绘制灵长类动物大脑长链非编码RNA时空表达地图

It is of great importance to perform primate-based in vivo study for exploring the roles of those long non-coding RNAs (lncRNAs) associated with neuronal development/function, due to the significant spatio-temporal manner and relatively low conservation of many lncRNAs. Recent work on primate genome sequencing, lncRNA knockout mice and successful CRISPR-Cas9-mediated gene targeting in a wide range of animal species have provided sufficient basis and feasibility to build lncRNA gene engineering model in non-human primate. However, the information about brain-associated lncRNAs and their spatio-temporal expression pattern is very limited in human and non-human primates. In this proposal, we will first use deep sequencing to uncover known/novel primate-expressed lncRNAs located in different brain areas. Next, we will investigate their temporal expression pattern during marmoset brain development by RT-qPCR, as well as their subcellular location via in situ. Furthermore, we will establish a novel marmoset-based primate model for lncRNA study using the recently developed CRISPR-Cas9 gene editing technology. Our work will provide a solid foundation for the further studies of lncRNA function in nervous system, as well as establish a suitable new animal model for the lncRNA field.

神经系统长链非编码RNA（lncRNA）具有显著的时空特异性，且物种间保守性较低，建立灵长类动物模型是该领域亟待解决的一个重要问题。近期在灵长类动物基因组测序、lncRNA基因敲除小鼠和利用CRISPR-Cas9基因编辑技术在多种动物上取得的成功经验为建立lncRNA研究的灵长类动物模型提供了充分依据和可行性。然而有关人和非人类灵长类动物大脑表达的lncRNA及其时空表达特征的信息十分匮乏。因此，本课题将首先利用深度测序发掘狨猴重要脑功能区/核团的新的lncRNA，进而通过RT-qPCR和RNA原位杂交技术探索其在大脑不同发育阶段的表达情况和亚细胞定位，由此获得狨猴lncRNA的脑功能区时空表达谱。本课题还将采用CRISPR-Cas9技术，建立新的以狨猴为基础的神经系统lncRNA基因定向敲除模型。该课题将为深入探索lncRNA在神经系统中的生理和病理学功能奠定坚实的工作基础。

“绘制灵长类动物大脑长链非编码RNA时空表达地图”这一项目是基于长链非编码RNA（long noncoding RNA, lncRNA）在灵长类动物、神经系统以及在体动物研究等方面亟需深入研究的基础上提出并开展的。在研究中，我们以更为丰富的内容全面开展了项目书中计划的研究内容，工作从以下3个方面开展：（1）绘制灵长类动物大脑lncRNA时空和性别表达谱；（2）可变启动子与RNA动态表达的相关性；（3）神经系统lncRNA功能的研究。取得了以下主要进展：（1）建立了非人灵长类动物大脑lncRNA的时空和性别表达谱；（2）发现可变启动子现象在lncRNA的动态调控中普遍存在；（3）发现了一系列在进化中高度保守的神经系统特异性表达lncRNA，并初步建立了其中部分lncRNA的CRISPR-Cas9动物模型。基于本项目，项目负责人以共同通讯或通讯作者身份发表论文一篇（Genome Research，2017），特邀综述和综述各一篇。项目的研究结果为进一步研究神经系统lncRNA的功能和作用分子机理奠定了基础，为进一步探索与脑发育、脑疾病相关的研究和治疗提供了新的方向，具有良好的学术推广价值、重要的科学意义和潜在的应用前景。