The escalating costs and high failure rates have decelerated the pace of drug development, which amplifies the research interests in developing combinatorial/repurposed drugs and understanding off-target adverse drug reaction (ADR). In other words, it is demanded to delineate the molecular atlas and pharma-information for the combinatorial/repurposed drugs and off-target interactions. However, such invaluable data were inadequately covered by existing databases. In this study, a major update was thus conducted to the DrugMAP, which accumulated (a) 20831 combinatorial drugs and their interacting atlas involving 1583 pharmacologically important molecules; (b) 842 repurposed drugs and their interacting atlas with 795 molecules; (c) 3260 off-targets relevant to the ADRs of 2731 drugs and (d) various types of pharmaceutical information, including diverse ADMET properties, versatile diseases, and various ADRs/off-targets. With the growing demands for discovering combinatorial/repurposed therapies and the rapidly emerging interest in AI-based drug discovery, DrugMAP was highly expected to act as an indispensable supplement to existing databases facilitating drug discovery, which was accessible at: https://idrblab.org/drugmap/.
{"title":"DrugMAP 2.0: molecular atlas and pharma-information of all drugs","authors":"Fengcheng Li, Minjie Mou, Xiaoyi Li, Weize Xu, Jiayi Yin, Yang Zhang, Feng Zhu","doi":"10.1093/nar/gkae791","DOIUrl":"https://doi.org/10.1093/nar/gkae791","url":null,"abstract":"The escalating costs and high failure rates have decelerated the pace of drug development, which amplifies the research interests in developing combinatorial/repurposed drugs and understanding off-target adverse drug reaction (ADR). In other words, it is demanded to delineate the molecular atlas and pharma-information for the combinatorial/repurposed drugs and off-target interactions. However, such invaluable data were inadequately covered by existing databases. In this study, a major update was thus conducted to the DrugMAP, which accumulated (a) 20831 combinatorial drugs and their interacting atlas involving 1583 pharmacologically important molecules; (b) 842 repurposed drugs and their interacting atlas with 795 molecules; (c) 3260 off-targets relevant to the ADRs of 2731 drugs and (d) various types of pharmaceutical information, including diverse ADMET properties, versatile diseases, and various ADRs/off-targets. With the growing demands for discovering combinatorial/repurposed therapies and the rapidly emerging interest in AI-based drug discovery, DrugMAP was highly expected to act as an indispensable supplement to existing databases facilitating drug discovery, which was accessible at: https://idrblab.org/drugmap/.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":14.9,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142233348","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}
Chin-Dian Wei, Hao-Yen Chang, Chia-Hua Lu, Chih-Chun Chang, Asako Furukohri, Stephen Mwaniki, Akira Shinohara, Peter Chi, Hung-Wen Li
Interhomolog recombination in meiosis requires a meiosis-specific recombinase, Dmc1. In Saccharomyces cerevisiae, the Mei5–Sae3 complex facilitates the loading of Dmc1 onto the replication protein A (RPA)-coated single-stranded DNA (ssDNA) to form nucleoprotein filaments. In vivo, Dmc1 and Mei5–Sae3 are interdependent in their colocalization on the chromosomes. However, the mechanistic role of Mei5–Sae3 in mediating Dmc1 activity remains unclear. We used single-molecule fluorescence resonance energy transfer and colocalization single-molecule spectroscopy experiments to elucidate how Mei5–Sae3 stimulates Dmc1 assembly on ssDNA and RPA-coated ssDNA. We showed that Mei5–Sae3 stabilized Dmc1 nucleating clusters with two to three molecules on naked DNA by preferentially reducing Dmc1 dissociation rates. Mei5–Sae3 also stimulated Dmc1 assembly on RPA-coated DNA. Using green fluorescent protein-labeled RPA, we showed the coexistence of an intermediate with Dmc1 and RPA on ssDNA before RPA dissociation. Moreover, the displacement efficiency of RPA depended on Dmc1 concentration, and its dependence was positively correlated with the stability of Dmc1 clusters on short ssDNA. These findings suggest a molecular model that Mei5–Sae3 mediates Dmc1 binding on RPA-coated ssDNA by stabilizing Dmc1 nucleating clusters, thus altering RPA dynamics on DNA to promote RPA dissociation.
