Sigma因子6促进拟南芥MIRNA基因转录
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摘要
本研究在筛选拟南芥miRNA水平异常的突变体的过程中,分离得到了一个新的突变体m112,随后通过图位克隆结合基因组测序发现M112的突变基因编码Sigma因子6(SIG6).通过Northern blot和小分子RNA测序,明确了sig6突变体中成熟miRNA的累积下调;通过荧光定量PCR检测pri-miRNA的表达,发现sig6突变体中pri-miRNA水平下调;进一步的MIRNA基因的启动子融合报告基因GUS染色结果显示在sig6突变体中,MIRNA基因启动子的活性减弱.这些实验结果表明SIG6通过影响MIRNA基因的转录参与了miRNA的生物合成,为人们进一步了解miRNA的生物合成途径提供了帮助.
Aiming to screen mutant defective in miRNA biogenesis,we isolated a novel mutant named m112.By map-based cloning combined the whole-genome sequencing,we cloned the M112 gene,which encodes Sigma factor6(SIG6).Northern blot and small RNA deep sequencing analysis show that miRNA levels are globally decreased in sig6 mutants.In order to understand the precise role of SIG6 in miRNA biogenesis,we then examined the accumulation of pri-miRNAs by qRT-PCR and show that pri-miRNA levels are reduced in the sig6 mutant.Furthermore,GUS staining analysis of the MIRNA gene promoter reporters shows that the activity of MIRNA gene promoters is compromised in sig6 mutants.Collectively,these results indicate that SIG6 is involved in miRNA biogenesis by regulating the transcription of MIRNA genes,and it is helpful to further understand the biogenesis pathway of miRNA.
Aiming to screen mutant defective in miRNA biogenesis,we isolated a novel mutant named m112.By map-based cloning combined the whole-genome sequencing,we cloned the M112 gene,which encodes Sigma factor6(SIG6).Northern blot and small RNA deep sequencing analysis show that miRNA levels are globally decreased in sig6 mutants.In order to understand the precise role of SIG6 in miRNA biogenesis,we then examined the accumulation of pri-miRNAs by qRT-PCR and show that pri-miRNA levels are reduced in the sig6 mutant.Furthermore,GUS staining analysis of the MIRNA gene promoter reporters shows that the activity of MIRNA gene promoters is compromised in sig6 mutants.Collectively,these results indicate that SIG6 is involved in miRNA biogenesis by regulating the transcription of MIRNA genes,and it is helpful to further understand the biogenesis pathway of miRNA.
引文
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[15]ZHANG S,XIE M,REN G,et al.CDC5,a DNA binding protein,positively regulates posttranscriptional processing and/or transcription of primary microRNA transcripts[J].Proc Nat Acad Sci USA,2013,110(43):17588-17593.
[16]ZHANG S,LIU Y,YU B.PRL1,an RNA-binding protein,positively regulates the accumulation of miRNAs and siRNAs in Arabidopsis[J].PLoS Genetics,2014,10(12):e1004841.
[17]JIA T,ZHANG B,YOU C,et al.The Arabidopsis MOS4-associated complex promotes microRNAbiogenesis and precursor messenger RNA splicing[J].Plant Cell,2017,29(10):2626-2643.
[18]LI S,LIU K,ZHOU B,et al.MAC3A and MAC3B,two core subunits of the MOS4-associated complex,positively influence miRNA biogenesis[J].Plant Cell,2018,30(2):481-494.
[19]KIM Y J,ZHENG B,YU Y,et al.The role of Mediator in small and long noncoding RNA production in Arabidopsis thaliana[J].Embo Journal,2011,30(5):814-822.
[20]WANG L,SONG X,GU L,et al.NOT2proteins promote polymerase II-dependent transcription and interact with multiple microRNA biogenesis factors in Arabidopsis[J].Plant Cell,2013,25(2):715-727.
[21]HAJHEIDARI M,FARRONA S,HUETTEL B,et al.CDKF;1and CDKD protein kinases regulate phosphorylation of serine residues in the C-terminal domain of Arabidopsis RNA polymeraseⅡ[J].Plant Cell,2012,24(4):1626-1642.
[22]ISHIZAKI Y,TSUNOYAMA Y,HATANO K,et al.A nuclear-encoded sigma factor,Arabidopsis SIG6,recognizes sigma-70type chloroplast promoters and regulates early chloroplast development in cotyledons[J].Plant Journal,2005,42(2):133-144.
