Functional studies of Arabidopsis HYL1, a dsRNA-binding protein required for microRNA metabolism

The Arabidopsis HYPONASTIC L&barbelow;EAVES 1&barbelow; (HYL1) gene encodes a dsRNA-binding protein containing two conserved dsRNA-binding motifs (dsRBMs) in its N-terminus, a central bipartite nuclear localization signal (NLS), and a C-terminal repeat structure (six near-perfect 28-aa repeats in Col0, and five in No0, Ler, and WS ecotypes). The hyl1 null mutant exhibits dramatic developmental defects and altered responses to several phytohormones and stresses, overlapping with those observed in dcl1 and hen1 mutants. The ABA-hypersensitivity of the hyl1 mutant is correlated with the accumulation of ABI5 and the activities of two ABA-induced MAPKs, in which the accumulation of ABI5 and the activities of the two MAPKs are higher in hyl1 than in wildtype plants at a lower ABA concentration in hyl1 mutant than in wildtype plants. Immunocomplex kinase activity assays using HA-tagged AtMPK3 and AtMPK6 suggested that the ABA-activated 42- and 46-kDa MAPKs in the in-gel kinase activity assay are AtMPK3 and AtMPK6, respectively. Two putative ABI5 kinases are activated transiently by ABA with similar sizes and activation kinetics as the two ABA-activated MAPKs, and the activities are higher in hyl1 mutant than in wildtype plants, suggesting that either the two putative ABI5 kinases are actually the ABA-induced MAPKs, or the ABA signal is transmitted to ABI5 kinases via MAPK cascades. HYL1 is a phosphoprotein and can be phosphorylated in vitro and in vivo. Two putative HYL1 kinases migrated at about 42- and 46-kDa were transiently activated by ABA in the in-gel kinase activity assay. Strikingly, they had very similar activation kinetics, albeit much weaker, as observed in the in-gel kinase activity assays using MBP or ABI5 as a substrate. HYL1 protein possesses seven putative MAPK phosphorylation sites and can be a substrate of AtMPK6 in vitro. The accumulation of several microRNAs (miR159, -167, and -171) are less abundant in the hyl1 mutant and more abundant in 35S::HYL1 plants as compared to wildtype plants, suggesting that HYL1 plays a role in the miRNA metabolism. The mRNA levels of MYB33 (target of miR159) and SCL6-III (target of miR171) are higher in the hyl1 mutant than in wildtype plants, indicating that the decreased accumulation of miRNAs in the hyl1 mutant reduces the target mRNA cleavages. The mRNA level of ARF8 (target of miR167) is unchanged in the hyl1 mutant, suggesting that the residual amount of miR167 in the hyl1 mutant is sufficient to maintain target cleavage. (Abstract shortened by UMI.)...