The recent development of gene transfer approaches in plants and animals has revealed that transgene can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the prese...The recent development of gene transfer approaches in plants and animals has revealed that transgene can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene. More and more investigations have demonstrated that double- stranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms and are assessing the role of methy- lation in transcriptional and posttranscriptional gene silencing. This review will focus on double-stranded RNA mediated mRNA degradation and gene inactivation in plants.展开更多
In our previous study, complete single DNA strands which were obtained from nuclei, chloroplasts and plant mitochondria obeyed Chargaff’s second parity rule, although those which were obtained from animal mitochondri...In our previous study, complete single DNA strands which were obtained from nuclei, chloroplasts and plant mitochondria obeyed Chargaff’s second parity rule, although those which were obtained from animal mitochondria deviated from the rule. On the other hand, plant mitochondria obeyed another different rule after their classification. Complete single DNA strand sequences obtained from chloroplasts, plant mitochondria, and animal mitochondria, were divided into the coding and non-coding regions. The non-coding region, which was the complementary coding region on the reverse strand, was incorporated as a coding region in the forward strand. When the nucleotide contents of the coding region or non-coding regions were plotted against the composition of the four nucleotides in the complete single DNA strand, it was determined that chloroplast and plant mitochondrial DNA obeyed Chargaff’s second parity rule in both the coding and non-coding regions. However, animal mitochondrial DNA deviated from this rule. In chloroplast and plant mitochondrial DNA, which obey Chargaff’s second parity rule, the lines of regression for G (purine) and C (pyrimidine) intersected with regression lines for A (purine) and T (pyrimidines), respectively, at around 0.250 in all cases. On the other hand, in animal mitochondrial DNA, which deviates from Chargaff’s second parity rule, only regression lines due to the content of homonucleotides or their analogs in the coding or non-coding region against those in the complete single DNA strand intersected at around 0.250 at the horizontal axis. Conversely, the intersection of the two lines of regression (G and A or C and T) against the contents of heteronucleotides or their analogs shifted from 0.25 in both coding and non-coding regions. Nucleotide alternations in chloroplasts and plant mitochondria are strictly regulated, not only by the proportion of homonucleotides and their analogs, but also by the heteronucleotides and their analogs. They are strictly regulated in animal mitochondria only by the content of homonucleotides and their analogs.展开更多
RNA interference(RNAi)targeting lethal genes in insects has great potential for sustainable crop protection.Compared with traditional double-stranded(ds)RNA delivery systems,nanoparticles such as chitosan,liposomes,an...RNA interference(RNAi)targeting lethal genes in insects has great potential for sustainable crop protection.Compared with traditional double-stranded(ds)RNA delivery systems,nanoparticles such as chitosan,liposomes,and cationic dendrimers offer advantages in delivering dsRNA/small interfering(si)RNA to improve RNAi efficiency,thus promoting the development and practice of RNAi-based pest management strategies.Here,we illustrate the limitations of traditional dsRNA delivery systems,reveal the mechanism of nanoparticle-mediated RNAi,summarize the recent progress and successful applications of nanoparticle-mediated RNAi in pest management,and finally address the prospects of nanoparticle-based RNA pesticides.展开更多
