[Objective] The aim was to establish the BHK-21 stable cell lines expressing T7 RNAP and GFP.[Method]T7 RNAP gene was amplified from E.coli BL21（DE3）and inserted into FG12 vector.The lenti-virus recombinant plasmid ...[Objective] The aim was to establish the BHK-21 stable cell lines expressing T7 RNAP and GFP.[Method]T7 RNAP gene was amplified from E.coli BL21（DE3）and inserted into FG12 vector.The lenti-virus recombinant plasmid FG12-T7 RNAP plasmid was obtained via identification with double enzymes digestion and gene sequencing.The transient expressed T7 RNAP protein was determined by WB in 293T cells transfected with FG12-T7 RNAP plasmid.The recombinant FG12-RNAP lenti-virus was packaged up by transfecting the 293 cells with the recombinant vector FG12-RNAP and the helper plasmids via lipofectamine-2000,which was then used to infect BHK-21 cells.The positive cell clones were obtained after continuous screening by GFP.The expression of T7 RNAP gene was confirmed by Western blot.[Result]The cell line stably expressing the T7 RNAP gene was established by Western blot.[Conclusion]T7 RNAP gene could be stably expressed in eukaryotic cells and it provided a good platform to rescue RNA virus in vivo.展开更多
新兴的染色质靶向切割和标签化(clevage under target and tagment,CUT&Tag)技术利用转座酶在目标蛋白结合的DNA附近进行切割并对切割下的DNA片段进行标签化,通过后续的二代测序可以快速鉴定蛋白质-DNA相互作用,极大的简化了染色质...新兴的染色质靶向切割和标签化(clevage under target and tagment,CUT&Tag)技术利用转座酶在目标蛋白结合的DNA附近进行切割并对切割下的DNA片段进行标签化,通过后续的二代测序可以快速鉴定蛋白质-DNA相互作用,极大的简化了染色质免疫共沉淀测序(chromatin immunoprecipitation sequencing,ChIP-seq)的实验过程。CUT&Tag中转座酶完成标签化后需要DNA回收或其他后处理才能进行建库PCR,不同的回收方法对CUT&Tag结果有着显著的影响。通过建立生物素化转座体-链霉亲和素磁珠体系(streptavidin beads recovery CUT&Tag,srCUT&Tag),可以快速便捷地完成CUT&Tag的产物回收。本文在K562细胞中展开H3K4me3、RNA聚合酶Ⅱ(RNA polymeraseⅡ,RNAPⅡ)、转录因子CTCF和HMGA1的CUT&Tag实验,并利用现有的乙醇沉淀、片段分选(solid-phase reversible immobilization,SPRI)磁珠回收和直接PCR法,以及本研究建立的srCUT&Tag方法对产物进行回收。结果表明,从整体上看,SPRI磁珠回收和srCUT&Tag方法着较高的回收效率,而乙醇沉淀法则回收效率低下。在全部4种CUT&Tag产物回收过程中,SPRI磁珠回收均会损失大部分小于150 bp的产物片段。在CTCF和HMGA1 CUT&Tag产物的回收中,直接PCR法则损失了大部分大于300 bp的片段并与其他回收方法的结果有较大的差别。因此,srCUT&Tag能够比其他三种回收方法提供更多更完整的测序信息。综上所述,新建立srCUT&Tag回收方法相比现有的CUT&Tag产物回收方法能提高建库效率并得到更好的数据质量,为表观遗传学研究提供了更好的技术选择。展开更多
生物体在正常生命过程中面临内/外因来源的DNA损伤,DNA损伤不仅影响基因正确复制,也阻碍其正常转录.为避免DNA损伤带来的灾难性后果,生物体进化出一整套修复机制,以保证复制和转录的正确性、基因组的完整性和遗传的稳定性.本文重点综述...生物体在正常生命过程中面临内/外因来源的DNA损伤,DNA损伤不仅影响基因正确复制,也阻碍其正常转录.为避免DNA损伤带来的灾难性后果,生物体进化出一整套修复机制,以保证复制和转录的正确性、基因组的完整性和遗传的稳定性.本文重点综述了RNA聚合酶监视(RNA polymerase-surveilled,RNAP-S)的DNA修复机制.首先从RNA聚合酶(RNA polymerase,RNAP)的结构出发介绍了RNAP对DNA损伤的感知机制;其次讨论了滞留RNAP的回溯、与其模板DNA的解离以及后续修复机制的启动,真核细胞科凯恩综合征B蛋白(Cockayne syndrome protein B,CSB)及其泛素化和8-氧代鸟嘌呤DNA糖基化酶1(8-oxoguanine DNA glycosylase1,OGG1)介导的RNAP-S修复;最后探讨了RNAP-S损伤修复的生物学意义并展望其前景.展开更多
Transcription-coupled nucleotide excision repair(TC-NER)is initiated by the stalling of elongating RNA polymerase II(RNAPIIo)at DNA lesions.The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily ...Transcription-coupled nucleotide excision repair(TC-NER)is initiated by the stalling of elongating RNA polymerase II(RNAPIIo)at DNA lesions.The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event,but its function in mammals is unknown.Here,we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268,which regulates transcription recovery and DNA damage resistance.Mechanistically,RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination.We developed a strand-specific ChIP-seq method,which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions.Finally,RPB1-K1268R knockin mice displayed a short life-span,premature aging,and neurodegeneration.Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and,in parallel,the processing of DNA damage-stalled RNAPIIo,which together prevent prolonged transcription arrest and protect against neurodegeneration.展开更多
