Drosha/DGCR8复合物调控microRNA加工成熟过程的结构与功能研究

MicroRNA (miRNA) offers an efficient way for gene regulation in eukaryotes. It is widely studied for its important roles in expression repression of many genes that may cause diseases. MicroRNAs regulate gene expression not only at the transcriptional level, but also at post-transcriptional and translational levels. In various diseases especially for cancers, dysregulation of certain types of miRNAs has been identified, as well as key protein components that function in miRNA processing regulation. Therefore, it is quite worthful to dissect the mechanism of how miRNA biogenesis is regulated and this will provide useful information for diagnosis and drug discovery..Pri-miRNA is primary transcribed from certain genomic regions. Generally, it will undergo two steps of processing to produce the mature miRNA, which occur in the nucleus and cytoplasm, respectively. The first processing step is catalyzed by an RNase III enzyme Drosha in complex with its partner DGCR8, which is called the microprocessor complex. However, the details of this site-specific processing reaction are still largely unknown. This project aims to investigate the mechanism of how Drosha/DGCR8 complex specifically recognizes and processes pri-miRNAs. Using X-ray crystallography, we plan to solve the structure of Drosha/DGCR8-miRNA complex. These findings will allow us to identify the key domains and RNA motifs that determine the complex formation and pri-miRNA recognition. Moreover, biochemical and functional studies will be performed to clarify the action mode of Drosha, thus lead us to gain insights into the molecular mechanism of pri-miRNA processing.

MicroRNA（miRNA）作为真核生物基因表达调控的重要途径一直是研究热点。其在转录水平、转录后修饰、翻译水平等各方面参与调节一些重要基因的表达。目前已在多种癌症中发现相关miRNA、以及miRNA加工成熟过程关键蛋白因子的异常表达。因此，深入了解miRNA表达调控的机制对于疾病诊疗、药物靶向研发等具有重要意义。.由基因组上特定区域转录生成的初级miRNA（pri-miRNA）需经过核内与胞质两步切割反应才能形成成熟miRNA。其中，核酶Drosha与伴侣蛋白DGCR8组成的microprocessor复合物催化完成第一步加工反应，然而其内在的机理并不明确。本项目拟采用X-射线晶体学手段，解析Drosha/DGCR8与pri-miRNA复合物三维结构，并结合生化功能实验，阐明Drosha/DGCR8蛋白复合物与底物miRNA相互作用的关键因素，从而揭示miRNA早期加工成熟的分子机制。

microRNA（miRNA）是为一类长度约22个碱基、具有重要生物学功能的非编码RNA，在基因表达调控等关键生命活动过程中发挥不可或缺的作用。它们一方面与细胞分化、个体发育等息息相关，另一方面与疾病之间存在密切关联，其中一些已被作为癌症等疾病的诊断标志物和药物研发靶点。因此，miRNA的功能及其自身的生成与调控机制一直都是研究热点。成熟miRNA是由一条包含一个茎环结构的更长转录本（又称pri-miRNA）经过两步切割反应而产生。第一步切割反应在细胞核内进行，由Drosha/DGCR8复合物催化切割pri-miRNA生成前体miRNA。随后前体miRNA出核，在细胞质由另一RNA酶Dicer完成第二步切割。目前关于miRNA加工的机制研究尚不全面，特别是核内切割步骤中，Drosha/DGCR8复合物如何对pri-miRNA进行特异性识别和精确加工的机制一直未被明确。本项目经过系统优化等方法，成功获得了纯度、均一性好的重组Drosha/DGCR8蛋白质复合物，并利用单颗粒冷冻电镜方法解析了Drosha/DGCR8与pri-miRNA的复合物结构。研究结果证实了Drosha在切割位点界定中具有决定性作用，并明确其通过多结构域协同作用完成pri-miRNA的识别和切割位点定位。此外，通过对比单独蛋白质与蛋白质-RNA复合物的结构信息，发现这些关键结构域在结合底物前后出现了显著的构象变化，形成了pri-miRNA关键特性识别的独特模式。此外，未结合底物时Drosha处于自抑制状态，结合pri-miRNA能促使其从抑制到活化状态的转换，这种活性调控机制有效保障了在体内复杂环境中对正确底物进行识别与切割。概括而言，本项目的研究成果揭示了Drosha/DGCR8如何特异性识别切割pri-miRNA的分子机制，阐明了pri-miRNA核内加工调控这一领域内困扰多年的科学问题，具有重要科学意义。