背景:非编码RNA作为一类不编码蛋白质的RNA分子,在细胞调控中发挥关键作用。同时,间充质干细胞具有多向分化潜能,对组织修复和再生至关重要。近年来,研究者们对非编码RNA在调控间充质干细胞功能中的作用展开了深入探讨。目的:通过文献...背景:非编码RNA作为一类不编码蛋白质的RNA分子,在细胞调控中发挥关键作用。同时,间充质干细胞具有多向分化潜能,对组织修复和再生至关重要。近年来,研究者们对非编码RNA在调控间充质干细胞功能中的作用展开了深入探讨。目的:通过文献计量学分析方法系统地了解非编码RNA与间充质干细胞相关研究的发展趋势与关键领域。方法:从Web of Science核心合集1990年至今的科学引文索引扩展获取非编码RNA与间充质干细胞相关研究的文献数据,运用VOSviewer和Cite Space计量学软件对年份、国家或地区、研究机构、被引文献和关键词进行发文量、聚类、被引频次、突增性和中介中心性进行分析,揭示该研究领域的知识基础及前沿热点。结果与结论:(1)最终纳入5348篇文献,发表在1997-2021年,相关文献呈现明显的增长趋势,尽管在2022年略有减少,其发文量仍保持较高水平,中国在该领域的研究中占据主导地位,其中上海交通大学为最活跃的机构。(2)对被引文献分析发现,高突增性、高中介中心性、高被引的文献主要与微小RNA、细胞外囊泡和骨代谢等内容相关,这些文献构成了非编码RNA与间充质干细胞研究领域的重要知识基础。(3)对关键词进行突增性分析发现,至今保持高突增性的关键词包括外泌体、环状RNA、细胞外囊泡和损伤。(4)对关键词进行聚类演变分析,发文量仍保持增长趋势的关键词聚类包括:肿瘤微环境、骨关节炎、氧化应激和细胞外囊泡,这些关键词反映了目前以及未来该领域的研究热点。(5)文章结果不仅展示了非编码RNA与间充质干细胞研究领域的研究态势,更重要的是有望为研究人员提供潜在的方向和启示。展开更多
RNA interference(RNAi)has emerged as a powerful technology for pest management.Previously,we have shown that plastid-mediated RNAi(PM-RNAi)can be utilized to control the Colorado potato beetle,an insect pest in the Ch...RNA interference(RNAi)has emerged as a powerful technology for pest management.Previously,we have shown that plastid-mediated RNAi(PM-RNAi)can be utilized to control the Colorado potato beetle,an insect pest in the Chrysomelidae family;however,whether this technology is suitable for controlling pests in the Coccinellidae remained unknown.The coccinellid 28-spotted potato ladybird(Henosepilachna vigintioctopunctata;HV)is a serious pest of solanaceous crops.In this study,we identified three efficient target genes(β-Actin,SRP54,and SNAP)for RNAi using in vitro double-stranded RNAs(dsRNAs)fed to HV,and found that dsRNAs targetingβ-Actin messenger RNA(dsACT)induced more potent RNAi than those targeting the other two genes.We next generated transplastomic and nuclear transgenic potato(Solanum tuberosum)plants expressing HV dsACT.Long dsACT stably accumulated to up to 0.7%of the total cellular RNA in the transplastomic plants,at least three orders of magnitude higher than in the nuclear transgenic plants.Notably,the transplastomic plants also exhibited a significantly stronger resistance to HV,killing all larvae within 6 d.Our data demonstrate the potential of PM-RNAi as an efficient pest control measure for HV,extending the application range of this technology to Coccinellidae pests.展开更多
Rhizoctonia solani is a soil-borne pathogenic fungus with several distinct isolates that have been classified based on their anastomosis groups （AG＇s）. Many isolates of these fungi contain double-stranded viral RNA...Rhizoctonia solani is a soil-borne pathogenic fungus with several distinct isolates that have been classified based on their anastomosis groups （AG＇s）. Many isolates of these fungi contain double-stranded viral RNA （dsRNA） that are cytoplasmic and viral in origin. Research in our laboratory has studied the epidemiology and molecular biology of viral RNA in R. solani, making it a useful biological model in the development of protocols for the rapid identification of biological agents. In the present study the dsRNA from the isolate EGR-4 which is characteristically large at 3.301 Kb was purified. Attempts to clone middle （M）-size dsRNA fragments from R, solani have been very difficult primarily due to artifacts that co-purify including large （L）-size dsRNA in the fungus. Various MgC12 concentrations were tested to optimize full length dsRNA PCR product. Magnesium is required for DNA polymerase, and EGR-4 requires a specific concentration; thus, several MgC1z concentrations were tested. The dsRNA was analyzed by gel electrophoresis. The gel-purified, nuclease-treated dsRNA was reverse transcribed into cDNA and ligated into the p-jet cloning vector and transformed using E. coli. All such clones were sequenced and forward and reverse primers were generated using BLAST sequence via Biosearch Technology. The plasmids were purified from transformed cultures and amplified using real-time PCR （RTqPCR） with the primers （reverse CCACCGGAAGAGGGAAATCC, forward AGCGCTGACCTTGCTATCGA ATC） and probe （5＇ Fam-AGTGCCGATCAGCCCTCCACCG-BHQ 1 3＇）. The ideal primer/probe concentration was determined through optimization by comparing the lowest threshold concentration （Ct） values using the plasmid cDNA as a template.展开更多
