A Role Of TRANSPORTIN1 In Regulating MicroRNA Activity In Arabidopsis

MicroRNAs(miRNAs)are a class of 21-to 24-nucleotide small RNAs(sRNAs)that regulate a wide range of biological processes in eukaryotes.In Arabidopsis thaliana,miRNA loci are transcribed by RNA polymerase II into primary miRNA transcripts(primiRNAs),which are cotranscriptionally processed by DICER-LIKE1(DCL1)into miRNA/miRNA* duplexes through two sequential cleavage steps.After processing,miRNA/miRNA* duplexes are predominantly loaded into ARGONAUTE1(AGO1).The passenger strand of the miRNA duplex is selectively displaced,whereas the guide strand remains associated with AGO1 to form the core of miRNA-induced silencing complex(miRISC).As directed by base complementarity,miRISC is guided to its target mRNA,resulting in mRNA cleavage or translational repression.Despite the progress in understanding the mechanism of miRNA loading,factors involved in this process and the mechanisms underlying their actions remain poorly defined.In a genetic screen for components involved in the miRNA pathway in Arabidopsis thaliana,we previously identified ENHANCED MicroRNA ACTIVITY1(EMA1),an importin ? family protein,as a negative regulator of miRNA activity.The Mutation of EMA1 leads to increased association of miRNAs with AGO1,thereby enhances miRNA activity.In this study,through a suppressor screening of ema1,we identified another importin ? protein,TRANSPORTIN1(TRN1),as a regulatory component in the miRNA pathway.TRN1 mutation impaired the activity of the majority of endogenous miRNAs without reducing miRNA accumulation,suggesting that TRN1 regulates miRNA activity not through regulating the biogenesis or stability of miRNAs.TRN1 could interact with AGO1 and mutation of the three conserved residues required for cargo recognition of TRN1 reduced its interaction with AGO1 and impaired its function in regulating miRNA activity.Intriguingly,TRN1 dysfunction did not change the cytoplasmic-nuclear distribution of miRNAs and AGO1 but reduced the amount of miRNAs associated with AGO1.These results indicate that TRN1 positively regulates miRNA activity by promoting the association of miRNAs with AGO1.In summary,this study reports the function of an importin ? family protein TRN1 in modulating miRNA activity and reveals the mechsnism of its action.The findings of this study also suggest the complex regulatory roles of importin ? family proteins in miRNA pathway.