利用CRISPR/Cas9技术建立敲除RNA结合蛋白(RALY)基因的PK-15细胞系PK-15-RALY^(-/-),并初步探究RALY对口蹄疫病毒(FMDV)复制的影响。根据GenBank中猪的RALY基因的外显子,设计合成sgRNA并克隆至PX459载体;将质粒转染PK-15细胞,并采用有...利用CRISPR/Cas9技术建立敲除RNA结合蛋白(RALY)基因的PK-15细胞系PK-15-RALY^(-/-),并初步探究RALY对口蹄疫病毒(FMDV)复制的影响。根据GenBank中猪的RALY基因的外显子,设计合成sgRNA并克隆至PX459载体;将质粒转染PK-15细胞,并采用有限稀释法筛选单克隆细胞株,经Western-blot及测序检测RALY基因的敲除效果。通过RT-qPCR检测FMDV感染PK-15-RALY^(-/-)和PK-15细胞后FMDV m RNA的转录水平,Western-blot检测FMDV-VP0、FMDV-VP3和FMDV-VP1的表达水平,最后检测子代病毒感染力。测序结果证实RALY基因发生了移码突变,且Western-blot无法检测到PK-15-RALY^(-/-)细胞中RALY蛋白的表达。FMDV感染两种细胞后,RT-PCR结果显示,FMDV在PK-15细胞中的m RNA水平显著低于PK-15-RALY^(-/-)细胞,Western-blot结果显示,VP0、VP3和VP1蛋白在PK-15-RALY^(-/-)细胞中的表达显著高于PK-15细胞,病毒感染力测定结果显示,PK-15-RALY^(-/-)细胞中子代病毒滴度显著高于PK-15细胞。上述结果表明,本研究成功构建了RALY基因敲除的PK-15细胞系,首次表明RALY可以抑制FMDV复制,为进一步开展RALY在细胞内调控病毒复制机制研究奠定了基础。展开更多
The interaction between m^(6)A-methylated RNA and chromatin modification remains largely unknown.We found that targeted inhibition of bromodomain-containing protein 4(BRD4)by siRNA or its inhibitor(JQ1)significantly d...The interaction between m^(6)A-methylated RNA and chromatin modification remains largely unknown.We found that targeted inhibition of bromodomain-containing protein 4(BRD4)by siRNA or its inhibitor(JQ1)significantly decreases mRNA m^(6)A levels and suppresses the malignancy of breast cancer(BC)cells via increased expression of demethylase AlkB homolog 5(ALKBH5).Mechanistically,inhibition of BRD4 increases the mRNA stability of ALKBH5 via enhanced binding between its 3′untranslated regions(3′UTRs)with RNA-binding protein RALY.Further,BRD4 serves as a scaffold for ubiquitin enzymes tripartite motif containing-21(TRIM21)and ALKBH5,resulting in the ubiquitination and degradation of ALKBH5 protein.JQ1-increased ALKBH5 then demethylates mRNA of extra spindle pole bodies like 1(ESPL1)and reduces binding between ESPL1 mRNA and m^(6)A reader insulin like growth factor 2 mRNA binding protein 3(IGF2BP3),leading to decay of ESPL1 mRNA.Animal and clinical studies confirm a critical role of BRD4/ALKBH5/ESPL1 pathway in BC progression.Further,our study sheds light on the crosstalks between histone modification and RNA methylation.展开更多
文摘利用CRISPR/Cas9技术建立敲除RNA结合蛋白(RALY)基因的PK-15细胞系PK-15-RALY^(-/-),并初步探究RALY对口蹄疫病毒(FMDV)复制的影响。根据GenBank中猪的RALY基因的外显子,设计合成sgRNA并克隆至PX459载体;将质粒转染PK-15细胞,并采用有限稀释法筛选单克隆细胞株,经Western-blot及测序检测RALY基因的敲除效果。通过RT-qPCR检测FMDV感染PK-15-RALY^(-/-)和PK-15细胞后FMDV m RNA的转录水平,Western-blot检测FMDV-VP0、FMDV-VP3和FMDV-VP1的表达水平,最后检测子代病毒感染力。测序结果证实RALY基因发生了移码突变,且Western-blot无法检测到PK-15-RALY^(-/-)细胞中RALY蛋白的表达。FMDV感染两种细胞后,RT-PCR结果显示,FMDV在PK-15细胞中的m RNA水平显著低于PK-15-RALY^(-/-)细胞,Western-blot结果显示,VP0、VP3和VP1蛋白在PK-15-RALY^(-/-)细胞中的表达显著高于PK-15细胞,病毒感染力测定结果显示,PK-15-RALY^(-/-)细胞中子代病毒滴度显著高于PK-15细胞。上述结果表明,本研究成功构建了RALY基因敲除的PK-15细胞系,首次表明RALY可以抑制FMDV复制,为进一步开展RALY在细胞内调控病毒复制机制研究奠定了基础。
基金supported by the National Key Research and Development Program of China(No.2022YFC2601800)the National Natural Science Foundation of China(Nos.82472761,32161143017,82173833,82372743,82173126,82373893,82341053,and 32100584)+10 种基金the Guangdong Basic and Applied Basic Research Foundation(Nos.2023B1515040006,China)the Key-Area Research and Development Program of Guangdong Province(No.2023B1111020007,China)the Guangzhou Science and Technology Program(No.2024A04J6480,China)the Guangdong Provincial Key Laboratory of Construction Foundation(No.2023B1212060022,China)the Shenzhen Bay Scholars Program,the Natural Science Foundation of Shandong Province(No.ZR2021QC061,China)the Natural Science Foundation of Hunan Province of China(No.2022JJ40413)the Outstanding Youth Project of Hunan Provincial Department of Education(No.22B0814,China)the Young Teachers Cultivation Program of Basic Research Operating Expenses of Universities at Sun Yat-sen University(No.23qnpy117,China)the Research Project of TCM Bureau of Guangdong Province(No.20231324,China)the Special Fund of Foshan Climbing Peak Plan(No.2020B018,China)the Basic and Applied Basic Research Foundation of Guangdong Province(No.2022A1515140091,China).
文摘The interaction between m^(6)A-methylated RNA and chromatin modification remains largely unknown.We found that targeted inhibition of bromodomain-containing protein 4(BRD4)by siRNA or its inhibitor(JQ1)significantly decreases mRNA m^(6)A levels and suppresses the malignancy of breast cancer(BC)cells via increased expression of demethylase AlkB homolog 5(ALKBH5).Mechanistically,inhibition of BRD4 increases the mRNA stability of ALKBH5 via enhanced binding between its 3′untranslated regions(3′UTRs)with RNA-binding protein RALY.Further,BRD4 serves as a scaffold for ubiquitin enzymes tripartite motif containing-21(TRIM21)and ALKBH5,resulting in the ubiquitination and degradation of ALKBH5 protein.JQ1-increased ALKBH5 then demethylates mRNA of extra spindle pole bodies like 1(ESPL1)and reduces binding between ESPL1 mRNA and m^(6)A reader insulin like growth factor 2 mRNA binding protein 3(IGF2BP3),leading to decay of ESPL1 mRNA.Animal and clinical studies confirm a critical role of BRD4/ALKBH5/ESPL1 pathway in BC progression.Further,our study sheds light on the crosstalks between histone modification and RNA methylation.