Salsolinol(1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline,Sal)is a catechol isoquinoline that causes neurotoxicity and shares structural similarity with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,an environme...Salsolinol(1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline,Sal)is a catechol isoquinoline that causes neurotoxicity and shares structural similarity with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,an environmental toxin that causes Parkinson's disease.However,the mechanism by which Sal mediates dopaminergic neuronal death remains unclear.In this study,we found that Sal significantly enhanced the global level of N~6-methyladenosine(m~6A)RNA methylation in PC12 cells,mainly by inducing the downregulation of the expression of m~6A demethylases fat mass and obesity-associated protein(FTO)and alk B homolog 5(ALKBH5).RNA sequencing analysis showed that Sal downregulated the Hippo signaling pathway.The m~6A reader YTH domain-containing family protein 2(YTHDF2)promoted the degradation of m~6A-containing Yes-associated protein 1(YAP1)mRNA,which is a downstream key effector in the Hippo signaling pathway.Additionally,downregulation of YAP1 promoted autophagy,indicating that the mutual regulation between YAP1 and autophagy can lead to neurotoxicity.These findings reveal the role of Sal on m~6A RNA methylation and suggest that Sal may act as an RNA methylation inducer mediating dopaminergic neuronal death through YAP1 and autophagy.Our results provide greater insights into the neurotoxic effects of catechol isoquinolines compared with other studies and may be a reference for assessing the involvement of RNA methylation in the pathogenesis of Parkinson's disease.展开更多
BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene ma...BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.展开更多
文摘The recent development of gene transfer approaches in plants and animals has revealed that transgene can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene. More and more investigations have demonstrated that double- stranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms and are assessing the role of methy- lation in transcriptional and posttranscriptional gene silencing. This review will focus on double-stranded RNA mediated mRNA degradation and gene inactivation in plants.
文摘In our previous study, complete single DNA strands which were obtained from nuclei, chloroplasts and plant mitochondria obeyed Chargaff’s second parity rule, although those which were obtained from animal mitochondria deviated from the rule. On the other hand, plant mitochondria obeyed another different rule after their classification. Complete single DNA strand sequences obtained from chloroplasts, plant mitochondria, and animal mitochondria, were divided into the coding and non-coding regions. The non-coding region, which was the complementary coding region on the reverse strand, was incorporated as a coding region in the forward strand. When the nucleotide contents of the coding region or non-coding regions were plotted against the composition of the four nucleotides in the complete single DNA strand, it was determined that chloroplast and plant mitochondrial DNA obeyed Chargaff’s second parity rule in both the coding and non-coding regions. However, animal mitochondrial DNA deviated from this rule. In chloroplast and plant mitochondrial DNA, which obey Chargaff’s second parity rule, the lines of regression for G (purine) and C (pyrimidine) intersected with regression lines for A (purine) and T (pyrimidines), respectively, at around 0.250 in all cases. On the other hand, in animal mitochondrial DNA, which deviates from Chargaff’s second parity rule, only regression lines due to the content of homonucleotides or their analogs in the coding or non-coding region against those in the complete single DNA strand intersected at around 0.250 at the horizontal axis. Conversely, the intersection of the two lines of regression (G and A or C and T) against the contents of heteronucleotides or their analogs shifted from 0.25 in both coding and non-coding regions. Nucleotide alternations in chloroplasts and plant mitochondria are strictly regulated, not only by the proportion of homonucleotides and their analogs, but also by the heteronucleotides and their analogs. They are strictly regulated in animal mitochondria only by the content of homonucleotides and their analogs.