[2]LEE R C,FEINBAUM R L,AMBROS V.The C.elegans heterochronic gene lin-4encodes small RNAs with antisense complementarity to lin-14[J].Cell,1993,75(5):843-854.
[3]PARK W,LI J J,SONG R T,et al.CARPEL FACTORY,a Dicer homolog,and HEN1,a novel protein,act in microRNA metabolism in Arabidopsis thaliana[J].Current Biology,2002,12(17):1484-1495.
[4]REINHART B J,WEINSTEIN E G,RHOADES M W,et al.MicroRNAs in plants[J].Genes&Development,2002,16(13):1616-1626.
[5]XIE Z X,ALLEN E,FAHLGREN N,et al.Expression of Arabidopsis MIRNA genes[J].Plant Physiology,2005,138(4):2145-2154.
[6]ZHANG S,LIU Y,YU B.New insights into pri-miRNA processing and accumulation in plants[J].Wiley Interdisciplinary Reviews-RNA,2015,6(5):533-545.
[7]KURIHARA Y,WATANABE Y.Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions[J].Proc Nat Acad Sci USA,2004,101(34):12753-12758.
[8]LIU C,AXTELL M J,FEDOROFF N V.The helicase and RNaseIIIa domains of Arabidopsis DicerLike1modulate catalytic parameters during microRNA biogenesis[J].Plant Physiology,2012,159(2):748-758.
[9]REN G,CHEN X,YU B.Uridylation of miRNAs by HEN1 SUPPRESSOR1in Arabidopsis[J].Current Biology,2012,22(8):695-700.
[10]MI S,CAI T,HU Y,et al.Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5'terminal nucleotide[J].Cell,2008,133(1):116-127.
[11]SCHWARZ D S,HUTVAGNER G,DU T,et al.Asymmetry in the assembly of the RNAi enzyme complex[J].Cell,2003,115(2):199-208.
[12]LUKOWITZ W,GILLMOR C S,SCHEIBLE W R.Positional cloning in Arabidopsis.Why it feels good to have a genome initiative working for you[J].Plant Physiology,2000,123(3):795-805.
[13]MEGRAW M,BAEV V,RUSINOV V,et al.MicroRNA promoter element discovery in Arabidopsis[J].Rna-a Publication of the Rna Society,2006,12(9):1612-1619.
[14]ZHAO X,ZHANG H,LI L.Identification and analysis of the proximal promoters of microRNA genes in Arabidopsis[J].Genomics,2013,101(3):187-194.
[15]ZHANG S,XIE M,REN G,et al.CDC5,a DNA binding protein,positively regulates posttranscriptional processing and/or transcription of primary microRNA transcripts[J].Proc Nat Acad Sci USA,2013,110(43):17588-17593.
[16]ZHANG S,LIU Y,YU B.PRL1,an RNA-binding protein,positively regulates the accumulation of miRNAs and siRNAs in Arabidopsis[J].PLoS Genetics,2014,10(12):e1004841.
[17]JIA T,ZHANG B,YOU C,et al.The Arabidopsis MOS4-associated complex promotes microRNAbiogenesis and precursor messenger RNA splicing[J].Plant Cell,2017,29(10):2626-2643.
[18]LI S,LIU K,ZHOU B,et al.MAC3A and MAC3B,two core subunits of the MOS4-associated complex,positively influence miRNA biogenesis[J].Plant Cell,2018,30(2):481-494.
[19]KIM Y J,ZHENG B,YU Y,et al.The role of Mediator in small and long noncoding RNA production in Arabidopsis thaliana[J].Embo Journal,2011,30(5):814-822.
[20]WANG L,SONG X,GU L,et al.NOT2proteins promote polymerase II-dependent transcription and interact with multiple microRNA biogenesis factors in Arabidopsis[J].Plant Cell,2013,25(2):715-727.
[21]HAJHEIDARI M,FARRONA S,HUETTEL B,et al.CDKF;1and CDKD protein kinases regulate phosphorylation of serine residues in the C-terminal domain of Arabidopsis RNA polymeraseⅡ[J].Plant Cell,2012,24(4):1626-1642.
[22]ISHIZAKI Y,TSUNOYAMA Y,HATANO K,et al.A nuclear-encoded sigma factor,Arabidopsis SIG6,recognizes sigma-70type chloroplast promoters and regulates early chloroplast development in cotyledons[J].Plant Journal,2005,42(2):133-144.
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