基于GAS5的长链非编码RNA转运出核机制的研究

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.

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。

LncRNA除了不编码蛋白外与mRNA非常相似，但其在胞内定位的机制不清。为了探讨lncRNA转运出核的机制，我们构建了三个lncRNA报告基因GAS5,ANCR和998。显微注射结合RNA-FISH表明这些报告基因转录产物定位于细胞质中，敲低mRNA出核关键因子UAP56、Thoc2、TAP和Xab2同样抑制这些lncRNA转运出核。为了进一步鉴定lncRNA转运出核反式因子，我们利用Flp-In技术构建了诱导型过表达3’端带发夹结构的lncRNA稳转细胞株，利用MS2-MBP与发夹机构的高亲和力纯化了在细胞核与细胞质内形成的lncRNP，并通过质谱鉴定其中组份。通过siRNA敲低结合RNA-FISH结果筛选发现NPC复合物组份nup98、RAE1及EJC复合物组份eIF4A3特异性影响lncRNA出核，但对mRNA β-globin和GADD45的出核无明显影响。进一步筛选发现NPC复合物中近半组份以及EJC复合物核心组份敲低后显著抑制lncRNA出核，表明这两个复合物在lncRNA出核中发挥重要作用。我们通过ANCR与β-globin报告基因序列嵌合结合RNA-FISH，通过系列截短实验发现nup98敲低后特异性抑制ANCR出核是由ANCR第三外显子中一段316nt的序列介导的，删除这一序列后敲低nup98不再能将ANCR滞留于细胞核中，而插入这一序列使得nup98敲低后原本定位于细胞质中的β-globin发生显著核滞留，表明这段序列在天然和异源背景下均能发挥功能。此外，敲低EJC组份Y14导致包括GAS5在内的四种lncRNA在细胞核中富集，表明Y14参与了内源性lncRNA转运出核。我们也探讨了lncRNA MEG3核滞留机制，发现MEG3中存在核滞留序列NRE，它通过招募U1 snRNP中多个组份介导MEG3的核滞留。本项目揭示了lncRNA出核以及核滞留的重要机制。