Arabidopsis thaliana strain:Columbia Transcriptome or Gene expression

MicroRNAs (miRNAs) are a class of endogenous regulatory RNAs that play an important role in many biological processes. In plants, HYPONASTIC LEAVES1 (HYL1) is a double-stranded RNA binding protein that facilitates DICER-LIKE1 (DCL1) to process primary miRNA (pri-miRNA) into mature miRNA. Recent studies showed that HYL1 is a short lived phospho-protein and is stabilized indirectly in the light by an E3 ligase, COP1, but whether the phosphorylation status is related to HYL1 stability and the mechanism by which HYL1 is constitutively stabilized remain unknown. Here, we show that SMEK1, as a suppressor of MEK, stabilizes HYL1 by inhibiting MPK3-dependent phosphorylation of HYL1 in plants. Loss-of-function of SMEK1 exhibit pleiotropic developmental abnormalities, resembling mutants that affect miRNA activities. In smek1 HYL1 was significantly reduced, which was concomitant with globally decreased levels of mature miRNAs. HYL1 is reduced in smek1 mutants either in the light or in the dark, indicating that SMEK1-mediated HYL1 stabilization is independent of the COP1 pathway. Treatments by kinase inhibitor or phosphatase inhibitor increases or reduces HYL1, respectively, suggesting that phosphorylation is anti-correlated with HYL1 stability. In vitro kinase assay showed that SMEK1 inhibits the activation of MPK3, a kinase of HYL1, indicating that SMEK1 acts as a suppressor of the MAPK pathway. Taken together, our work thus uncovers how the stability of HYL1 is regulated by the phosphorylation events, and identifies that SMEK1 acts as a negative regulator of the MAPK pathway to promote miRNA biogenesis.

微小RNA（MicroRNAs，miRNAs）是一类内源性调控RNA，在诸多生物学过程中发挥关键调控作用。在植物中，下卷叶1（HYPONASTIC LEAVES1，HYL1）是一种双链RNA结合蛋白，可协助DICER样蛋白1（DICER-LIKE1，DCL1）将初级微小RNA（pri-miRNA）加工为成熟miRNA。已有研究表明，HYL1是一种半衰期较短的磷酸化蛋白，在光照条件下可通过E3泛素连接酶COP1间接维持其稳定性，但磷酸化状态是否与HYL1稳定性相关，以及HYL1组成型稳定的具体分子机制仍未明确。本研究发现，SMEK1作为丝裂原活化蛋白激酶激酶（MEK）的抑制因子，在植物中通过抑制丝裂原活化蛋白激酶3（MPK3）依赖的HYL1磷酸化来稳定HYL1。SMEK1功能缺失突变体表现出多效性发育异常，其表型与影响miRNA活性的突变体相似。在smek1突变体中，HYL1蛋白水平显著降低，同时伴随全基因组范围内成熟miRNA水平的普遍下降。无论在光照还是黑暗条件下，smek1突变体中的HYL1水平均出现降低，这表明SMEK1介导的HYL1稳定不依赖于COP1通路。激酶抑制剂处理可提升HYL1水平，而磷酸酶抑制剂处理则会降低HYL1水平，这表明磷酸化状态与HYL1稳定性呈负相关。体外激酶实验表明，SMEK1可抑制HYL1的激酶MPK3的激活，这提示SMEK1作为丝裂原活化蛋白激酶（MAPK）通路的负调控因子发挥作用。综上，本研究阐明了磷酸化事件对HYL1稳定性的调控机制，并证实SMEK1作为MAPK通路的负调控因子，可促进miRNA的生物发生。