microRNAs (miRNAs) encode a novel class of small, non-coding RNAs that regulate gene expression post-trancriptionally. miRNAs comprise one of the major non-coding RNA families, whose diverse bio- logical functions and...microRNAs (miRNAs) encode a novel class of small, non-coding RNAs that regulate gene expression post-trancriptionally. miRNAs comprise one of the major non-coding RNA families, whose diverse bio- logical functions and unusual capacity for gene regulation have attracted enormous interests in the RNA world. Over the past 16 years, genetic, biochemical and computational approaches have greatly shaped the growth of the field, leading to the identification of thousands of miRNA genes in nearly all metazoans. The key molecular machinery for miRNA biogenesis and silencing has been identified, yet the precise biochemical and regulatory mechanisms still remain elusive. However, recent findings have shed new light on how miRNAs are generated and how they function to repress gene expression. miRNAs provide a paradigm for endogenous small RNAs that mediate gene silencing at a genome-wide level. The gene silencing mediated by these small RNAs constitutes a major component of gene regu- lation during various developmental and physiological processes. The accumulating knowledge about their biogenesis and gene silencing mechanism will add a new dimension to our understanding about the complex gene regulatory networks.展开更多
Eukaryotic gene expression is controlled by different levels of biological events, such as transcription factors regulating the timing and strength of transcripts production, alteration of transcription rate by RNA pr...Eukaryotic gene expression is controlled by different levels of biological events, such as transcription factors regulating the timing and strength of transcripts production, alteration of transcription rate by RNA processing, and mRNA stability during RNA processing and translation. RNAs, especially mRNAs, are relatively vulnerable molecules in living cells for ribonucleases （RNases）. The maintenance of quality and quantity of transcripts is a key issue for many biological processes. Extensive studies draw the conclusion that the stability of RNAs is dedicated-regulated, occurring co- and post-transcriptionally, and translation-coupled as well, either in the nucleus or cytoplasm. Recently, RNA stability in the nucleus has aroused much research interest, especially the stability of newly-made transcripts. In this article, we summarize recent progresses on mRNA stability in the nucleus, especially focusing on quality control of newly-made RNA by RNA polymerase Ⅱ in eukaryotes.展开更多
MicroRNAs(miRNAs)are regulatory RNA molecules that bind to target messenger RNAs(mRNAs)and affect the stability or translational efficiency of the bound mRNAs.Single or dual-luciferase reporter systems have been succe...MicroRNAs(miRNAs)are regulatory RNA molecules that bind to target messenger RNAs(mRNAs)and affect the stability or translational efficiency of the bound mRNAs.Single or dual-luciferase reporter systems have been successfully used to identify miRNA target genes in mammalian cells.These reporter systems,however,are not sensitive enough to verify miRNA-target gene relationships in insect cell lines because the promoters of the target luciferase(usually Renilla)used in these reporter systems are too weak to drive sufficient expression of the target luciferase in insect cells.In this study,we replaced the SV40 promoter in the psiCHECK-2 reporter vector,which is widely used with mammalian cell lines,with the HSV-TK or AC5.1 promoter to yield two new dual-luciferase reporter vectors,designated psiCHECK-2-TK and psiCHECK-2-AC5.1,respectively.Only psiCHECK-2 and psiCHECK-2-AC5.1 had suitable target(7?enz7/a)/reference(firefly)luciferase activity ratios in mammalian(HeLa and HEK293)and insect(Sf9,S2,Helicoverpa zea fat body and ovary)cell lines,while psiCHECK-2-TK had suitable Renilla/firefly luciferase activity ratios regardless of the cell line.Moreover,psiCHECK-2-TK successfully detected the interaction between Helicoverpa armigera miRNA9a and its target,the 3'-untranslated region of heat shock protein 90,in both mammalian and H.zea cell lines,but psiCHECK-2 failed to do so in IT.zea cell lines.Furthermore,psiCHECK-2-TK with the target sequence,HzMasc(H.zea Masculinizer),accurately differentiated between H.zea cell lines with or without the negative regulation factor(miRNA or piRNA)of HzMasc.These data demonstrate that psiCHECK-2-TK can be used to functionally characterize small RNA target genes in both mammalian and insect cells.展开更多
Suppression of the effects of an spt10 mutation on ADH2 expression is a phenotype shared by a small number of genes whose protein products are either components of the CCR4-NOT complex required for mRNA deadenylation ...Suppression of the effects of an spt10 mutation on ADH2 expression is a phenotype shared by a small number of genes whose protein products are either components of the CCR4-NOT complex required for mRNA deadenylation and degradation (CCR4, CAF1, NOT4) or have been shown to interact with the complex (DBF2, SRB9, SRB10). In this work, we conducted a screen for additional suppressors of spt10 at ADH2 to identify new factors related to CCR4 function. In addition to reisolating ccr4 and caf1 alleles, three previously unidentified suppressors of spt10 were obtained: ebs1, lsm6, and nup159. These three genes are known or presumed to affect mRNA export or degradation. Mutations in EBS1, LSM6 and NUP159 not only suppressed spt10-induced ADH2 expression but also, like ccr4 and caf1 defects, reduced the ability of ADH2 to derepress. None of these defects affected the expression of CCR4-NOT complex components or the formation of the CCR4-NOT complex. The reduced ADH2 expression was also not the result of increased degradation of ADH2 mRNA, as the lsm6 and nup159 alleles, like that of a ccr4 deletion, actually slowed ADH2 degradation. Our results indicate that alterations in factors that slow mRNA degradation or affect mRNA transport may also interfere with the synthesis of mRNA and suggest an integration of such events in gene expression.展开更多
