB TA, rather than the StbDE TA, acts as a crucial addiction module to increase horizontal plasmid–plasmid competition. In contrast to the canonical type II TA systems in which the TA genes are cotranscribed and/or the antitoxin gene has an additional promoter to allow for an increased antitoxin/toxin ratio, the HicAB TA system with the toxin gene preceding the antitoxin gene employs internal transcription termination to allow for a higher toxin production. This intrinsic terminator, featuring a G/C-rich hairpin with a UUU tract, lies upstream of the antitoxin gene, introducing a unique mechanism for the enhancing toxin/antitoxin ratio. Critically, the hicAB TA significantly contributes to plasmid competition and plasmid persistence in the absence of antibiotic selection, and deleting this intrinsic terminator alone diminishes this function. These findings align with the observed high occurrence of hicAB in IncI2 plasmids and the persistence of these plasmids after banning colistin as a feed additive. This study reveals how reprogramming the regulatory circuits of TA operons impacts plasmid occupancy in the microbial community and provides critical targets for combating antibiotic resistance.
{"title":"A noncanonical intrinsic terminator in the HicAB toxin–antitoxin operon promotes the transmission of conjugative antibiotic resistance plasmids","authors":"Jianzhong Lin, Songwei Ni, Baiyuan Li, Yunxue Guo, Xinyu Gao, Yabo Liu, Lingxian Yi, Pengxia Wang, Ran Chen, Jianyun Yao, Thomas K Wood, Xiaoxue Wang","doi":"10.1093/nar/gkaf125","DOIUrl":"https://doi.org/10.1093/nar/gkaf125","url":null,"abstract":"Conjugative plasmids, major vehicles for the spread of antibiotic resistance genes, often contain multiple toxin–antitoxin (TA) systems. However, the physiological functions of TA systems remain obscure. By studying two TA families commonly found on colistin-resistant IncI2 mcr-1-bearing plasmids, we discovered that the HicAB TA, rather than the StbDE TA, acts as a crucial addiction module to increase horizontal plasmid–plasmid competition. In contrast to the canonical type II TA systems in which the TA genes are cotranscribed and/or the antitoxin gene has an additional promoter to allow for an increased antitoxin/toxin ratio, the HicAB TA system with the toxin gene preceding the antitoxin gene employs internal transcription termination to allow for a higher toxin production. This intrinsic terminator, featuring a G/C-rich hairpin with a UUU tract, lies upstream of the antitoxin gene, introducing a unique mechanism for the enhancing toxin/antitoxin ratio. Critically, the hicAB TA significantly contributes to plasmid competition and plasmid persistence in the absence of antibiotic selection, and deleting this intrinsic terminator alone diminishes this function. These findings align with the observed high occurrence of hicAB in IncI2 plasmids and the persistence of these plasmids after banning colistin as a feed additive. This study reveals how reprogramming the regulatory circuits of TA operons impacts plasmid occupancy in the microbial community and provides critical targets for combating antibiotic resistance.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"98 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143546404","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}
Kathryn Langenfeld, Bridget Hegarty, Santiago Vidaurri, Emily Crossette, Melissa B Duhaime, Krista R Wigginton
Quantitative metagenomic methods are maturing but continue to lack clearly-defined analytical limits. Here, we developed a computational tool, QuantMeta, to determine the absolute abundance of targets in metagenomes spiked with synthetic DNA standards. The tool establishes (i) entropy-based detection thresholds to confidently determine the presence of targets, and (ii) an approach to identify and correct read mapping or assembly errors and thus improve the quantification accuracy. Together this allows for an approach to confidently quantify absolute abundance of targets, be they microbial populations, genes, contigs, or metagenome-assembled genomes. We applied the approach to quantify single- and double-stranded DNA viruses in wastewater viral metagenomes, including pathogens and bacteriophages. Concentrations of total DNA viruses in wastewater influent and effluent were >108 copies/ml using QuantMeta. Human-associated DNA viruses were detected and quantifiable with QuantMeta thresholds, including polyomavirus, papillomavirus, and crAss-like phages, at concentrations similar to previous reports that utilized quantitative polymerase chain reaction (PCR)-based assays. Our results highlight the higher detection thresholds of quantitative metagenomics (approximately 500 copies/μl) as compared to PCR-based quantification (approximately 10 copies/μl) despite a sequencing depth of 200 million reads per sample. The QuantMeta approach, applicable to both viral and cellular metagenomes, advances quantitative metagenomics by improving the accuracy of measured target absolute abundances.
