CapRNA在表观遗传调控中的作用与机制

Human genome produces a large number of long non-coding RNAs (lncRNAs) which are 200 to a few thousand nucleotides (nt) long and are generally expressed in low abundance and in a tissue specific manner. Among these lncRNAs, only a few dozen have been functionally characterized. Epigenetic marking especially DNA methylation is an important mechanism of gene regulation. Despite intensive study in the past two decades, a fundamental question that remains unanswered is how a particular epigenetic pattern in normal cells and disease is established and maintained. In our preliminary study we identified a potentially new class of lncRNAs which we termed CpG-associated promoter RNAs (CapRNAs). CapRNAs are transcribed from gene promoters in close proximity to CpG islands with a size around 500 nt and are potentially exported by XPO5, a karyopherin protein, to the cytoplasm to be processed into small RNAs or for disposal. We hypothesize that CapRNAs participate in epigenetic regulation of their associated protein coding genes and dysregulation of CapRNAs underlies aberrant epigenetic regulation in cancer cells. In this application, we will apply the CRISPR technique combined with RNA-seq, and biochemical and molecular biology assays to identify and characterize genome-wide CapRNAs and to determine the mechanisms by which CapRNAs regulate the expression of associated genes. Accomplishment of the proposed study will allow us to build a fundamentally important mechanistic model for the origin of aberrant cancer epigenetics. This study will also provide an unprecedented novel method for identifying and functionally characterizing lncRNAs which are otherwise difficult to detect by conventional methods due to their low abundance.

人基因组存在广泛的源自启动子序列的转录本，但对其功能知之甚少。本课题前期研究发现一类转录于基因启动子CpG岛附近的长链非编码RNA，我们称之为CapRNA（CpG island-associated promoter RNA）。CapRNA的主要特征是其丰度受RNA转运蛋白XPO5的影响，在XPO5敲低细胞其表达显著上升，并改变下游基因表达。我们假设CapRNA可能参与下游基因启动子的表观遗传调控，XPO5及其他RNA转运蛋白如XPO1可能介导CapRNA的降解或出核。该通路异常导致CapRNA在胞核的聚集，进而触发异常的表观遗传信号。本研究拟通过基因敲除技术结合深度测序与生化、分子生物学分析在基因组水平系统发现CapRNA，阐明其生物学特征及其对蛋白编码基因的表观遗传调控作用与机制。该研究有望建立一个重要的关于异常表观遗传起源的新模式，同时建立一种发现低表达长链非编码RNA的新方法。

人基因组存在广泛的源自启动子序列的转录本，但对其功能知之甚少。本课题前期研究发现一类转录于基因启动子CpG岛附近的长链非编码RNA，我们称之为CapRNA（CpG island-associated promoter RNA）。CapRNA的主要特征是其丰度受RNA转运蛋白XPO5的影响，在XPO5敲低细胞其表达显著上升，并改变下游基因表达。我们假设CapRNA可能参与下游基因启动子的表观遗传调控，XPO5及其他RNA转运蛋白如XPO1（CRM1）可能介导CapRNA的降解或出核。该通路异常导致CapRNA在胞核的聚集，进而触发异常的表观遗传信号。本研究通过基因敲除技术结合深度测序与生化、分子生物学分析等手段发现（1）CapRNA是大量存在于细胞内的一类特殊的参与蛋白编码基因表观遗传调控的lncRNA；（2）XPO5敲低后CapRNA水平改变的形式，即上调同时出现于胞浆与胞核，与早前我们的假设（XPO5敲低后导致CapRNA在胞核内聚集）存在偏差；（3）XPO5敲低并不影响CapRNA的稳定性，因此XPO5并不参与调控CapRNA的降解；（4）CRM1也可能参与某些CapRNA的调控并与CapRNA存在互作；CMR1也有可能通过调控XPO5 mRNA或者蛋白间接参与CapRNA的调控；（5）XPO5及CRM1与部分CapRNA存在互作；（6）CapRNA通过表观遗传机制如组蛋白修饰而参与下游蛋白编码基因的调控，多表现为正向调控。因此，本项目创新性地提出了在肿瘤细胞中参与表观遗传调控过程的CapRNA的概念。在肿瘤细胞中，参与CapRNA生物加工途径的基因的异常有可能在肿瘤的发生发展中起重要作用。此外，敲低或者功能抑制与CapRNA表达及生物生成有关的基因如XPO5和CRM1可作为一种发现低丰度CapRNA的手段。本项目的成果有望建立一个重要的关于异常表观遗传起源的新模式。