RNA在胞内的正确定位对于其转录后加工、稳定性及功能发挥至关重要。新近发现的长链非编码RNA（lncRNA）数量庞大且多数位于细胞质中，但其转运出核机制尚未阐明。申请人曾对人无内含子mRNA出核机制进行深入研究，纯化鉴定了出核元件RNP主要蛋白组份（PNAS，2011；NAR，2013），并在预实验中确定lncRNA GAS5同时分布于细胞核与细胞质中。 据此，将以GAS5为模型对lncRNA出核机制进行深入探讨。首先通过MS2-MBP亲和纯化技术分离并鉴定GAS5核内RNP组份，从中筛选GAS5出核关键蛋白；然后通过显微注射和RNA-FISH探讨5’帽端结构、剪接与3’polyA尾在GAS5出核中的作用，并探讨GAS5中是否存在顺式出核元件；最后利用基因芯片技术检测GAS5出核关键蛋白过表达或敲低对内源性lncRNAs在核质中分布的影响。本研究将建立lncRNA出核通路并阐明其出核机制。

The correct localization of RNAs in cell is essential for their post-transcriptional processing, stability and function. The number of lncRNA identified in recent years expands rapidly with most of them located in the cytoplasm, however, mechanism of lncRNA export has still been elusive. We previously examined the mechanism on the export of naturally intronless mRNAs and purified the RNP assembled on export element (PNAS, 2011; NAR, 2013). GAS5 is one of the lncRNAs located in both nucleus and cytoplasm as showed in our preliminary study. Thus we plan to study the mechanism on lncRNA export using GAS5 as a model in this project. We will first identify components in GAS5 RNP assembled in the nucleus using MS2-MBP affinity purification followed by mass spectrometry, and then screen for key factors involved in the export of GAS5. Next we will investigate the role of 5’ cap, splicing and 3’ polyA tail in the export of GAS5 using microinjection and RNA-FISH. We will also explore whether there is cis-element that functions in GAS5 export. Finally we will compare the nucleo-cytoplasmic distribution of endogenous lncRNAs using genechip technology under the condition of over-expression or knockdown of key factors for GAS5 export. The project aims to build the export pathway for lncRNAs and gain deep insight into the mechanism of lncRNA export.