Objective:To investigate the role of RPRD1B in the progression of diffuse large B-cell lymphoma(DLBCL)and its potential as a therapeutic target.Methods:This study analyzed RPRD1B expression in DLBCL and normal tissues...Objective:To investigate the role of RPRD1B in the progression of diffuse large B-cell lymphoma(DLBCL)and its potential as a therapeutic target.Methods:This study analyzed RPRD1B expression in DLBCL and normal tissues using public databases and assessed its prognostic impact through survival analysis.In vitro and in vivo experiments were conducted to explore the mechanisms by which RPRD1B influences tumor growth and apoptosis.Results:RPRD1B expression was significantly elevated in DLBCL compared to normal tissues and was associated with poor prognosis.In vitro and in vivo experiments demonstrated that RPRD1B promoted lymphoma cell proliferation and inhibited apoptosis through the NF-κB signaling pathway.Conclusions:RPRD1B plays a critical role in the progression of DLBCL by modulating apoptosis and cellular proliferation.Targeting RPRD1B may offer a novel therapeutic strategy for DLBCL,suggesting its potential as a prognostic marker and therapeutic target in hematological malignancies.展开更多
RNA polymerases(RNAPs)are the enzymes responsible for transcribing genetic information from DNA into RNA.Gene transcription can be divided into three stages:initiation,elongation,and termination(Cramer et al.,2008;Roe...RNA polymerases(RNAPs)are the enzymes responsible for transcribing genetic information from DNA into RNA.Gene transcription can be divided into three stages:initiation,elongation,and termination(Cramer et al.,2008;Roeder,2019).Transcription initiation is the first and most-regulated stage.During gene transcription,RNAPs.展开更多
T7 RNA polymerase(T7 RNAP)-catalyzed in vitro transcription(IVT)is the gold standard manufacturing process for large-scale production of therapeutic mRNA molecules.However,the undesired catalytic activity of T7 RNAP c...T7 RNA polymerase(T7 RNAP)-catalyzed in vitro transcription(IVT)is the gold standard manufacturing process for large-scale production of therapeutic mRNA molecules.However,the undesired catalytic activity of T7 RNAP concomitantly generates deleterious impurities,such as double-stranded RNAs,that can exacerbate the downstream purification burden and engender safety concerns.The aim of this study was to engineer T7 RNAP thermostability for high-temperature IVT to reduce the dsRNA.The web server PROSS was utilized to predict thermostable mutation sites from the intermediate and elongation structure of T7 RNAP.Through systematic evaluation of individual mutation sites followed by greedy-accumulation optimization of multi-site combinatorial mutants,we successfully overcame the inherent activity-stability trade-off during the evolution and obtained a thermostable variant,M10(Tm:49.5℃),which exhibits robust catalytic activity at elevated temperatures and significantly reduced dsRNA byproduct formation.Structural analysis using homology modelling and molecular dynamics(MD)simulation revealed that the accumulated mutations increased the local rigidity of T7 RNAP with a compacted conformation,enhanced the helical propensity,and allowed the formation of new salt bridges.The enhanced mutant has the potential to act as an effective biocatalyst for high-temperature IVT,adding in high-quality mRNA production,which is a prerequisite for optimizing the downstream purification processes and improving the clinical viability of such therapeutic agents.展开更多
The presence of actin in the nucleus as well as its functions in various nuclear processes has been made clear in the past few years. Actin is known to be a part of chromatin-remodeling complexes BAF, which are requir...The presence of actin in the nucleus as well as its functions in various nuclear processes has been made clear in the past few years. Actin is known to be a part of chromatin-remodeling complexes BAF, which are required for maximal ATPase activity of the Brg1 component of the BAF complex. Moreover, the essential roles of actin in transcription mediated by RNA polymerases Ⅰ,Ⅱ and Ⅲ have been demonstrated recently. On the other hand, a myosin Ⅰ isoform, which contains a unique NH2-terminal extension for nucleus localization, has been specifically localized in nucleus. As is well known, myosin Ⅰ is an actin-binding protein and plays an important role in various