{"title":"Mei5–Sae3 stabilizes Dmc1 nucleating clusters for efficient Dmc1 assembly on RPA-coated single-stranded DNA","authors":"Chin-Dian Wei, Hao-Yen Chang, Chia-Hua Lu, Chih-Chun Chang, Asako Furukohri, Stephen Mwaniki, Akira Shinohara, Peter Chi, Hung-Wen Li","doi":"10.1093/nar/gkae780","DOIUrl":"https://doi.org/10.1093/nar/gkae780","url":null,"abstract":"Interhomolog recombination in meiosis requires a meiosis-specific recombinase, Dmc1. In Saccharomyces cerevisiae, the Mei5–Sae3 complex facilitates the loading of Dmc1 onto the replication protein A (RPA)-coated single-stranded DNA (ssDNA) to form nucleoprotein filaments. In vivo, Dmc1 and Mei5–Sae3 are interdependent in their colocalization on the chromosomes. However, the mechanistic role of Mei5–Sae3 in mediating Dmc1 activity remains unclear. We used single-molecule fluorescence resonance energy transfer and colocalization single-molecule spectroscopy experiments to elucidate how Mei5–Sae3 stimulates Dmc1 assembly on ssDNA and RPA-coated ssDNA. We showed that Mei5–Sae3 stabilized Dmc1 nucleating clusters with two to three molecules on naked DNA by preferentially reducing Dmc1 dissociation rates. Mei5–Sae3 also stimulated Dmc1 assembly on RPA-coated DNA. Using green fluorescent protein-labeled RPA, we showed the coexistence of an intermediate with Dmc1 and RPA on ssDNA before RPA dissociation. Moreover, the displacement efficiency of RPA depended on Dmc1 concentration, and its dependence was positively correlated with the stability of Dmc1 clusters on short ssDNA. These findings suggest a molecular model that Mei5–Sae3 mediates Dmc1 binding on RPA-coated ssDNA by stabilizing Dmc1 nucleating clusters, thus altering RPA dynamics on DNA to promote RPA dissociation.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":14.9,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142233442","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}
Transposable elements (TEs) are mobile DNA repeats known to shape the evolution of eukaryotic genomes. In complex organisms, they exhibit tissue-specific transcription. However, understanding their role in cellular diversity across most tissues remains a challenge, when employing single-cell RNA sequencing (scRNA-seq), due to their widespread presence and genetic similarity. To address this, we present IRescue (Interspersed Repeats single-cell quantifier), a software capable of estimating the expression of TE subfamilies at the single-cell level. IRescue incorporates a unique UMI deduplication algorithm to rectify sequencing errors and employs an Expectation-Maximization procedure to effectively redistribute the counts of multi-mapping reads. Our study showcases the precision of IRescue through analysis of both simulated and real single cell and nuclei RNA-seq data from human colorectal cancer, brain, skin aging, and PBMCs during SARS-CoV-2 infection and recovery. By linking the expression patterns of TE signatures to specific conditions and biological contexts, we unveil insights into their potential roles in cellular heterogeneity and disease progression.
可转座元件（Transposable elements，TEs）是一种可移动的 DNA 重复序列，已知可影响真核生物基因组的进化。在复杂生物体中，它们表现出组织特异性转录。然而，由于它们的广泛存在和遗传相似性，在使用单细胞 RNA 测序（scRNA-seq）时，了解它们在大多数组织的细胞多样性中的作用仍然是一个挑战。为了解决这个问题，我们推出了 IRescue（穿插重复序列单细胞定量器），这是一款能够在单细胞水平估算 TE 亚家族表达量的软件。IRescue 采用了独特的 UMI 重复数据删除算法来纠正测序错误，并采用期望最大化程序来有效地重新分配多映射读数的计数。我们的研究通过分析模拟和真实的单细胞和细胞核 RNA-seq 数据，展示了 IRescue 的精确性，这些数据来自 SARS-CoV-2 感染和恢复期间的人类结直肠癌、大脑、皮肤老化和 PBMCs。通过将 TE 特征的表达模式与特定条件和生物环境联系起来，我们揭示了它们在细胞异质性和疾病进展中的潜在作用。
{"title":"IRescue: uncertainty-aware quantification of transposable elements expression at single cell level","authors":"Benedetto Polimeni, Federica Marasca, Valeria Ranzani, Beatrice Bodega","doi":"10.1093/nar/gkae793","DOIUrl":"https://doi.org/10.1093/nar/gkae793","url":null,"abstract":"Transposable elements (TEs) are mobile DNA repeats known to shape the evolution of eukaryotic genomes. In complex organisms, they exhibit tissue-specific transcription. However, understanding their role in cellular diversity across most tissues remains a challenge, when employing single-cell RNA sequencing (scRNA-seq), due to their widespread presence and genetic similarity. To address this, we present IRescue (Interspersed Repeats single-cell quantifier), a software capable of estimating the expression of TE subfamilies at the single-cell level. IRescue incorporates a unique UMI deduplication algorithm to rectify sequencing errors and employs an Expectation-Maximization procedure to effectively redistribute the counts of multi-mapping reads. Our study showcases the precision of IRescue through analysis of both simulated and real single cell and nuclei RNA-seq data from human colorectal cancer, brain, skin aging, and PBMCs during SARS-CoV-2 infection and recovery. By linking the expression patterns of TE signatures to specific conditions and biological contexts, we unveil insights into their potential roles in cellular heterogeneity and disease progression.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":14.9,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142233350","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}
