The Molecular Mechanism Of PRL1 Suppressor IYO In Regulating MiRNA Synthesis

Micro RNA(miRNA)is non-coding RNA with a length of 18-25 nt in eukaryotes,which can regulate gene expression at the post-transcriptional levels,and play important roles in plant development.miRNAs bind to target genes through base complementary pairing,degrade target genes or inhibit their translation process,thus regulating plant physiological processes.PLEIOTROPIC REGULATORY LOCUS 1(PRL1)is a RNA binding protein with conserved WD40 domain,which participates in the synthesis and processing of microRNAs.PRL1 positively regulates the accumulation of microRNAs and siRNAs in Arabidopsis thaliana,and regulates the processing of pri-microRNAs and dsRNAs by affecting the activity of DICER-LIKE 1(DCL1).prl1-2,a T-DNA insertion mutant,its pri-miRNA and miRNA accumulation were significantly reduced,showing severe developmental defects,such as short roots,short plants,stunted development.In this study,prl1-2 was used as the material,and EMS mutagenesis was adopted to screen forwardly double mutant plants that could restore the phenotype of prl1-2 mutants.We aimed to obtain new factors that could affect the miRNA pathway,and reveal the correlation between PRL1 and new factors,and further clarify the fine regulation mechanism of PRL1 and PRL1 suppressors on miRNA level.The main results are as follows:(1)We obtained 11 prl1-2 Suppressors.The seeds of prl1-2 mutant induced by EMS were planted in M2 generation for phenotypic analysis and screening.Eleven suppressor plants of prl1-2 mutant were obtained.The phenotype of P27S1 was able to restore the mutant phenotype of prl1-2.Phenotype analysis showed that there was no significant difference in P27S1 compared with the Col(wild type),and the development of leaves and the root were completely restored relative to the prl1-2.The expression of mature miRNA in P27S1 detected by Stem loop RT-PCR showed that the accumulation of miR156,miR167,miR171 and miR173 in P27S1 was significantly higher than that in prl1-2,which was close to Col.The expression of miRNA primary transcripts(pri-miRNA)and miRNA targets detected by qRT-PCR in P27S1 showed that the expression of pri-miRNA(pri-mir167,pri-mir171,pri-mir172)increased,and the expression of miRNA targets decreased significantly.The expression of pri-miRNA and miRNA targets almost completely restored the decrease of pri-miRNA and the increased expression of miRNA targets caused by PRL1 mutation.(2)The P27S1 was hybridized with the prl1 mutant(Ler background)to construct the map-based clone population.Preliminary positioning results showed that the mutant gene was located on the first or fourth chromosome.P27S1 was backcrossed with prl1-2 to construct backcrossing isolates.About 100 plants with P27S1 phenotype were selected for mixed pool whole genome resequencing,and the resequencing results were analyzed.We obtained 4 candidate mutant genes,namely At4g38440,At1g53360,At4g33210 and At5g04590.The mutation site was confirmed to be At4g38440 by sequencing and genotype identification.At4g38440 corresponds to IYO gene in Arabidopsis database,which encodes transcriptional elongator related to initiation of differentiation.After cloning full-length genome sequence and transforming P27S1,all plants showed nearly prl1-2 phenotype,which verified the results of gene cloning.(3)Interaction of IYO with PRL1 and SE affects the processing of pri-microRNA.The interaction between IYO and PRL1 was verified by bimolecule fluorescence complementation.The results showed that IYO could interact with PRL1,and at the same time,IYO interacted with SERRATE,a key component of the splicing complex,indicating that IYO might affect the processing of miRNA.