{"title":"Development of a quantitative metagenomic approach to establish quantitative limits and its application to viruses","authors":"Kathryn Langenfeld, Bridget Hegarty, Santiago Vidaurri, Emily Crossette, Melissa B Duhaime, Krista R Wigginton","doi":"10.1093/nar/gkaf118","DOIUrl":"https://doi.org/10.1093/nar/gkaf118","url":null,"abstract":"Quantitative metagenomic methods are maturing but continue to lack clearly-defined analytical limits. Here, we developed a computational tool, QuantMeta, to determine the absolute abundance of targets in metagenomes spiked with synthetic DNA standards. The tool establishes (i) entropy-based detection thresholds to confidently determine the presence of targets, and (ii) an approach to identify and correct read mapping or assembly errors and thus improve the quantification accuracy. Together this allows for an approach to confidently quantify absolute abundance of targets, be they microbial populations, genes, contigs, or metagenome-assembled genomes. We applied the approach to quantify single- and double-stranded DNA viruses in wastewater viral metagenomes, including pathogens and bacteriophages. Concentrations of total DNA viruses in wastewater influent and effluent were >108 copies/ml using QuantMeta. Human-associated DNA viruses were detected and quantifiable with QuantMeta thresholds, including polyomavirus, papillomavirus, and crAss-like phages, at concentrations similar to previous reports that utilized quantitative polymerase chain reaction (PCR)-based assays. Our results highlight the higher detection thresholds of quantitative metagenomics (approximately 500 copies/μl) as compared to PCR-based quantification (approximately 10 copies/μl) despite a sequencing depth of 200 million reads per sample. The QuantMeta approach, applicable to both viral and cellular metagenomes, advances quantitative metagenomics by improving the accuracy of measured target absolute abundances.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"131 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143546407","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}
Christian Marx, Xiaobing Qing, Yamin Gong, Joanna Kirkpatrick, Kanstantsin Siniuk, Galina V Beznoussenko, Gururaj Rao Kidiyoor, Murat Kirtay, Katrin Buder, Philipp Koch, Martin Westermann, Christopher Bruhn, Eric J Brown, Xingzhi Xu, Marco Foiani, Zhao-Qi Wang
Defective DNA damage response (DDR) and mitochondrial dysfunction are a major etiology of tissue impairment and aging. Mitochondrial autophagy (mitophagy) is a mitochondrial quality control (MQC) mechanism to selectively eliminate dysfunctional mitochondria. ATR (ataxia-telangiectasia and Rad3-related) is a key DDR regulator playing a pivotal role in DNA replication stress response and genomic stability. Paradoxically, the human Seckel syndrome caused by ATR mutations exhibits premature aging and neuropathies, suggesting a role of ATR in nonreplicating tissues. Here, we report a previously unknown yet direct role of ATR at mitochondria. We find that ATR and PINK1 (PTEN-induced kinase 1) dock at the mitochondrial translocase TOM/TIM complex, where ATR interacts directly with and thereby stabilizes PINK1. ATR deletion silences mitophagy initiation thereby altering oxidative phosphorylation functionality resulting in reactive oxygen species overproduction that attack cytosolic macromolecules, in both cells and brain tissues, prior to nuclear DNA. This study discloses ATR as an integrated component of the PINK1-mediated MQC program to ensure mitochondrial fitness. Together with its DDR function, ATR safeguards mitochondrial and genomic integrity under physiological and genotoxic conditions.
