Regulatory Roles Of CYP71 And CUE1 In Arabidopsis MicroRNA Biogenesis

MicroRNAs(miRNAs)are endogenous small non-coding RNAs that regulate gene expression at the post-transcriptional level.In plants,two consecutive cleavages of RNA Polymerase II(Pol II)-transcribed primary transcripts by Dicer-Like 1(DCL1)produce miRNA duplexes.The guide strand of miRNA duplex is incorporated into ARGONAUTE1(AGO1)to form RNA-induced Silencing Complex(RISC).miRNAs,by binding target m RNAs,direct target m RNA degradation or translational repression.miRNA biogenesis and activity are delicately regulated,however,knowledge about the underlying mechanisms remains limited.To identify regulatory factors and mechanisms in the miRNA pathway,we previously conducted a suppressor screen of ema1-1,a mutant that has highly clustered trichome due to enhanced activity of the artificial miRNA amiR-trichome and identified46 soe(suppressor of ema1)mutants.Here we investigated soe5 and soe6.soe6 carries a mutation in CYP71,which encodes a Peptidyl-Prolyl cis/trans Isomerase(PPIase)family protein.In soe6 and cyp71 mutants,miRNA levels are decreased and target m RNA levels are increased.Complementation of cyp71 mutants with wild-type CYP71 could restore miRNA and target m RNA level,suggesting that CYP71 regulates miRNA biogenesis.CYP71 does not regulate transcription of MIR genes into pri-miRNAs.It associates with Dicing complex and promotes pri-miRNA processing by DCL1.PPIase enzymes catalyze the transition between cis and trans Proline residues and can modulate protein conformational dynamics.Complementation of cyp71 mutants with CYP71 variants with diminished proline cis/trans isomerase activity could not restore miRNA and target m RNA levels,suggesting that CYP71 enhances pri-miRNA processing through catalyzing DCL1 conformational transition.These findings reveal regulation of miRNA biogenesis by structural rearrangement of DCL1.soe5 also displays severe miRNA defiency.soe5 carries a mutation in CUE1,which encodes a phosphoenolpyruvate(PEP)translocator that can import PEP into chloroplasts.PEP is metabolized to produce aromatic amino acids including Tyrosine(Tyr),Phenylalanine(Phe)and Tryptophan(Trp).We demonstrate that Tyr but not the other two positively regulates miRNA biogenesis,because tocopherols,the lipid-soluble antioxidants derived from Tyr are required for the miRNA accumulation.Mutants lack of tocopherol biosynthesis show accelerated pri-miRNA decay,suggesting a role of tocopherols in preventing pri-miRNA degradation.These findings reveal regulation of miRNA biogenesis by a chloroplast-to-nucleus retrograde signaling pathway.