Precursor mRNA(pre-mRNA)splicing is essential for gene expression in most eukaryotic organisms.Previous studies from mammals,Drosophila,and yeast show that the majority of splicing events occurs co-transcriptionally.I...Precursor mRNA(pre-mRNA)splicing is essential for gene expression in most eukaryotic organisms.Previous studies from mammals,Drosophila,and yeast show that the majority of splicing events occurs co-transcriptionally.In plants,however,the features of co-transcriptional splicing(CTS)and its regulation still remain largely unknown.Here,we used chromatin-bound RNA sequencing to study CTS in Arabidopsis thaliana.We found that CTS is widespread in Arabidopsis seedlings,with a large proportion of alternative splicing events determined co-transcriptionally.CTS efficiency correlated with gene expression level,the chromatin landscape and,most surprisingly,the number of introns and exons of individual genes,but is independent of gene length.In combination with enhanced crosslinking and immunoprecipitation sequencing analysis,we further showed that the hnRNP-like proteins RZ-1B and RZ-1C promote efficient CTS globally through direct binding,frequently to exonic sequences.Notably,this general effect of RZ-1B/1C on splicing promotion is mainly observed at the chromatin level,not at the mRNA level.RZ-1C promotes CTS of multiple-exon genes in association with its binding to regions both proximal and distal to the regulated introns.We propose that RZ-1C promotes efficient CTS of genes with multiple exons through cooperative interactions with many exons,introns,and splicing factors.Our work thus reveals important features of CTS in plants and provides methodologies for the investigation of CTS and RNA-binding proteins in plants.展开更多
RNA splicing and spliceosome assembly in eukaryotes occur mainly during transcription.However,co-transcriptional splicing has not yet been explored in plants.Here,we built transcriptomes of nascent chromatin RNAs in A...RNA splicing and spliceosome assembly in eukaryotes occur mainly during transcription.However,co-transcriptional splicing has not yet been explored in plants.Here,we built transcriptomes of nascent chromatin RNAs in Arabidopsis thaliana and showed that nearly all introns undergo co-transcriptional splicing,which occurs with higher efficiency for introns in protein-coding genes than for those in noncoding RNAs.Total intron number and intron position are two predominant features that correlate with co-transcriptional splicing efficiency,and introns with alternative 5′or 3′splice sites are less efficiently spliced.Furthermore,we found that mutations in genes encoding trans-acting proteins lead to more introns with increased splicing defects in nascent RNAs than in mature RNAs,and that introns with increased splicing defects in mature RNAs are inefficiently spliced at the co-transcriptional level.Collectively,our results not only uncovered widespread co-transcriptional splicing in Arabidopsis but also identified features that may affect or be affected by co-transcriptional splicing efficiency.展开更多
目的确定贾第虫滋养体内核糖核酸酶P(RNase P)RNA 3′端序列及其存在形式。方法提取贾第虫滋养体总RNA,大肠埃希菌(E.coli)Poly(A)聚合酶加polyA尾后,进行反转录,扩增出加polyA尾后的cDNA,经PCR及测序进行鉴定,确定其3′端序列。应用实...目的确定贾第虫滋养体内核糖核酸酶P(RNase P)RNA 3′端序列及其存在形式。方法提取贾第虫滋养体总RNA,大肠埃希菌(E.coli)Poly(A)聚合酶加polyA尾后,进行反转录,扩增出加polyA尾后的cDNA,经PCR及测序进行鉴定,确定其3′端序列。应用实时荧光定量PCR(RT-qPCR)分别检测RNase P-GLsR15共转录体与RNase PRNA成熟体的总表达量,两者之差即为RNase P RNA成熟体的表达量,确定贾第虫RNase P RNA的存在形式。结果 RNase P RNA 3′cDNA大小约300 nt,3′端序列与GLsR15 3′端序列一致;RNase P-GLsR15共转录体和RNaseP RNA成熟体的总表达量与RNase P-GLsR15共转录体表达量差异无统计学意义(P>0.05)。结论已成功克隆了RNase P RNA 3′端序列,证实RNase P RNA和GlsR15的共转录体即为RNase P RNA成熟体的存在形式。展开更多
目的探讨姜黄素对B细胞淋巴瘤细胞系Raji细胞转录共激活因子P300的影响及对Raji细胞的作用及其机制。方法以不同浓度的姜黄素作用于体外培养Raji细胞,MTT法检测细胞生长抑制率,应用RT-PCR和蛋白印迹(W estern b lot)法检测Raji细胞中P30...目的探讨姜黄素对B细胞淋巴瘤细胞系Raji细胞转录共激活因子P300的影响及对Raji细胞的作用及其机制。方法以不同浓度的姜黄素作用于体外培养Raji细胞,MTT法检测细胞生长抑制率,应用RT-PCR和蛋白印迹(W estern b lot)法检测Raji细胞中P300的表达。结果①姜黄素具有明显的抑制Raji细胞生长作用,并呈明显的量效关系。②姜黄素作用24 h后,随着浓度剂量的增加P300的mRNA和蛋白表达而逐渐降低。低剂量(6.25μmol.L-1)组P300表达有一定的降低,但与对照组相比差异无显著性(P>0.05),而中、高剂量(12.5、25及50μmol.L-1)则抑制P300的表达(P<0.05)。结论姜黄素对B细胞淋巴瘤细胞系Raji细胞具有抗肿瘤细胞的增殖作用,并呈浓度依赖性。姜黄素能够抑制转录共激活因子P300的表达,可能是其重要的机制。展开更多
基金supported by Guangdong Innovative and Entrepreneurial Research Team Program(2016ZT06S172)the Shenzhen Sci-Tech Fund No.KYTDPT20181011104005the National Natural Science Foundation of China(31771365 to Z.W.and 31800268 to D.Z.).