Anowarul Kabir, Manish Bhattarai, Selma Peterson, Yonatan Najman-Licht, Kim Ø Rasmussen, Amarda Shehu, Alan R Bishop, Boian Alexandrov, Anny Usheva
It was previously shown that DNA breathing, thermodynamic stability, as well as transcriptional activity and transcription factor (TF) bindings are functionally correlated. To ascertain the precise relationship between TF binding and DNA breathing, we developed the multi-modal deep learning model EPBDxDNABERT-2, which is based on the Extended Peyrard-Bishop-Dauxois (EPBD) nonlinear DNA dynamics model. To train our EPBDxDNABERT-2, we used chromatin immunoprecipitation sequencing (ChIP-Seq) data comprising 690 ChIP-seq experimental results encompassing 161 distinct TFs and 91 human cell types. EPBDxDNABERT-2 significantly improves the prediction of over 660 TF-DNA, with an increase in the area under the receiver operating characteristic (AUROC) metric of up to 9.6% when compared to the baseline model that does not leverage DNA biophysical properties. We expanded our analysis to in vitro high-throughput Systematic Evolution of Ligands by Exponential enrichment (HT-SELEX) dataset of 215 TFs from 27 families, comparing EPBD with established frameworks. The integration of the DNA breathing features with DNABERT-2 foundational model, greatly enhanced TF-binding predictions. Notably, EPBDxDNABERT-2, trained on a large-scale multi-species genomes, with a cross-attention mechanism, improved predictive power shedding light on the mechanisms underlying disease-related non-coding variants discovered in genome-wide association studies.
{"title":"DNA breathing integration with deep learning foundational model advances genome-wide binding prediction of human transcription factors","authors":"Anowarul Kabir, Manish Bhattarai, Selma Peterson, Yonatan Najman-Licht, Kim Ø Rasmussen, Amarda Shehu, Alan R Bishop, Boian Alexandrov, Anny Usheva","doi":"10.1093/nar/gkae783","DOIUrl":"https://doi.org/10.1093/nar/gkae783","url":null,"abstract":"It was previously shown that DNA breathing, thermodynamic stability, as well as transcriptional activity and transcription factor (TF) bindings are functionally correlated. To ascertain the precise relationship between TF binding and DNA breathing, we developed the multi-modal deep learning model EPBDxDNABERT-2, which is based on the Extended Peyrard-Bishop-Dauxois (EPBD) nonlinear DNA dynamics model. To train our EPBDxDNABERT-2, we used chromatin immunoprecipitation sequencing (ChIP-Seq) data comprising 690 ChIP-seq experimental results encompassing 161 distinct TFs and 91 human cell types. EPBDxDNABERT-2 significantly improves the prediction of over 660 TF-DNA, with an increase in the area under the receiver operating characteristic (AUROC) metric of up to 9.6% when compared to the baseline model that does not leverage DNA biophysical properties. We expanded our analysis to in vitro high-throughput Systematic Evolution of Ligands by Exponential enrichment (HT-SELEX) dataset of 215 TFs from 27 families, comparing EPBD with established frameworks. The integration of the DNA breathing features with DNABERT-2 foundational model, greatly enhanced TF-binding predictions. Notably, EPBDxDNABERT-2, trained on a large-scale multi-species genomes, with a cross-attention mechanism, improved predictive power shedding light on the mechanisms underlying disease-related non-coding variants discovered in genome-wide association studies.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":14.9,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142233464","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}
Tao Wang, Fan He, Ting He, Chen Lin, Xin Guan, Zhongjun Qin, Xiaoli Xue
Chromosomal DNA replication is a fundamental process of life, involving the assembly of complex machinery and dynamic regulation. In this study, we reconstructed a bacterial replication module (pRC) by artificially clustering 23 genes involved in DNA replication and sequentially deleting these genes from their naturally scattered loci on the chromosome of Escherichia coli. The integration of pRC into the chromosome, moving from positions farther away to close to the replication origin, leads to an enhanced efficiency in DNA synthesis, varying from lower to higher. Strains containing replication modules exhibited increased DNA replication by accelerating the replication fork movement and initiating chromosomal replication earlier in the replication cycle. The minimized module pRC16, containing only replisome and elongation encoding genes, exhibited chromosomal DNA replication efficiency comparable to that of pRC. The replication module demonstrated robust and rapid DNA replication, regardless of growth conditions. Moreover, the replication module is plug-and-play, and integrating it into Mb-sized extrachromosomal plasmids improves their genetic stability. Our findings indicate that DNA replication, being a fundamental life process, can be artificially reconstructed into replication functional modules. This suggests potential applications in DNA replication and the construction of synthetic modular genomes.