cellular activities. Though actin and nuclear myosin Ⅰ (NM Ⅰ) have been implicated to play distinct roles in gene expression, there has been no evidence for the actin-myosin interaction that might be involved in gene transcription mediated by RNA polymerase Ⅱ (RNAP Ⅱ). Here we show evidence that both actin and NM Ⅰ are associated with RNAP Ⅱ in nucleus by using co-localization and co-IP assays, and they may act together on gene transcription. The antibodies against β-actin or NM Ⅰ can block RNA synthesis in a eukaryotic in vitro transcription system with template DNA comprising the promoter and the coding region of human autocrine motility factor receptor (hAMFR) gene; the antibodies pre-adsorbed with purified actin and NM Ⅰ have no effect in transcriptional inhibition, indicating that the inhibition of transcription by anti-actin and anti-NM I is specific. These results suggest a direct involvement of actin-myosin complexes in regulating transcription. It also implicates that actin and NM Ⅰ may co-exist in a same complex with RNAP Ⅱ and the interaction of RNAP Ⅱ with actin and NM Ⅰ functions in the RNAP Ⅱ-mediated transcription.展开更多
基金Supported by National Transgenic Major Program of China(2009ZX08007-006B)the National Natural Science Foundation of China(31072160)+2 种基金Science and Technique Foundation of Shandong Province(2009GG20002032)Natural Science Foundation of Shandong Province(Y2008D20)an Open Issue of State Key Laboratory of Veterinary Biotechnology Fund(SKLVBF200806)~~
文摘[Objective] The aim was to establish the BHK-21 stable cell lines expressing T7 RNAP and GFP.[Method]T7 RNAP gene was amplified from E.coli BL21（DE3）and inserted into FG12 vector.The lenti-virus recombinant plasmid FG12-T7 RNAP plasmid was obtained via identification with double enzymes digestion and gene sequencing.The transient expressed T7 RNAP protein was determined by WB in 293T cells transfected with FG12-T7 RNAP plasmid.The recombinant FG12-RNAP lenti-virus was packaged up by transfecting the 293 cells with the recombinant vector FG12-RNAP and the helper plasmids via lipofectamine-2000,which was then used to infect BHK-21 cells.The positive cell clones were obtained after continuous screening by GFP.The expression of T7 RNAP gene was confirmed by Western blot.[Result]The cell line stably expressing the T7 RNAP gene was established by Western blot.[Conclusion]T7 RNAP gene could be stably expressed in eukaryotic cells and it provided a good platform to rescue RNA virus in vivo.
文摘生物体在正常生命过程中面临内/外因来源的DNA损伤,DNA损伤不仅影响基因正确复制,也阻碍其正常转录.为避免DNA损伤带来的灾难性后果,生物体进化出一整套修复机制,以保证复制和转录的正确性、基因组的完整性和遗传的稳定性.本文重点综述了RNA聚合酶监视(RNA polymerase-surveilled,RNAP-S)的DNA修复机制.首先从RNA聚合酶(RNA polymerase,RNAP)的结构出发介绍了RNAP对DNA损伤的感知机制;其次讨论了滞留RNAP的回溯、与其模板DNA的解离以及后续修复机制的启动,真核细胞科凯恩综合征B蛋白(Cockayne syndrome protein B,CSB)及其泛素化和8-氧代鸟嘌呤DNA糖基化酶1(8-oxoguanine DNA glycosylase1,OGG1)介导的RNAP-S修复;最后探讨了RNAP-S损伤修复的生物学意义并展望其前景.
文摘Transcription-coupled nucleotide excision repair(TC-NER)is initiated by the stalling of elongating RNA polymerase II(RNAPIIo)at DNA lesions.The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event,but its function in mammals is unknown.Here,we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268,which regulates transcription recovery and DNA damage resistance.Mechanistically,RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination.We developed a strand-specific ChIP-seq method,which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions.Finally,RPB1-K1268R knockin mice displayed a short life-span,premature aging,and neurodegeneration.Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and,in parallel,the processing of DNA damage-stalled RNAPIIo,which together prevent prolonged transcription arrest and protect against neurodegeneration.
基金funded by Hainan Provincial Natural Science Foundation of China(820QN401,822QN468)Science and Technology Special Fund of Hainan Province,China,(ZDYF2024SHFZ114)+1 种基金Health Science and Technology Innovation Joint Project of Hainan Province,China(WSJK2024MS231)Hainan Province Clinical Medical Center Construction(Project[2022]276).
