The Molecular Mechanism Of Histone Demethylase JMJ14 Regulates MiRNA Synthesis In Arabidopsis Thaliana

Micro RNA(mi RNA)is a class of non-coding single stranded RNA molecules with a length of about 22 nt encoded by endogenous genes.Mi RNA binds m RNA of the target gene in the form of base complementary pairing,which can eventually lead to m RNA degradation or translation initiation inhibition of the target gene,thus regulates gene expression by gene silencing.PRL1,an RNA-binding protein,which belongs to the conserved WD-40 protein,can affect the transcription and processing of pri-mi RNAs and positively regulate the accumulation of mi RNAs and si RNAs.PRL1 mutant prl1-2 has severe growth and development defects,the pri-mi RNA level and mi RNA level show significantly decrease in prl1-2.Through the EMS mutagenesis of prl1-2 and suppressor screening,we obtained the double mutant p35s1 that can restore the developmental phenotype of prl1-2,aiming to explore the new regulatory factors that co-regulate the small RNA biosynthetic pathway with PRL1.Through map cloning and genome resequencing,the mutated gene JMJ14 of double mutant p35s1 was identified and its function was studied.The association between PRL1 and JMJ14 was explored by means of biochemistry,molecular and genetic methods,and the mechanism of PRL1 regulating the metabolism of small RNA was further analyzed.The main research results are as follows:(1)Double mutant p35s1 obtained by EMS mutagenesis a suppressor of the prl1-2.By means of forward genetics,we obtained double mutant p35s1,which can partially restore prl1-2 defects in the developmental phenotype.Its growth status is similar to that of Col-0,and all organs including root system,rosette leaf,inflorescence and silique are normally developed.The results of Stem-loop q RT-PCR experiment showed that the accumulation level of major mi RNAs in p35s1 was significantly higher than that of prl1-2,which was close to the accumulation level of mi RNA in Col-0,indicating that the accumulation of mature mi RNAs in prl1-2 was partially complemented by p35s1.By means of q RT-PCR analysis,It was found that the expressions of major pri-mi RNAs in p35s1 could partially restore the decreased expression of pri-mi RNA in prl1-2 mutant.In addition,p35s1 can also restore part of the mi RNA target genes’ expression level to the wild-type stage.(2)Suppressor p35s1 encodes H3K4me2/3 demethylase JMJ14.A total of 103 Arabidopsis with p35s1 phenotype were selected for genome resequencing.The resequencing analysis of the whole genome data initially identified the candidate genes with point mutation:AT4G20440,AT4G21660,AT4G22900,AT4G19490,AT4G32880,and AT3G63300.Through further experimental verification and sequencing,we finally determined that the gene at the mutation site was AT4G20400.AT4G20400 corresponds to the JMJ14 gene in the Arabidopsis database,encoding histone H3K4me2/3 demethylase.By cloning the CDS sequence of JMJ14 gene and transforming it into p35s1,the transgenic plants can complement the phenotype of p35s1,then shows a phenotype similar to prl1-2.The prl1-2jmj14 double mutant was similar to p35s1.The above two results verified the correctness of gene cloning.(3)JMJ14 regulates flowering in a non-FLC-dependent manner.Under long day conditions,mutants jmj14 and p35s1 showed early flowering phenotype,while prl1-2 showed late flowering phenotype.The results of q RT-PCR showed that the flowering phenotypes of prl1-2,p35s1,jmj14,prl1-2 jmj14 were not regulated by FLC,and the early flowering phenotypes of mutants p35s1,jmj14,prl1-2 jmj14 were caused by the changed expression of LFY,SOC1,AP1,FT and so on.Therefore,JMJ14 regulates flowering by affecting the expression of the above regulation genes,rather than regulating the flowering of Arabidopsis thaliana in a FLC-dependent manner.(4)The interaction between JMJ14 and Dicer complex further affects the processing and maturation of pri-mi RNA,the primary transcript of mi RNA.The subcellular localization of JMJ14 showed that it was located in the nucleus,and the interaction between JMJ14 and key factors in the mi RNA pathway was verified by the Bi FC.The results showed that JMJ14 interacts with PRL1,CDC5(Cell Division Cycle 5),SE(SERRATE),DDL(DAWDLE),DCL3(DICLER-LIKE 3).Meanwhile,the interaction of JMJ14 with PRL1 and CDC5 was further confirmed by Co-IP.JMJ14 may affect the processing and maturation of pri-mi RNA by regulating the activity of pri-mi RNA processing complex.(5)The mutant jmj14-1 was genetically hybridized with HYL1-YFP,p MIR167a::GUS transgenic lines and L1 line respectively,to identify and screen the transgenic lines in the background of jmj14-1 or Col-0.The YFP signal of Arabidopsis root tip cells was observed under the ultra-high resolution fluorescence microscope,and it was found that the mutation of JMJ14 affected the localization of HYL1,with small fluorescent spots located on cytoplasm and cell membrane.GUS staining and q RT-PCR showed that there was no significant difference in the expression of p MIR167a::GUS in the mutant and wild type,so jmj14-1 did not affect the promoter activity of MIR167 a.GUS expression in L1/jmj14-1 was significantly higher than that in L1/Col-0,indicating that jmj14-1 was involved in the accumulation of transgene-induced si RNAs.(6)PRL1 mediated the demethylation of H3K4me3/me2.Since JMJ14 is a histone H3K4 demethylase,the accumulation of H3K4me3/me2 in jmj14 mutant was significantly higher than that in Col-0.Western blot showed that the level of H3K4me3/me2 in prl1-2 and p35s1 was also significantly higher than that in Col-0 though lower than that in jmj14-1.Therefore,it can be concluded that PRL1 plays a regulatory role in the upstream of JMJ14 and mediates the demethylation of histone H3K4me3/me2.In brief,histone demethylase JMJ14,as an suppressor of PRL1,can participate in the regulation of the anabolism of small RNA in Arabidopsis thaliana together with PRL1.This study analyzed the molecular mechanism of Arabidopsis thaliana histone demmethylase JMJ14 in regulating mi RNA synthesis,providing a theoretical foundation for the exploration of key proteins in the regulation pathway of plant mi RNA anabolism and the study of fine regulation mechanism of mi RNA.