染色体 DNA 复制是生命的基本过程，涉及复杂机器的组装和动态调控。在这项研究中，我们人为地将 23 个参与 DNA 复制的基因聚集在一起，并从大肠杆菌染色体上自然分散的基因位点上依次删除这些基因，从而重建了细菌复制模块（pRC）。pRC 集成到染色体上后，从离复制源较远的位置移到离复制源较近的位置，从而提高了 DNA 合成的效率，从较低到较高不等。含有复制模块的菌株通过加速复制叉的移动，在复制周期的早期启动染色体复制，从而提高了 DNA 复制的效率。最小化模块 pRC16 只含有复制体和延伸编码基因，其染色体 DNA 复制效率与 pRC 相当。无论生长条件如何，该复制模块都表现出强大而快速的 DNA 复制能力。此外，复制模块是即插即用的，将其整合到 Mb 大小的染色体外质粒中可提高其遗传稳定性。我们的研究结果表明，DNA 复制作为一个基本的生命过程，可以被人为地重建为复制功能模块。这为 DNA 复制和合成模块基因组的构建提供了潜在的应用前景。
{"title":"Reconstruction of a robust bacterial replication module","authors":"Tao Wang, Fan He, Ting He, Chen Lin, Xin Guan, Zhongjun Qin, Xiaoli Xue","doi":"10.1093/nar/gkae786","DOIUrl":"https://doi.org/10.1093/nar/gkae786","url":null,"abstract":"Chromosomal DNA replication is a fundamental process of life, involving the assembly of complex machinery and dynamic regulation. In this study, we reconstructed a bacterial replication module (pRC) by artificially clustering 23 genes involved in DNA replication and sequentially deleting these genes from their naturally scattered loci on the chromosome of Escherichia coli. The integration of pRC into the chromosome, moving from positions farther away to close to the replication origin, leads to an enhanced efficiency in DNA synthesis, varying from lower to higher. Strains containing replication modules exhibited increased DNA replication by accelerating the replication fork movement and initiating chromosomal replication earlier in the replication cycle. The minimized module pRC16, containing only replisome and elongation encoding genes, exhibited chromosomal DNA replication efficiency comparable to that of pRC. The replication module demonstrated robust and rapid DNA replication, regardless of growth conditions. Moreover, the replication module is plug-and-play, and integrating it into Mb-sized extrachromosomal plasmids improves their genetic stability. Our findings indicate that DNA replication, being a fundamental life process, can be artificially reconstructed into replication functional modules. This suggests potential applications in DNA replication and the construction of synthetic modular genomes.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":14.9,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142233307","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}
Genís Campoy-Campos, Julia Solana-Balaguer, Anna Guisado-Corcoll, Almudena Chicote-González, Pol Garcia-Segura, Leticia Pérez-Sisqués, Adrian Gabriel Torres, Mercè Canal, Laura Molina-Porcel, Joaquín Fernández-Irigoyen, Enrique Santamaria, Lluís Ribas de Pouplana, Jordi Alberch, Eulàlia Martí, Albert Giralt, Esther Pérez-Navarro, Cristina Malagelada
RTP801/REDD1 is a stress-responsive protein overexpressed in neurodegenerative diseases such as Alzheimer’s disease (AD) that contributes to cognitive deficits and neuroinflammation. Here, we found that RTP801 interacts with HSPC117, DDX1 and CGI-99, three members of the tRNA ligase complex (tRNA-LC), which ligates the excised exons of intron-containing tRNAs and the mRNA exons of the transcription factor XBP1 during the unfolded protein response (UPR). We also found that RTP801 modulates the mRNA ligase activity of the complex in vitro since RTP801 knockdown promoted XBP1 splicing and the expression of its transcriptional target, SEC24D. Conversely, RTP801 overexpression inhibited the splicing of XBP1. Similarly, in human AD postmortem hippocampal samples, where RTP801 is upregulated, we found that XBP1 splicing was dramatically decreased. In the 5xFAD mouse model of AD, silencing RTP801 expression in hippocampal neurons promoted Xbp1 splicing and prevented the accumulation of intron-containing pre-tRNAs. Finally, the tRNA-enriched fraction obtained from 5xFAD mice promoted abnormal dendritic arborization in cultured hippocampal neurons, and RTP801 silencing in the source neurons prevented this phenotype. Altogether, these results show that elevated RTP801 impairs RNA processing in vitro and in vivo in the context of AD and suggest that RTP801 inhibition could be a promising therapeutic approach.