To investigate the influence of miRNAs on target gene at transcription levels, we have systematically analyzed the expression profiles by a regression method. The results indicated that the down-regulation level of ta...To investigate the influence of miRNAs on target gene at transcription levels, we have systematically analyzed the expression profiles by a regression method. The results indicated that the down-regulation level of target mRNA is determined by multiple factors including the length and secondary structure of target mRNA, the structural accessibility of the target site and so on. Furthermore, by comparison of different miRNAs, the results indicated that, in animals, all miRNAs regulate their targets in a similar way.展开更多
The CCR4-NOT complex has been shown to have multiple roles in mRNA metabolism, including that of transcriptional elongation, mRNA transport, and nuclear exosome function, but the primary function of CCR4 and CAF1 is i...The CCR4-NOT complex has been shown to have multiple roles in mRNA metabolism, including that of transcriptional elongation, mRNA transport, and nuclear exosome function, but the primary function of CCR4 and CAF1 is in the deadenylation and degradation of cytoplasmic mRNA. As previous genetic analysis supported an interaction between SPT5, known to be involved in transcriptional elongation, and that of CCR4, the physical association of SPT5 with CCR4 was examined. A two-hybrid screen utilizing the deadenylase domain of CCR4 as a bait identified SPT5 as a potential interacting protein. SPT5 at its physiological concentration was shown to immunoprecipitate CCR4 and CAF1, and in vitro purified SPT5 specifically could bind to CAF1 and the deadenylase domain of CCR4. We additionally demonstrated that mutations in SPT5 or an spt4 deletion slowed the rate of mRNA degradation, a phenotype associated with defects in the CCR4 mRNA deadenylase complex. Yet, unlike ccr4 and caf1 deletions, spt5 and spt4 defects displayed little effect on the rate of deadenylation. They also did not affect decapping or 5' - 3' degradation of mRNA. These results suggest that the interactions between SPT5/SPT4 and the CCR4-NOT complex are probably the consequences of effects involving nuclear events and do not involve the primary role of CCR4 in mRNA deadenylation and turnover.展开更多
文摘microRNAs (miRNAs) encode a novel class of small, non-coding RNAs that regulate gene expression post-trancriptionally. miRNAs comprise one of the major non-coding RNA families, whose diverse bio- logical functions and unusual capacity for gene regulation have attracted enormous interests in the RNA world. Over the past 16 years, genetic, biochemical and computational approaches have greatly shaped the growth of the field, leading to the identification of thousands of miRNA genes in nearly all metazoans. The key molecular machinery for miRNA biogenesis and silencing has been identified, yet the precise biochemical and regulatory mechanisms still remain elusive. However, recent findings have shed new light on how miRNAs are generated and how they function to repress gene expression. miRNAs provide a paradigm for endogenous small RNAs that mediate gene silencing at a genome-wide level. The gene silencing mediated by these small RNAs constitutes a major component of gene regu- lation during various developmental and physiological processes. The accumulating knowledge about their biogenesis and gene silencing mechanism will add a new dimension to our understanding about the complex gene regulatory networks.
基金Project supported by the Talented Scientist Program from South China Agricultural University (No.4600-K14013)the National Natural Science Foundation of China (No.81301901)
文摘Eukaryotic gene expression is controlled by different levels of biological events, such as transcription factors regulating the timing and strength of transcripts production, alteration of transcription rate by RNA processing, and mRNA stability during RNA processing and translation. RNAs, especially mRNAs, are relatively vulnerable molecules in living cells for ribonucleases （RNases）. The maintenance of quality and quantity of transcripts is a key issue for many biological processes. Extensive studies draw the conclusion that the stability of RNAs is dedicated-regulated, occurring co- and post-transcriptionally, and translation-coupled as well, either in the nucleus or cytoplasm. Recently, RNA stability in the nucleus has aroused much research interest, especially the stability of newly-made transcripts. In this article, we summarize recent progresses on mRNA stability in the nucleus, especially focusing on quality control of newly-made RNA by RNA polymerase Ⅱ in eukaryotes.
基金This work was supported by the National Science Foundation of China(NSFC)-Henan Joint major grant(U2004206)the State Key Laboratory of Cotton Biology(CB2020A06).
