一类内含子来源的长非编码RNA的加工成熟机制及功能研究

Long noncoding RNAs (lncRNAs) are mRNA-like, non-protein-coding RNAs that are pervasively transcribed throughout eukaryotic genomes. Rather than silently accumulating in the nucleus, we now know that many lncRNAs play important roles in biological processes. We have recently used deep sequencing to explore the repertoire of non-polyadenylated RNAs from HeLa cells and H9 human embryonic stem cells (hESCs), and have discovered a new class of non-polyadenylated lncRNAs that is derived from introns (we name them id-lncRNAs). This proposal is to study them in greater detail. Such RNAs can be found in many cell types examined so far, and appear to be widely expressed in human tissues. Further, the genomic region encoding some of the most abundant of these RNAs is highly enriched in hESCs and is also specifically deleted in human genetic disorder, Prader-Willi Syndrome (PWS), thus implicating these molecules in an important human disease. We will characterize how id-lncRNAs are made and what proteins associate with them. In addition, we will ask what they do in cells and begin to address their possible roles in PWS. In the first aim, we will characterize the sequences and motifs that are required for id-lncRNAs formation and also examine the expression of id-lncRNAs. We will also investigate their sub-nuclear localization and stability, and efforts will be made to follow their synthesis and dynamic localization throughout the cell cycle. The second aim is to investigate the mechanism of action of id-lncRNAs by loss-of-function and gain-of-function studies followed by the bioinformatics analysis of any effect resulted at the transcriptome level. Of particular interest of our analysis is the connection of these id-lncRNAs to Prader-Willi pathology. In addition, as id-lncRNAs accumulate to specific nuclear architectures in cells, we will also ask whether some id-lncRNAs can alter chromatin organization like a number of other lncRNAs do. Taken together, our studies will not only provide mechanistic studies into the nature and expression of long noncoding transcripts that lack poly(A) tails in eukaryotic cells, but also have the potential to offer new insights into stem cell biology and Prader-Willi pathology.

长非编码RNA（lncRNA）是以长链RNA（>200核苷酸）形式发挥功能的非编码生物大分子，广泛参与调控一系列细胞的重要功能。我们最近发现一类新型内含子来源的lncRNAs（id-lncRNAs）。已有研究结果表明，它们在干细胞中高表达，其基因所在染色体区域提示与遗传疾病Prader-Willy Syndrome（PWS）相关；同时它们富集并形成特殊的细胞核亚结构，提示具有重要的生物学功能。本申请项目主要是在以上研究基础上，深入探索这些新型id-lncRNAs的分子特性(结构、细胞核内定位等变化特性)、转录加工机制（特异性转录、抗RNA酶降解等特征机制）和相应的生物学功能（核亚结构、干细胞干性维持等相关功能），为进一步了解长非编RNA的来源、加工等提供新的理论依据和对真核生物转录组提供更丰富的认识，为干细胞多能性维持研究提供一种新的RNA水平调控思路，为PWS综合症研究拓展新的方向。

长非编码RNA（lncRNA）是以长链RNA（>200核苷酸）形式发挥功能的非编码生物大分子，广泛参与调控一系列细胞的重要功能。我们最近发现一类新型内含子来源的lncRNAs（id-lncRNAs）。已有研究结果表明，它们在干细胞中高表达，其基因所在染色体区域提示与遗传疾病小胖威利症（PWS）相关；同时它们富集并形成特殊的细胞核亚结构，提示具有重要的生物学功能。在以上研究基础上，本项目深入探索这些新型id-lncRNAs的分子特性、转录加工机制和相应的生物学功能。在本项目的支持下，出色的完成了预定的总体目标，还进一步拓展了新的前沿领域：发现内含子衍生的两类长非编码RNA家族，即sno-lncRNAs (Mol Cell, 2012, 通讯作者。注：该研究在项目提出申请时就着手展开，在获批前顺利完成。)和ciRNAs (Mol Cell, 2013, 通讯作者)；依据sno-lncRNA分子特性创建细胞核内lncRNA功能的新型表达载体snoVector系统(Nucleic Acids Res，2015)；发现人源胚胎干细胞中多内含子snoRNAs通过可变剪接产生sno-lncRNA并具有物种特异性(BMC Genomics，2014)；进一步揭示多聚顺反子加工产生的SPA新型分子家族 及其缺失与PWS 疾病紧密关联 (Mol Cell, 2016)；此外还拓展研究发现人源胚胎干细胞中具有调控基因转录功能的内含子来源环形RNA (Mol Cell, 2013)，外显子环化来源的环形RNA (Cell, 2014)；发现细胞核内lncRNA及RNA修饰的调控新机制,包括揭示CCAT1-L调控染色体构象 (Cell Res, 2014)、发现RNA编辑酶ADAR1调控miRNA前体加工影响人源干细胞神经分化的新功能 (Cell Res, 2015)等。.研究工作系统性挖掘了人源胚胎干细胞基因组中的新型长非编码RNA分子，并深入解析了这些新型分子的特性、转录加工机制和相应的生物学功能，为进一步了解长非编RNA 的来源、加工等提供新的理论依据和对真核生物转录组提供更丰富的认识，为干细胞多能性维持研究提供一种新的RNA 水平调控思路，为PWS 综合症研究拓展新的方向。