{"title":"RTP801 interacts with the tRNA ligase complex and dysregulates its RNA ligase activity in Alzheimer’s disease","authors":"Genís Campoy-Campos, Julia Solana-Balaguer, Anna Guisado-Corcoll, Almudena Chicote-González, Pol Garcia-Segura, Leticia Pérez-Sisqués, Adrian Gabriel Torres, Mercè Canal, Laura Molina-Porcel, Joaquín Fernández-Irigoyen, Enrique Santamaria, Lluís Ribas de Pouplana, Jordi Alberch, Eulàlia Martí, Albert Giralt, Esther Pérez-Navarro, Cristina Malagelada","doi":"10.1093/nar/gkae776","DOIUrl":"https://doi.org/10.1093/nar/gkae776","url":null,"abstract":"RTP801/REDD1 is a stress-responsive protein overexpressed in neurodegenerative diseases such as Alzheimer’s disease (AD) that contributes to cognitive deficits and neuroinflammation. Here, we found that RTP801 interacts with HSPC117, DDX1 and CGI-99, three members of the tRNA ligase complex (tRNA-LC), which ligates the excised exons of intron-containing tRNAs and the mRNA exons of the transcription factor XBP1 during the unfolded protein response (UPR). We also found that RTP801 modulates the mRNA ligase activity of the complex in vitro since RTP801 knockdown promoted XBP1 splicing and the expression of its transcriptional target, SEC24D. Conversely, RTP801 overexpression inhibited the splicing of XBP1. Similarly, in human AD postmortem hippocampal samples, where RTP801 is upregulated, we found that XBP1 splicing was dramatically decreased. In the 5xFAD mouse model of AD, silencing RTP801 expression in hippocampal neurons promoted Xbp1 splicing and prevented the accumulation of intron-containing pre-tRNAs. Finally, the tRNA-enriched fraction obtained from 5xFAD mice promoted abnormal dendritic arborization in cultured hippocampal neurons, and RTP801 silencing in the source neurons prevented this phenotype. Altogether, these results show that elevated RTP801 impairs RNA processing in vitro and in vivo in the context of AD and suggest that RTP801 inhibition could be a promising therapeutic approach.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":14.9,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142231564","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}
Perturb-Seq combines CRISPR (clustered regularly interspaced short palindromic repeats)-based genetic screens with single-cell RNA sequencing readouts for high-content phenotypic screens. Despite the rapid accumulation of Perturb-Seq datasets, there remains a lack of a user-friendly platform for their efficient reuse. Here, we developed PerturbDB (http://research.gzsys.org.cn/perturbdb), a platform to help users unveil gene functions using Perturb-Seq datasets. PerturbDB hosts 66 Perturb-Seq datasets, which encompass 4 518 521 single-cell transcriptomes derived from the knockdown of 10 194 genes across 19 different cell lines. All datasets were uniformly processed using the Mixscape algorithm. Genes were clustered by their perturbed transcriptomic phenotypes derived from Perturb-Seq data, resulting in 421 gene clusters, 157 of which were stable across different cellular contexts. Through integrating chemically perturbed transcriptomes with Perturb-Seq data, we identified 552 potential inhibitors targeting 1409 genes, including an mammalian target of rapamycin (mTOR) signaling inhibitor, retinol, which was experimentally verified. Moreover, we developed a ‘Cancer’ module to facilitate the understanding of the regulatory role of genes in cancer using Perturb-Seq data. An interactive web interface has also been developed, enabling users to visualize, analyze and download all the comprehensive datasets available in PerturbDB. PerturbDB will greatly drive gene functional studies and enhance our understanding of the regulatory roles of genes in diseases such as cancer.