文摘Precursor mRNA(pre-mRNA)splicing is essential for gene expression in most eukaryotic organisms.Previous studies from mammals,Drosophila,and yeast show that the majority of splicing events occurs co-transcriptionally.In plants,however,the features of co-transcriptional splicing(CTS)and its regulation still remain largely unknown.Here,we used chromatin-bound RNA sequencing to study CTS in Arabidopsis thaliana.We found that CTS is widespread in Arabidopsis seedlings,with a large proportion of alternative splicing events determined co-transcriptionally.CTS efficiency correlated with gene expression level,the chromatin landscape and,most surprisingly,the number of introns and exons of individual genes,but is independent of gene length.In combination with enhanced crosslinking and immunoprecipitation sequencing analysis,we further showed that the hnRNP-like proteins RZ-1B and RZ-1C promote efficient CTS globally through direct binding,frequently to exonic sequences.Notably,this general effect of RZ-1B/1C on splicing promotion is mainly observed at the chromatin level,not at the mRNA level.RZ-1C promotes CTS of multiple-exon genes in association with its binding to regions both proximal and distal to the regulated introns.We propose that RZ-1C promotes efficient CTS of genes with multiple exons through cooperative interactions with many exons,introns,and splicing factors.Our work thus reveals important features of CTS in plants and provides methodologies for the investigation of CTS and RNA-binding proteins in plants.
基金supported by the Fundamental Research Funds for the Central Universities,China and National Science Foundation of China(91740202).Y.W.was supported by a fellowship from Shenzhen Un iversity.
文摘RNA splicing and spliceosome assembly in eukaryotes occur mainly during transcription.However,co-transcriptional splicing has not yet been explored in plants.Here,we built transcriptomes of nascent chromatin RNAs in Arabidopsis thaliana and showed that nearly all introns undergo co-transcriptional splicing,which occurs with higher efficiency for introns in protein-coding genes than for those in noncoding RNAs.Total intron number and intron position are two predominant features that correlate with co-transcriptional splicing efficiency,and introns with alternative 5′or 3′splice sites are less efficiently spliced.Furthermore,we found that mutations in genes encoding trans-acting proteins lead to more introns with increased splicing defects in nascent RNAs than in mature RNAs,and that introns with increased splicing defects in mature RNAs are inefficiently spliced at the co-transcriptional level.Collectively,our results not only uncovered widespread co-transcriptional splicing in Arabidopsis but also identified features that may affect or be affected by co-transcriptional splicing efficiency.
文摘目的确定贾第虫滋养体内核糖核酸酶P(RNase P)RNA 3′端序列及其存在形式。方法提取贾第虫滋养体总RNA,大肠埃希菌(E.coli)Poly(A)聚合酶加polyA尾后,进行反转录,扩增出加polyA尾后的cDNA,经PCR及测序进行鉴定,确定其3′端序列。应用实时荧光定量PCR(RT-qPCR)分别检测RNase P-GLsR15共转录体与RNase PRNA成熟体的总表达量,两者之差即为RNase P RNA成熟体的表达量,确定贾第虫RNase P RNA的存在形式。结果 RNase P RNA 3′cDNA大小约300 nt,3′端序列与GLsR15 3′端序列一致;RNase P-GLsR15共转录体和RNaseP RNA成熟体的总表达量与RNase P-GLsR15共转录体表达量差异无统计学意义(P>0.05)。结论已成功克隆了RNase P RNA 3′端序列,证实RNase P RNA和GlsR15的共转录体即为RNase P RNA成熟体的存在形式。
文摘目的探讨姜黄素对B细胞淋巴瘤细胞系Raji细胞转录共激活因子P300的影响及对Raji细胞的作用及其机制。方法以不同浓度的姜黄素作用于体外培养Raji细胞,MTT法检测细胞生长抑制率,应用RT-PCR和蛋白印迹(W estern b lot)法检测Raji细胞中P300的表达。结果①姜黄素具有明显的抑制Raji细胞生长作用,并呈明显的量效关系。②姜黄素作用24 h后,随着浓度剂量的增加P300的mRNA和蛋白表达而逐渐降低。低剂量(6.25μmol.L-1)组P300表达有一定的降低,但与对照组相比差异无显著性(P>0.05),而中、高剂量(12.5、25及50μmol.L-1)则抑制P300的表达(P<0.05)。结论姜黄素对B细胞淋巴瘤细胞系Raji细胞具有抗肿瘤细胞的增殖作用,并呈浓度依赖性。姜黄素能够抑制转录共激活因子P300的表达,可能是其重要的机制。