基金the Beijing Natural Science Foundation(6204043)National Natural Science Foundation of China(31900363).
文摘RNA interference(RNAi)targeting lethal genes in insects has great potential for sustainable crop protection.Compared with traditional double-stranded(ds)RNA delivery systems,nanoparticles such as chitosan,liposomes,and cationic dendrimers offer advantages in delivering dsRNA/small interfering(si)RNA to improve RNAi efficiency,thus promoting the development and practice of RNAi-based pest management strategies.Here,we illustrate the limitations of traditional dsRNA delivery systems,reveal the mechanism of nanoparticle-mediated RNAi,summarize the recent progress and successful applications of nanoparticle-mediated RNAi in pest management,and finally address the prospects of nanoparticle-based RNA pesticides.
文摘背景:非编码RNA作为一类不编码蛋白质的RNA分子,在细胞调控中发挥关键作用。同时,间充质干细胞具有多向分化潜能,对组织修复和再生至关重要。近年来,研究者们对非编码RNA在调控间充质干细胞功能中的作用展开了深入探讨。目的:通过文献计量学分析方法系统地了解非编码RNA与间充质干细胞相关研究的发展趋势与关键领域。方法:从Web of Science核心合集1990年至今的科学引文索引扩展获取非编码RNA与间充质干细胞相关研究的文献数据,运用VOSviewer和Cite Space计量学软件对年份、国家或地区、研究机构、被引文献和关键词进行发文量、聚类、被引频次、突增性和中介中心性进行分析,揭示该研究领域的知识基础及前沿热点。结果与结论:(1)最终纳入5348篇文献,发表在1997-2021年,相关文献呈现明显的增长趋势,尽管在2022年略有减少,其发文量仍保持较高水平,中国在该领域的研究中占据主导地位,其中上海交通大学为最活跃的机构。(2)对被引文献分析发现,高突增性、高中介中心性、高被引的文献主要与微小RNA、细胞外囊泡和骨代谢等内容相关,这些文献构成了非编码RNA与间充质干细胞研究领域的重要知识基础。(3)对关键词进行突增性分析发现,至今保持高突增性的关键词包括外泌体、环状RNA、细胞外囊泡和损伤。(4)对关键词进行聚类演变分析,发文量仍保持增长趋势的关键词聚类包括:肿瘤微环境、骨关节炎、氧化应激和细胞外囊泡,这些关键词反映了目前以及未来该领域的研究热点。(5)文章结果不仅展示了非编码RNA与间充质干细胞研究领域的研究态势,更重要的是有望为研究人员提供潜在的方向和启示。
基金supported by grants from the Natural Science Foundation of Hubei Province(2020CFA012)the National Natural Science Foundation of China(32271912)to J.Z。
文摘RNA interference(RNAi)has emerged as a powerful technology for pest management.Previously,we have shown that plastid-mediated RNAi(PM-RNAi)can be utilized to control the Colorado potato beetle,an insect pest in the Chrysomelidae family;however,whether this technology is suitable for controlling pests in the Coccinellidae remained unknown.The coccinellid 28-spotted potato ladybird(Henosepilachna vigintioctopunctata;HV)is a serious pest of solanaceous crops.In this study,we identified three efficient target genes(β-Actin,SRP54,and SNAP)for RNAi using in vitro double-stranded RNAs(dsRNAs)fed to HV,and found that dsRNAs targetingβ-Actin messenger RNA(dsACT)induced more potent RNAi than those targeting the other two genes.We next generated transplastomic and nuclear transgenic potato(Solanum tuberosum)plants expressing HV dsACT.Long dsACT stably accumulated to up to 0.7%of the total cellular RNA in the transplastomic plants,at least three orders of magnitude higher than in the nuclear transgenic plants.Notably,the transplastomic plants also exhibited a significantly stronger resistance to HV,killing all larvae within 6 d.Our data demonstrate the potential of PM-RNAi as an efficient pest control measure for HV,extending the application range of this technology to Coccinellidae pests.