{"title":"PerturbDB for unraveling gene functions and regulatory networks","authors":"Bing Yang, Man Zhang, Yanmei Shi, Bing-Qi Zheng, Chuanping Shi, Daning Lu, Zhi-Zhi Yang, Yi-Ming Dong, Liwen Zhu, Xingyu Ma, Jingyuan Zhang, Jiehua He, Yin Zhang, Kaishun Hu, Haoming Lin, Jian-You Liao, Dong Yin","doi":"10.1093/nar/gkae777","DOIUrl":"https://doi.org/10.1093/nar/gkae777","url":null,"abstract":"Perturb-Seq combines CRISPR (clustered regularly interspaced short palindromic repeats)-based genetic screens with single-cell RNA sequencing readouts for high-content phenotypic screens. Despite the rapid accumulation of Perturb-Seq datasets, there remains a lack of a user-friendly platform for their efficient reuse. Here, we developed PerturbDB (http://research.gzsys.org.cn/perturbdb), a platform to help users unveil gene functions using Perturb-Seq datasets. PerturbDB hosts 66 Perturb-Seq datasets, which encompass 4 518 521 single-cell transcriptomes derived from the knockdown of 10 194 genes across 19 different cell lines. All datasets were uniformly processed using the Mixscape algorithm. Genes were clustered by their perturbed transcriptomic phenotypes derived from Perturb-Seq data, resulting in 421 gene clusters, 157 of which were stable across different cellular contexts. Through integrating chemically perturbed transcriptomes with Perturb-Seq data, we identified 552 potential inhibitors targeting 1409 genes, including an mammalian target of rapamycin (mTOR) signaling inhibitor, retinol, which was experimentally verified. Moreover, we developed a ‘Cancer’ module to facilitate the understanding of the regulatory role of genes in cancer using Perturb-Seq data. An interactive web interface has also been developed, enabling users to visualize, analyze and download all the comprehensive datasets available in PerturbDB. PerturbDB will greatly drive gene functional studies and enhance our understanding of the regulatory roles of genes in diseases such as cancer.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":14.9,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142231565","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 efficient refactoring of natural product biosynthetic gene clusters (BGCs) for activating silent BGCs is a central challenge for the discovery of new bioactive natural products. Herein, we have developed a simple and robust CRISETR (CRISPR/Cas9 and RecET-mediated Refactoring) technique, combining clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 and RecET, for the multiplexed refactoring of natural product BGCs. By this approach, natural product BGCs can be refactored through the synergistic interaction between RecET-mediated efficient homologous recombination and the CRISPR/Cas9 system. We first performed a proof-of-concept validation of the ability of CRISETR, and CRISETR can achieve simultaneous replacement of four promoter sites and marker-free replacement of single promoter site in natural product BGCs. Subsequently, we applied CRISETR to the promoter engineering of the 74-kb daptomycin BGC containing a large number of direct repeat sequences for enhancing the heterologous production of daptomycin. We used combinatorial design to build multiple refactored daptomycin BGCs with diverse combinations of promoters different in transcriptional strengths, and the yield of daptomycin was improved 20.4-fold in heterologous host Streptomyces coelicolor A3(2). In general, CRISETR exhibits enhanced tolerance to repetitive sequences within gene clusters, enabling efficient refactoring of diverse and complex BGCs, which would greatly accelerate discovery of novel bioactive metabolites present in microorganism.
