拟南芥剪切因子RBM25参与pri-miRNA加工过程的机制研究

MicroRNAs (miRNAs) are short noncoding RNAs (20-24 nt) that regulate plant growth and adaptation through silencing of their target genes that are associated with various biological processes. Despite the great progress, there are still many key scientific questions regarding the regulation of miRNA biogenesis needed to be addressed. Recently, pri-miRNA splicing during pri-miRNA has drawn great attention, however, the molecular mechanism by which pri-miRNA processing is regulated remains largely unknown. Most recently, we have identified a new splicing factor RBM25 that regulates alternative splicing of HAB1 pre-miRNA and switch on/off of the ABA signaling. Interestingly, RBM25 mutation also affects miRNA biogenesis. Furthermore, we found that RBM25 affects pri-miRNA processing but not transcription; and intriguingly, RBM25 physically interacts with HYL1, a key component of the pri-miRNA processing complex. Together, these results point to a critical role of RBM25 in pri-miRNA processing. In this study, we will adopt a combinatorial research approach to decipher the molecular mechanism underlying the RBM25-mediated pri-miRNA processing. The objectives include: to determine whether RBM25 directly binds to the double strand/single strand pri-miRNAs and its action mode; to search for the splicing enhancers (the recognition and binding site) for RBM25; to elucidate the functional relationship between RBM25 and the pri-miRNA processing complex; to explore the potential regulatory network of RBM25-mediated splicing/processing of pri-miRNAs through screening and functional analysis of the RBM25 interacting proteins. The ultimate goal is to uncover the molecular mechanism through which the splicing machinery and pri-miRNA processing is integrated during miRNA biogenesis. The study will not only reveal a novel role of RBM25 in miRNA biogenesis, but also provide direct evidence that the splicing machinery is involved in pri-miRNA processing. The results obtained will provide novel insights into the mechanism of pri-miRNA splicing/processing and further our understanding of miRNA biogenesis, in particular pri-miRNA splicing.

miRNA是单链非编码小RNA，通过沉默其靶基因控制植物发育和适应。miRNA生物合成调控机制已取得重要进展，但很多问题急需解决。在前期研究中，我们发现了一个剪切因子RBM25，调控PP2C家族基因HAB1 pre-mRNA可变剪切体的比例和植物对ABA的响应。有意思的是RBM25也影响pri-miRNA稳定性。更重要的是RBM25与pri-miRNA加工核心组分HYL1互作，表明它在pri-miRNA加工剪切中的重要作用，但背后机制尚不清楚。在本项目中，我们拟采用综合研究手段，明确RBM25参与pri-miRNA加工的方式，阐明它与pri-miRNA加工复合体的关系和作用机制，并通过寻找其互作蛋白，深入探索剪切体在pri-miRNA加工中的机制。研究结果不仅能揭示RBM25在pri-miRNA加工中的新功能，还将明确剪切机制和pri-miRNA加工间的关系，具有重要理论意义。

microRNA (miRNA)是单链非编码小RNA，通过沉默其靶基因控制植物发育和适应。miRNA生物合成的调控机制已取得重要进展，但miRNA转录后加工过程，如pri-miRNA的剪切详尽的调控机制还不清楚。前期我们发现了一个pre-mRNA剪切复合体中的一个重要组分RBM25作为剪切因子在调控ABA信号转导负向调控因子PP2C磷酸酶HAB1编码基因的pre-mRNA的可变剪切中发挥重要功能，同时发现RBM25功能缺失造成植物生长迟滞、开化异常等表型与miRNA生物合成相关突变体非常类似，暗示RBM25有可能参与miRNA的生物加工。在本项目中，我们确定了RBM25的确在拟南芥miRNA的加工中发挥重要的调控功能，并初步阐明了其作用机制。为了确定RBM25是否参与miRNA的生物加工，我们首先通过RNA-seq比较了野生型Col-0和rbm25突变体中miRNA的丰度, 发现大多miRNA在rbm25中的丰度和野生型相比都大幅降低, 确定了RBM25的确调控miRNA的生物合成。为了确定RBM25调控miRNA生物加工的具体过程，我们采用定量PCR和启动子-GUS转录活性分析,发现RBM25并不影响miRNA的转录活性，而是影响miRNA前体含量。基于这些结果和RBM25剪切因子的特性，我们推测RBM25可能参加了pri-miRNA加工。为了进一步明确RBM25的作用机制，我们开展了酵母双杂交和BiFC蛋白质互作研究方法检测了RBM25与miRNA加工核心成员互作。结果发现RBM25与DCL1、HYL1、CBP20、CBP80和SE蛋白均可以直接互作，而且RBM25还影响CBP20在植物细胞核中的分布模式，从而为RBM25参与pri-miRNA的生物加工提供了直接证据。最后，为了进一步鉴定RBM25参与的复合物成员，我们还通过IP-MS筛选了RBM25的互作蛋白，发现RBM25复合体的蛋白中有很多前体RNA加工复合体中的多个成员，进一步确定了RBM25在前提RNA加工中的重要作用。这些研究结果说明RBM25除了参与HAB1的pre-miRNA可变剪切外，很可能是pri-miRNA转录后加工复合体的一个新组分，同时也为pre-mRNA前体剪切复合体参与pri-miRNA的加工提供了重要依据，对深入解析miRNA转录后的加工过程具有重要理论意义。