蛋白磷酸酶4调控植物miRNA合成的分子机制

MicroRNA (miRNA) is an important regulator of gene expression in eukaryotes, our understanding of miRNA biogenesis, mode of action and regulation in plants has been more advanced than that in animals. An Arabidopsis mutant carrying mutation in the gene encoding a regulation subunit of protein phosphatase 4 complex (PP4) was screened out from our mutant population with artificial miRNA defects, the genes encoding catalytic subunit of protein phosphatase 4 were also knocked-out using CRISPR system. Accumulation of transgenic and endogenous pri-miRNAs and mature miRNAs is obviously reduced in the pp4 mutants, confirming promotion of PP4 on the miRNA biogenesis. In this study, we are focusing on 1) differential regulation of rice PP4 in development and miRNA biogenesis through mutation of PP4 subunits by strategy of CRISPR-editing genome; 2) gene cloning and characterization of suppressors of pp4 mutant already screened out from EMS-mutagenized pp4 population, acquiring knowledge on regulation and action of the PP4; 3)identification of the substrate(s) in the miRNA biogenesis pathway through combination of PP4 co-immunoprecipitation/mass spectrometry (Co-IP MS) result with the changed protein phosphorylation level, verification of the substrate(s) by protein interaction and mutation on putative phosphorylation sites, introduce of hyper-/hypo-phosphorylated substrate(s) into their loss-of-function mutants and pp4 mutants, observation of phenotypic changes of the mutants and examination of protein phosphorylation level of candidate substrate(s) in the pp4 mutants, uncovering molecular mechanism of protein phosphatase 4 in plant miRNA biogenesis and development.

MicroRNA (miRNA) 是一类真核生物基因表达的重要调控因子，植物中对miRNA产生、作用方式及调控机制的很多方面研究领先于动物。在筛出拟南芥蛋白磷酸酶4复合体调节亚基功能缺陷的突变体并成功用CRISPR敲除催化亚基基因，明确蛋白磷酸酶4作用于miRNA合成的基础上，本次研究通过CRISPR突变水稻蛋白磷酸酶4亚基的基因，对蛋白磷酸酶4调控水稻生长发育和miRNA合成有新认识；利用已经筛出蛋白磷酸酶4突变的抑制突变体，对抑制子基因的克隆和功能分析阐述蛋白磷酸酶4的调控和作用机制；通过Co-IP联合质谱鉴定蛋白磷酸酶4的候选底物，选取其中在miRNA合成中起关键作用的候选底物进行磷酸化位点突变后转入相应的突变体和蛋白磷酸酶4突变体，观察转基因植株的表型和检测磷酸化水平，确定蛋白磷酸酶4的作用底物，从而阐明蛋白磷酸酶4调控植物miRNA合成和生长发育的分子机制。

蛋白磷酸酶4（protein phosphatase 4, PP4）是酵母、植物和动物中高度保守的丝氨酸/苏氨酸蛋白磷酸酶，然而植物中PP4的组成和功能知之甚少。该研究揭示了拟南芥PP4的复杂性，并鉴定出含有调节亚基PP4R3A（protein phosphatase 4 regulatory subunit 3A）的一种形式的PP4组成。研究发现PP4R3A与两个高度冗余的催化亚基基因PPX1和PPX2之一共同促进microRNA (miRNA) 的生物合成。PP4R3A是一种染色质结合蛋白，与RNA聚合酶II（DNA-dependent RNA polymerase II, Pol II）相互作用，并将Pol II募集到MIR基因的启动上以促进其转录。PP4R3A可能还促进miRNA前体的共转录加工，它将microprocessor组分HYL1募集至MIR基因和细胞核剪切体中。此外研究还发现数百个内含子在pp4r3a突变体中表现出剪切缺陷。总之，该项目研究了拟南芥PP4复合体的组成和功能，揭示了PP4能够协调miRNA转录和加工，并且在细胞核RNA的调节方面具有广泛的作用。