文摘MicroRNAs(miRNAs)are regulatory RNA molecules that bind to target messenger RNAs(mRNAs)and affect the stability or translational efficiency of the bound mRNAs.Single or dual-luciferase reporter systems have been successfully used to identify miRNA target genes in mammalian cells.These reporter systems,however,are not sensitive enough to verify miRNA-target gene relationships in insect cell lines because the promoters of the target luciferase(usually Renilla)used in these reporter systems are too weak to drive sufficient expression of the target luciferase in insect cells.In this study,we replaced the SV40 promoter in the psiCHECK-2 reporter vector,which is widely used with mammalian cell lines,with the HSV-TK or AC5.1 promoter to yield two new dual-luciferase reporter vectors,designated psiCHECK-2-TK and psiCHECK-2-AC5.1,respectively.Only psiCHECK-2 and psiCHECK-2-AC5.1 had suitable target(7?enz7/a)/reference(firefly)luciferase activity ratios in mammalian(HeLa and HEK293)and insect(Sf9,S2,Helicoverpa zea fat body and ovary)cell lines,while psiCHECK-2-TK had suitable Renilla/firefly luciferase activity ratios regardless of the cell line.Moreover,psiCHECK-2-TK successfully detected the interaction between Helicoverpa armigera miRNA9a and its target,the 3'-untranslated region of heat shock protein 90,in both mammalian and H.zea cell lines,but psiCHECK-2 failed to do so in IT.zea cell lines.Furthermore,psiCHECK-2-TK with the target sequence,HzMasc(H.zea Masculinizer),accurately differentiated between H.zea cell lines with or without the negative regulation factor(miRNA or piRNA)of HzMasc.These data demonstrate that psiCHECK-2-TK can be used to functionally characterize small RNA target genes in both mammalian and insect cells.
文摘Suppression of the effects of an spt10 mutation on ADH2 expression is a phenotype shared by a small number of genes whose protein products are either components of the CCR4-NOT complex required for mRNA deadenylation and degradation (CCR4, CAF1, NOT4) or have been shown to interact with the complex (DBF2, SRB9, SRB10). In this work, we conducted a screen for additional suppressors of spt10 at ADH2 to identify new factors related to CCR4 function. In addition to reisolating ccr4 and caf1 alleles, three previously unidentified suppressors of spt10 were obtained: ebs1, lsm6, and nup159. These three genes are known or presumed to affect mRNA export or degradation. Mutations in EBS1, LSM6 and NUP159 not only suppressed spt10-induced ADH2 expression but also, like ccr4 and caf1 defects, reduced the ability of ADH2 to derepress. None of these defects affected the expression of CCR4-NOT complex components or the formation of the CCR4-NOT complex. The reduced ADH2 expression was also not the result of increased degradation of ADH2 mRNA, as the lsm6 and nup159 alleles, like that of a ccr4 deletion, actually slowed ADH2 degradation. Our results indicate that alterations in factors that slow mRNA degradation or affect mRNA transport may also interfere with the synthesis of mRNA and suggest an integration of such events in gene expression.
基金Supported by the Natural Science Foundation of Jiangsu Province, China (BK2010500)
文摘To investigate the influence of miRNAs on target gene at transcription levels, we have systematically analyzed the expression profiles by a regression method. The results indicated that the down-regulation level of target mRNA is determined by multiple factors including the length and secondary structure of target mRNA, the structural accessibility of the target site and so on. Furthermore, by comparison of different miRNAs, the results indicated that, in animals, all miRNAs regulate their targets in a similar way.
文摘The CCR4-NOT complex has been shown to have multiple roles in mRNA metabolism, including that of transcriptional elongation, mRNA transport, and nuclear exosome function, but the primary function of CCR4 and CAF1 is in the deadenylation and degradation of cytoplasmic mRNA. As previous genetic analysis supported an interaction between SPT5, known to be involved in transcriptional elongation, and that of CCR4, the physical association of SPT5 with CCR4 was examined. A two-hybrid screen utilizing the deadenylase domain of CCR4 as a bait identified SPT5 as a potential interacting protein. SPT5 at its physiological concentration was shown to immunoprecipitate CCR4 and CAF1, and in vitro purified SPT5 specifically could bind to CAF1 and the deadenylase domain of CCR4. We additionally demonstrated that mutations in SPT5 or an spt4 deletion slowed the rate of mRNA degradation, a phenotype associated with defects in the CCR4 mRNA deadenylase complex. Yet, unlike ccr4 and caf1 deletions, spt5 and spt4 defects displayed little effect on the rate of deadenylation. They also did not affect decapping or 5' - 3' degradation of mRNA. These results suggest that the interactions between SPT5/SPT4 and the CCR4-NOT complex are probably the consequences of effects involving nuclear events and do not involve the primary role of CCR4 in mRNA deadenylation and turnover.