文摘Objective:To investigate the role of RPRD1B in the progression of diffuse large B-cell lymphoma(DLBCL)and its potential as a therapeutic target.Methods:This study analyzed RPRD1B expression in DLBCL and normal tissues using public databases and assessed its prognostic impact through survival analysis.In vitro and in vivo experiments were conducted to explore the mechanisms by which RPRD1B influences tumor growth and apoptosis.Results:RPRD1B expression was significantly elevated in DLBCL compared to normal tissues and was associated with poor prognosis.In vitro and in vivo experiments demonstrated that RPRD1B promoted lymphoma cell proliferation and inhibited apoptosis through the NF-κB signaling pathway.Conclusions:RPRD1B plays a critical role in the progression of DLBCL by modulating apoptosis and cellular proliferation.Targeting RPRD1B may offer a novel therapeutic strategy for DLBCL,suggesting its potential as a prognostic marker and therapeutic target in hematological malignancies.
文摘RNA polymerases(RNAPs)are the enzymes responsible for transcribing genetic information from DNA into RNA.Gene transcription can be divided into three stages:initiation,elongation,and termination(Cramer et al.,2008;Roeder,2019).Transcription initiation is the first and most-regulated stage.During gene transcription,RNAPs.
基金supported by the Social Development Key Project of Jiangsu Province(BE2023682)supported by the Open lab foundation of the Key Laboratory of Carbohydrate Chemistry&Biotechnology(No.KLCCB-KF202304)the 111 Project(No.111-2-06).
文摘T7 RNA polymerase(T7 RNAP)-catalyzed in vitro transcription(IVT)is the gold standard manufacturing process for large-scale production of therapeutic mRNA molecules.However,the undesired catalytic activity of T7 RNAP concomitantly generates deleterious impurities,such as double-stranded RNAs,that can exacerbate the downstream purification burden and engender safety concerns.The aim of this study was to engineer T7 RNAP thermostability for high-temperature IVT to reduce the dsRNA.The web server PROSS was utilized to predict thermostable mutation sites from the intermediate and elongation structure of T7 RNAP.Through systematic evaluation of individual mutation sites followed by greedy-accumulation optimization of multi-site combinatorial mutants,we successfully overcame the inherent activity-stability trade-off during the evolution and obtained a thermostable variant,M10(Tm:49.5℃),which exhibits robust catalytic activity at elevated temperatures and significantly reduced dsRNA byproduct formation.Structural analysis using homology modelling and molecular dynamics(MD)simulation revealed that the accumulated mutations increased the local rigidity of T7 RNAP with a compacted conformation,enhanced the helical propensity,and allowed the formation of new salt bridges.The enhanced mutant has the potential to act as an effective biocatalyst for high-temperature IVT,adding in high-quality mRNA production,which is a prerequisite for optimizing the downstream purification processes and improving the clinical viability of such therapeutic agents.
基金Supported by the National Basic Research Program of China (Grant No.2005CB522400)the National Natural Science Foundation of China (Grant Nos.90608021 and 30670689)
文摘The presence of actin in the nucleus as well as its functions in various nuclear processes has been made clear in the past few years. Actin is known to be a part of chromatin-remodeling complexes BAF, which are required for maximal ATPase activity of the Brg1 component of the BAF complex. Moreover, the essential roles of actin in transcription mediated by RNA polymerases Ⅰ,Ⅱ and Ⅲ have been demonstrated recently. On the other hand, a myosin Ⅰ isoform, which contains a unique NH2-terminal extension for nucleus localization, has been specifically localized in nucleus. As is well known, myosin Ⅰ is an actin-binding protein and plays an important role in various cellular activities. Though actin and nuclear myosin Ⅰ (NM Ⅰ) have been implicated to play distinct roles in gene expression, there has been no evidence for the actin-myosin interaction that might be involved in gene transcription mediated by RNA polymerase Ⅱ (RNAP Ⅱ). Here we show evidence that both actin and NM Ⅰ are associated with RNAP Ⅱ in nucleus by using co-localization and co-IP assays, and they may act together on gene transcription. The antibodies against β-actin or NM Ⅰ can block RNA synthesis in a eukaryotic in vitro transcription system with template DNA comprising the promoter and the coding region of human autocrine motility factor receptor (hAMFR) gene; the antibodies pre-adsorbed with purified actin and NM Ⅰ have no effect in transcriptional inhibition, indicating that the inhibition of transcription by anti-actin and anti-NM I is specific. These results suggest a direct involvement of actin-myosin complexes in regulating transcription. It also implicates that actin and NM Ⅰ may co-exist in a same complex with RNAP Ⅱ and the interaction of RNAP Ⅱ with actin and NM Ⅰ functions in the RNAP Ⅱ-mediated transcription.
