RNA核滞留的功能元件及分子机制

The RNA export from the nucleus to the cytoplasm is a key step for RNA quality control and gene expression regulation in eukaryotic organisms. Many long non-coding RNAs and introns are selectively detained in the nucleus. However, critical cis-elements that mediate such nuclear detention and the underlying molecular mechanisms remain elusive. Our recent studies show that hundreds of sequence-conserved introns in many RNAs are detained in nuclear matrix, hinting their functional significance in nuclear detention. We have identified numerous RNA binding proteins (RBPs) that bind those matrix-associated RNAs. To systematically investigate the mechanisms regulating RNA detention, we propose to apply and develop a few high-throughput methods for decoding RNA-RBP interactions to define essential RNA Detaining Elements (RDE) and their associated RBPs. Increasing evidence also suggests roles of RNA modification m6A in this process, therefore we will systematically map m6A modifications in detained RNAs and investigate their functional relevance. Finally, combining with the existing knowledge on RBP-RDE and RBP-RBP interactions, we will integrate the multiple-dimensional high-throughput information to decipher the RNA “detaining code”. The results would help us better understand the mechanisms of RNA quality control in nucleus and pathogenesis of diseases caused by defects in RNA nuclear export.

真核生物RNA的出核是RNA质量控制及基因表达调控的关键步骤。很多长非编码RNA和内含子RNA被选择性地滞留于细胞核内，但介导核滞留的关键顺式元件和机制尚不清楚。我们最近的研究发现大量RNA的特定内含子滞留于核基质组分，它们在序列上非常保守，提示这些序列可能发挥滞留RNA的功能；同时鉴定了一批特异结合在这类核基质RNA上的蛋白（RBPs）。为系统地研究介导RNA核滞留的机制，我们将利用和开发多种解析RNA-RBP互作的高通量方法，鉴定发挥滞留作用的关键顺式元件（RNA Detaining Element, RDE）及与其特异结合的蛋白。许多证据提示m6A修饰也可能参与调控，我们将系统鉴定核滞留RNA的m6A图谱并研究其功能和作用方式。最后整合RBP-RDE、RBP-RBP等多维高通量信息，系统解析RNA核滞留调控密码。研究结果将有助于理解RNA核内质量控制以及RNA出核缺陷导致的疾病发生。

真核生物RNA的出核是RNA质量控制及基因表达调控的关键步骤。RNA 聚合酶II转录产生mRNA和大部分非编码RNA，两者之间有很多不同点，其中一个重要区别是非编码RNA更多的定位于细胞核内。目前对于非编码RNA的核滞留元件已经有一些系统性研究，尽管有报道mRNA的核滞留也是一个普遍的现象，但对于mRNA的核滞留机制仍然缺乏系统性研究，只有个别报道如mRNA 3’UTR上的inverted Alu repeats介导的核滞留。在本项目中，我们基于Massive parallel reporter assay开发了Frag-seq的高通量方法，系统地鉴定了非编码RNA和mRNA的核滞留元件，并分析了这些元件的特征。通过系统整合已有的RNA结合蛋白的CLIP数据，筛选了潜在的有RNA核滞留作用的蛋白因子，并对部分因子进行了验证，进一步阐明了核滞留的作用机理。此外，我们发现很多的核滞留元件位于RNA的可变加工区域，如alternative 3’UTR中。我们深入研究发现RNA选择性加工偶联的RNA核滞留不仅是被动式RNA质量控制体系的一部分，它更可能主动参与了广泛的基因表达调控。RNA异常加工与RNA核滞留偶联可导致蛋白表达量下降，这可能是相关疾病发生发展的一种新机制。