文摘Rhizoctonia solani is a soil-borne pathogenic fungus with several distinct isolates that have been classified based on their anastomosis groups （AG＇s）. Many isolates of these fungi contain double-stranded viral RNA （dsRNA） that are cytoplasmic and viral in origin. Research in our laboratory has studied the epidemiology and molecular biology of viral RNA in R. solani, making it a useful biological model in the development of protocols for the rapid identification of biological agents. In the present study the dsRNA from the isolate EGR-4 which is characteristically large at 3.301 Kb was purified. Attempts to clone middle （M）-size dsRNA fragments from R, solani have been very difficult primarily due to artifacts that co-purify including large （L）-size dsRNA in the fungus. Various MgC12 concentrations were tested to optimize full length dsRNA PCR product. Magnesium is required for DNA polymerase, and EGR-4 requires a specific concentration; thus, several MgC1z concentrations were tested. The dsRNA was analyzed by gel electrophoresis. The gel-purified, nuclease-treated dsRNA was reverse transcribed into cDNA and ligated into the p-jet cloning vector and transformed using E. coli. All such clones were sequenced and forward and reverse primers were generated using BLAST sequence via Biosearch Technology. The plasmids were purified from transformed cultures and amplified using real-time PCR （RTqPCR） with the primers （reverse CCACCGGAAGAGGGAAATCC, forward AGCGCTGACCTTGCTATCGA ATC） and probe （5＇ Fam-AGTGCCGATCAGCCCTCCACCG-BHQ 1 3＇）. The ideal primer/probe concentration was determined through optimization by comparing the lowest threshold concentration （Ct） values using the plasmid cDNA as a template.
基金supported by the National Natural Science Foundation of China,Nos.82271283(to XC),91854115(to JW),31970044(to JW)the Natural Science Foundation of Beijing,No.7202001(to XC)the Scientific Research Project of Beijing Educational Committee,No.KM202010005022(to XC)。
文摘Salsolinol(1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline,Sal)is a catechol isoquinoline that causes neurotoxicity and shares structural similarity with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,an environmental toxin that causes Parkinson's disease.However,the mechanism by which Sal mediates dopaminergic neuronal death remains unclear.In this study,we found that Sal significantly enhanced the global level of N~6-methyladenosine(m~6A)RNA methylation in PC12 cells,mainly by inducing the downregulation of the expression of m~6A demethylases fat mass and obesity-associated protein(FTO)and alk B homolog 5(ALKBH5).RNA sequencing analysis showed that Sal downregulated the Hippo signaling pathway.The m~6A reader YTH domain-containing family protein 2(YTHDF2)promoted the degradation of m~6A-containing Yes-associated protein 1(YAP1)mRNA,which is a downstream key effector in the Hippo signaling pathway.Additionally,downregulation of YAP1 promoted autophagy,indicating that the mutual regulation between YAP1 and autophagy can lead to neurotoxicity.These findings reveal the role of Sal on m~6A RNA methylation and suggest that Sal may act as an RNA methylation inducer mediating dopaminergic neuronal death through YAP1 and autophagy.Our results provide greater insights into the neurotoxic effects of catechol isoquinolines compared with other studies and may be a reference for assessing the involvement of RNA methylation in the pathogenesis of Parkinson's disease.
文摘BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.
