Identification And Characterization Of Novel Components In The Arabidopsis Microrna Pathway

MicroRNAs (miRNAs) are a class of small RNAs (sRNAs) that regulate various biological processes in eukaryotes. The biogenesis of miRNAs involves the transcription of primary miRNAs and their subsequent processing by Dicer-like1(DCL1) into mature miRNAs. Mature miRNAs are loaded into Argonaute1(AG01) to form RISC (RNA induced silencing complex) to regulate target genes through mRNA cleavage and/or translational repression.From a genetic screen that is based on an artificial miRNA causing trichome clustering when overexpressed, we have obtained dozens of mutants with enhanced miRNA activity (ema) or compromised miRNA activity (cma). In this study, we analyzed cma33. CMA33encodes a nuclear localized protein XCT (XAP5ORCADIAN TIMEKEEPER). XCT has been previously shown to be involved in circadian clock and ethylene response. The cma33/xct mutation led to reduced accumulation of miRNAs as well as DCL3-dependent heterochromatic siRNAs (hc-siRNAs) and DCL4-dependent trans-acting siRNAs (ta-siRNAs). Surprisingly, we found that the expression of DCL1, DCL3and DCL4, but not other genes involved in sRNA pathways, was decreased in cma33/xct mutant. Consistent with this, the occupancy of Pol II at DCL1, DCL3and DCL4, was reduced upon the loss of CMA33/XCT, suggesting that CMA33/XCT regulates the biogenesis of sRNAs through regulating the transcription of DCLs.We have previously charactererized EMA1, which encodes an importin β protein that negatively regulates the loading of miRNAs into AGO1. Here, we further performed a suppressor screening of emal (soe) to more sensitively identify new factors in the miRNA pathway. Here, we characterized two of the mutants, soel and soe2. SOE1and SOE2encode ELP2and ELP5, two subunits of the evolutionarily conserved Elongator complex, respectively. Disruption of Elongator complex reduced the occupancy of RNA polymerase II at miRNA genes and the levels of a subset of pri-miRNAs, indicating that Elongator complex is involved in the transcription of pri-miRNAs. Moreover, Elongator interacted with the Dicing complex and the lack of Elongator impaired the localization of DCLl. More importantly, we found that pri-miRNA transcripts as well as DCL1were enriched on chromatin, and the association of DCL1with chromatin was compromised in the absence of Elongator. These results together suggest that the transcription and processing of pri-miRNAs are coupled by Elongator.