An Importin β Protein Negatively Regulates MicroRNA Activity In Arabidopsis

In plants, microRNAs (miRNAs) are a class of～21nt small RNAs processed from pri-miRNA with stem-loop structure by Dicer like protein1(DCL1). The mature miRNA can be loaded into Argonautel (AGO1) to form the RISC (RNA induced silencing complex) which guides the target transcript cleavage and translational repression. MiRNAs play an important role in modulating many biological progresses, including phase transition, adaptive stresses responses and maintenance of genome stability. As key components in the eukaryotic gene regulatory network, miRNAs themselves are subjected to fine regulation.To identify new factors involved in modulating miRNA activity, we took advantage of an Arabidopsis thaliana transgenic line amiR-triOX that overexpresses an artificial miRNA (amiR-trichome), amiR-trichome is designed to target three R3MYB genes, CPC, TRY and ETC2. These genes suppress trichome initiation and differentiation in a redundant manner, decrease of them causes an increase in trichome density and/or trichome clustering. The overexpression of amiR-trichome in the transgenic plants resulted in clustered trichomes on leaf blades utilized as a visible selection mark. Then we carried out a forward genetic screen for mutants with compromised miRNA activity (cma mutants) or enhanced miRNA activity (ema mutants). From this screen, we identified dozens of cma and ema mutants. Among the cma mutants, we isolated two new mutant alleles of SERRATE (SE) known to be required for miRNA biogenesis, validating the efficacy of the screen.In this study, we analyzed an ema mutant, emal. In emal-1mutant, the clustering of trichomes was significantly enhanced and the accumulation of amiRNA-trichome targets and endogenous miRNAs targets were decreased, suggesting EMA1generally regulates miRNA activity. Map-based cloning revealed that emal-1carries a G to A transition in the junction between the19th exon and the18th intron of At2g31660, which leads to a splicing defect. EMA1encoded an Importin β protein, which may be involved in nuclear transport. Overexpression of EMA1in plant cells could lead to decreased miRNA activity. Taken together, these results demonstrate that EMA1is a negative regulator of the miRNA pathway. Genetic analysis indicated that emal mutation can partially rescues the phenotype of hyl1-2mutant but not ago1-25mutant. This suggests that EMA1functions through AGO1to modulate miRNA activity. The emal mutation had no effects on the accumulation of miRNAs and AGO1or their cytoplasmic and nuclear distributions. Intriguingly, we found that the miRNA effector complexes purified from emal contained a larger amount of miRNAs and displayed elevated mRNA cleavage activities, indicating that EMA1modulates miRNA activity through influencing the loading of miRNAs into AGO1complexes. These results implicate EMA1as a negative regulator of the miRNA pathway and reveal a new layer of miRNA activity modulation.