{"title":"DNA damage response regulator ATR licenses PINK1-mediated mitophagy.","authors":"Christian Marx, Xiaobing Qing, Yamin Gong, Joanna Kirkpatrick, Kanstantsin Siniuk, Galina V Beznoussenko, Gururaj Rao Kidiyoor, Murat Kirtay, Katrin Buder, Philipp Koch, Martin Westermann, Christopher Bruhn, Eric J Brown, Xingzhi Xu, Marco Foiani, Zhao-Qi Wang","doi":"10.1093/nar/gkaf178","DOIUrl":"10.1093/nar/gkaf178","url":null,"abstract":"<p><p>Defective DNA damage response (DDR) and mitochondrial dysfunction are a major etiology of tissue impairment and aging. Mitochondrial autophagy (mitophagy) is a mitochondrial quality control (MQC) mechanism to selectively eliminate dysfunctional mitochondria. ATR (ataxia-telangiectasia and Rad3-related) is a key DDR regulator playing a pivotal role in DNA replication stress response and genomic stability. Paradoxically, the human Seckel syndrome caused by ATR mutations exhibits premature aging and neuropathies, suggesting a role of ATR in nonreplicating tissues. Here, we report a previously unknown yet direct role of ATR at mitochondria. We find that ATR and PINK1 (PTEN-induced kinase 1) dock at the mitochondrial translocase TOM/TIM complex, where ATR interacts directly with and thereby stabilizes PINK1. ATR deletion silences mitophagy initiation thereby altering oxidative phosphorylation functionality resulting in reactive oxygen species overproduction that attack cytosolic macromolecules, in both cells and brain tissues, prior to nuclear DNA. This study discloses ATR as an integrated component of the PINK1-mediated MQC program to ensure mitochondrial fitness. Together with its DDR function, ATR safeguards mitochondrial and genomic integrity under physiological and genotoxic conditions.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"53 5","pages":""},"PeriodicalIF":16.6,"publicationDate":"2025-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11920799/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143658070","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}
Lena K Träger, Morris Degen, Joana Pereira, Janani Durairaj, Raphael Dias Teixeira, Sebastian Hiller, Nicolas Huguenin-Dezot
Protein-primed DNA replication is a unique mechanism, bioorthogonal to other known DNA replication modes. It relies on specialised single-stranded DNA (ssDNA)-binding proteins (SSBs) to stabilise ssDNA intermediates by unknown mechanisms. Here, we present the structural and biochemical characterisation of P12, an SSB from bacteriophage PRD1. High-resolution cryo-electron microscopy reveals that P12 forms a unique, cooperative filament along ssDNA. Each protomer binds the phosphate backbone of 6 nucleotides in a sequence-independent manner, protecting ssDNA from nuclease degradation. Filament formation is driven by an intrinsically disordered C-terminal tail, facilitating cooperative binding. We identify residues essential for ssDNA interaction and link the ssDNA-binding ability of P12 to toxicity in host cells. Bioinformatic analyses place the P12 fold as a distinct branch within the OB-like fold family. This work offers new insights into protein-primed DNA replication and lays a foundation for biotechnological applications.
{"title":"Structural basis for cooperative ssDNA binding by bacteriophage protein filament P12.","authors":"Lena K Träger, Morris Degen, Joana Pereira, Janani Durairaj, Raphael Dias Teixeira, Sebastian Hiller, Nicolas Huguenin-Dezot","doi":"10.1093/nar/gkaf132","DOIUrl":"10.1093/nar/gkaf132","url":null,"abstract":"<p><p>Protein-primed DNA replication is a unique mechanism, bioorthogonal to other known DNA replication modes. It relies on specialised single-stranded DNA (ssDNA)-binding proteins (SSBs) to stabilise ssDNA intermediates by unknown mechanisms. Here, we present the structural and biochemical characterisation of P12, an SSB from bacteriophage PRD1. High-resolution cryo-electron microscopy reveals that P12 forms a unique, cooperative filament along ssDNA. Each protomer binds the phosphate backbone of 6 nucleotides in a sequence-independent manner, protecting ssDNA from nuclease degradation. Filament formation is driven by an intrinsically disordered C-terminal tail, facilitating cooperative binding. We identify residues essential for ssDNA interaction and link the ssDNA-binding ability of P12 to toxicity in host cells. Bioinformatic analyses place the P12 fold as a distinct branch within the OB-like fold family. This work offers new insights into protein-primed DNA replication and lays a foundation for biotechnological applications.</p>","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"53 5","pages":""},"PeriodicalIF":16.6,"publicationDate":"2025-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11886824/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143573526","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}
{"title":"Correction 'LncPepAtlas: a comprehensive resource for exploring the translational landscape of long non-coding RNA'.","authors":"","doi":"10.1093/nar/gkaf186","DOIUrl":"10.1093/nar/gkaf186","url":null,"abstract":"","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"53 5","pages":""},"PeriodicalIF":16.6,"publicationDate":"2025-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11886797/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143573470","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}
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