HYL1 (HYPONASTIC LEAVES 1) in Arabidopsis thaliana encodes a double-stranded RNA-binding protein needed for proper miRNA maturation, and its null mutant hyl1 shows a typical leaf-incurvature phenotype. In Chinese cabbage, BcpLH (Brassica rapa ssp. pekinensis LEAFY HEADS), a close homolog of HYL1, is differentially expressed in juvenile leaves, which are flat, and in adult leaves, which display extreme incurvature. BcpLH lacks protein–protein interaction domains and is much shorter than HYL1. To test whether BcpLH is associated with defects in microRNA (miRNA) biogenesis and leaf flatness, we enhanced and repressed the activity of BcpLH by transgenics and investigated BcpLH-dependent miRNAs and plant morphology. BcpLH promoted miRNA biogenesis by the proper processing of primary miRNAs. BcpLH downregulation via antisense decreased a specific subset of miRNAs and increased the activities of their target genes, causing upward curvature of rosette leaves and early leaf incurvature, concurrent with the enlargement, earliness, and round-to-oval shape transition of leafy heads. Moreover, BcpLH-dependent miRNAs in Chinese cabbage are not the same as HYL1-dependent miRNAs in Arabidopsis. We suggest that BcpLH controls a specific subset of miRNAs in Chinese cabbage and coordinates the direction, extent, and timing of leaf curvature during head formation in Brassica rapa.
MicroRNA (miRNA) plays an important role in the control of gene expression. HYPONASTIC LEAVES1 (HYL1) is a double-stranded RNA-binding protein that forms a complex with DICER-LIKE1 (DCL1) and SERRATE (SE) to process primary miRNA (pri-miRNA) into mature miRNA. Although HYL1 has been shown to partner with DCL1 to enhance miRNA accuracy, the mechanism by which HYL1 selects the DCL1-targeted cleavage sites in pri-miRNA has remained unknown. By mutagenesis of HYL1 and analysis of in vivo pri-miRNA processing, we investigated the role of HYL1 in pri-miRNA cleavage. HYL1 forms homodimers in which the residues Gly147 and Leu165 in the dsRBD2 domain are shown to be critical. Disruption of HYL1 homodimerization causes incorrect cleavage at sites in pri-miRNA without interrupting the interaction of HYL1 with DCL1 and accumulation of pri-miRNAs in HYL1/pri-miRNA complexes, leading to a reduction in the efficiency and accuracy of miRNAs that results in strong mutant phenotypes of the plants. HYL1 homodimers may function as a molecular anchor for DCL1 to cleave at a distance from the ssRNA–dsRNA junction in pri-miRNA. These results suggest that HYL1 ensures the correct selection of pri-miRNA cleavage sites through homodimerization and thus contributes to gene silencing and plant development.
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