{"title":"CRISETR: an efficient technology for multiplexed refactoring of biosynthetic gene clusters.","authors":"Fuqiang He,Xinpeng Liu,Min Tang,Haiyi Wang,Yun Wu,Shufang Liang","doi":"10.1093/nar/gkae781","DOIUrl":"https://doi.org/10.1093/nar/gkae781","url":null,"abstract":"The efficient refactoring of natural product biosynthetic gene clusters (BGCs) for activating silent BGCs is a central challenge for the discovery of new bioactive natural products. Herein, we have developed a simple and robust CRISETR (CRISPR/Cas9 and RecET-mediated Refactoring) technique, combining clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 and RecET, for the multiplexed refactoring of natural product BGCs. By this approach, natural product BGCs can be refactored through the synergistic interaction between RecET-mediated efficient homologous recombination and the CRISPR/Cas9 system. We first performed a proof-of-concept validation of the ability of CRISETR, and CRISETR can achieve simultaneous replacement of four promoter sites and marker-free replacement of single promoter site in natural product BGCs. Subsequently, we applied CRISETR to the promoter engineering of the 74-kb daptomycin BGC containing a large number of direct repeat sequences for enhancing the heterologous production of daptomycin. We used combinatorial design to build multiple refactored daptomycin BGCs with diverse combinations of promoters different in transcriptional strengths, and the yield of daptomycin was improved 20.4-fold in heterologous host Streptomyces coelicolor A3(2). In general, CRISETR exhibits enhanced tolerance to repetitive sequences within gene clusters, enabling efficient refactoring of diverse and complex BGCs, which would greatly accelerate discovery of novel bioactive metabolites present in microorganism.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":14.9,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142233480","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-Cas12a, an RNA-guided nuclease, has been repurposed for genome editing and molecular diagnostics due to its capability of cis-cleavage on target DNA and trans-cleavage on non-target single-strand DNA (ssDNA). However, the mechanisms underlying the activation of trans-cleavage activity of Cas12a, particularly in the context of split DNA activators, remain poorly understood. We elucidate the synergistic effect of these activators and introduce the concepts of induced targeting effect and exon-unwinding to describe the phenomenon. We demonstrate that upon binding of split DNA activators adjacent to the Protospacer Adjacent Motif (PAM) to the Cas12a ribonucleoprotein (Cas12a-RNP), a ternary complex form that can capture and interact with distal split DNA activators to achieve synergistic effects. Notably, if the distal activator is double-strand DNA (dsDNA), the complex initiates exon-unwinding, facilitating the RNA-guide sequence's access. Our findings provide a mechanistic insight into action of Cas12a and propose a model that could significantly advance our understanding of its function.
CRISPR-Cas12a是一种RNA引导的核酸酶，由于其能够顺式裂解靶DNA和反式裂解非靶单链DNA（ssDNA），已被重新用于基因组编辑和分子诊断。然而，人们对 Cas12a 反式裂解活性的激活机制，尤其是在 DNA 激活剂分裂的情况下，仍然知之甚少。我们阐明了这些激活剂的协同效应，并引入了诱导靶向效应和外显子解旋的概念来描述这一现象。我们证明，与原位相邻结构（PAM）相邻的分裂 DNA 激活剂与 Cas12a 核糖核蛋白（Cas12a-RNP）结合后，形成的三元复合物可捕获远端分裂 DNA 激活剂并与之相互作用，从而实现协同效应。值得注意的是，如果远端激活剂是双链 DNA（dsDNA），该复合物就会启动外显子解旋，从而促进 RNA 引导序列的进入。我们的研究结果从机理上揭示了 Cas12a 的作用，并提出了一个模型，该模型可极大地促进我们对 Cas12a 功能的理解。
{"title":"Synergistic effect of split DNA activators of Cas12a with exon-unwinding and induced targeting effect.","authors":"Shen Huang,Yongliang Lou,Laibao Zheng","doi":"10.1093/nar/gkae766","DOIUrl":"https://doi.org/10.1093/nar/gkae766","url":null,"abstract":"CRISPR-Cas12a, an RNA-guided nuclease, has been repurposed for genome editing and molecular diagnostics due to its capability of cis-cleavage on target DNA and trans-cleavage on non-target single-strand DNA (ssDNA). However, the mechanisms underlying the activation of trans-cleavage activity of Cas12a, particularly in the context of split DNA activators, remain poorly understood. We elucidate the synergistic effect of these activators and introduce the concepts of induced targeting effect and exon-unwinding to describe the phenomenon. We demonstrate that upon binding of split DNA activators adjacent to the Protospacer Adjacent Motif (PAM) to the Cas12a ribonucleoprotein (Cas12a-RNP), a ternary complex form that can capture and interact with distal split DNA activators to achieve synergistic effects. Notably, if the distal activator is double-strand DNA (dsDNA), the complex initiates exon-unwinding, facilitating the RNA-guide sequence's access. Our findings provide a mechanistic insight into action of Cas12a and propose a model that could significantly advance our understanding of its function.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":14.9,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142170877","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}
Sooin Jang,Gregory J Bedwell,Satya P Singh,Hyun Jae Yu,Bjarki Arnarson,Parmit K Singh,Rajalingam Radhakrishnan,AidanDarian W Douglas,Zachary M Ingram,Christian Freniere,Onno Akkermans,Stefan G Sarafianos,Zandrea Ambrose,Yong Xiong,Praju V Anekal,Paula Montero Llopis,Vineet N KewalRamani,Ashwanth C Francis,Alan N Engelman
HIV-1 integration favors nuclear speckle (NS)-proximal chromatin and viral infection induces the formation of capsid-dependent CPSF6 condensates that colocalize with nuclear speckles (NSs). Although CPSF6 displays liquid-liquid phase separation (LLPS) activity in vitro, the contributions of its different intrinsically disordered regions, which includes a central prion-like domain (PrLD) with capsid binding FG motif and C-terminal mixed-charge domain (MCD), to LLPS activity and to HIV-1 infection remain unclear. Herein, we determined that the PrLD and MCD both contribute to CPSF6 LLPS activity in vitro. Akin to FG mutant CPSF6, infection of cells expressing MCD-deleted CPSF6 uncharacteristically arrested at the nuclear rim. While heterologous MCDs effectively substituted for CPSF6 MCD function during HIV-1 infection, Arg-Ser domains from related SR proteins were largely ineffective. While MCD-deleted and wildtype CPSF6 proteins displayed similar capsid binding affinities, the MCD imparted LLPS-dependent higher-order binding and co-aggregation with capsids in vitro and in cellulo. NS depletion reduced CPSF6 puncta formation without significantly affecting integration into NS-proximal chromatin, and appending the MCD onto a heterologous capsid binding protein partially restored virus nuclear penetration and integration targeting in CPSF6 knockout cells. We conclude that MCD-dependent CPSF6 condensation with capsids underlies post-nuclear incursion for viral DNA integration and HIV-1 pathogenesis.
{"title":"HIV-1 usurps mixed-charge domain-dependent CPSF6 phase separation for higher-order capsid binding, nuclear entry and viral DNA integration.","authors":"Sooin Jang,Gregory J Bedwell,Satya P Singh,Hyun Jae Yu,Bjarki Arnarson,Parmit K Singh,Rajalingam Radhakrishnan,AidanDarian W Douglas,Zachary M Ingram,Christian Freniere,Onno Akkermans,Stefan G Sarafianos,Zandrea Ambrose,Yong Xiong,Praju V Anekal,Paula Montero Llopis,Vineet N KewalRamani,Ashwanth C Francis,Alan N Engelman","doi":"10.1093/nar/gkae769","DOIUrl":"https://doi.org/10.1093/nar/gkae769","url":null,"abstract":"HIV-1 integration favors nuclear speckle (NS)-proximal chromatin and viral infection induces the formation of capsid-dependent CPSF6 condensates that colocalize with nuclear speckles (NSs). Although CPSF6 displays liquid-liquid phase separation (LLPS) activity in vitro, the contributions of its different intrinsically disordered regions, which includes a central prion-like domain (PrLD) with capsid binding FG motif and C-terminal mixed-charge domain (MCD), to LLPS activity and to HIV-1 infection remain unclear. Herein, we determined that the PrLD and MCD both contribute to CPSF6 LLPS activity in vitro. Akin to FG mutant CPSF6, infection of cells expressing MCD-deleted CPSF6 uncharacteristically arrested at the nuclear rim. While heterologous MCDs effectively substituted for CPSF6 MCD function during HIV-1 infection, Arg-Ser domains from related SR proteins were largely ineffective. While MCD-deleted and wildtype CPSF6 proteins displayed similar capsid binding affinities, the MCD imparted LLPS-dependent higher-order binding and co-aggregation with capsids in vitro and in cellulo. NS depletion reduced CPSF6 puncta formation without significantly affecting integration into NS-proximal chromatin, and appending the MCD onto a heterologous capsid binding protein partially restored virus nuclear penetration and integration targeting in CPSF6 knockout cells. We conclude that MCD-dependent CPSF6 condensation with capsids underlies post-nuclear incursion for viral DNA integration and HIV-1 pathogenesis.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":null,"pages":null},"PeriodicalIF":14.9,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142170878","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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