Noncoding RNAs at the heart of the Prader-Willi locus

描述(申请人提供)：Prader-Willi综合征(PWS)是一种神经发育障碍，具有已知的遗传病因，但具有复杂的表观遗传学基础。PWS是一种印迹疾病，这意味着只在父亲染色体上表达的基因，而不是母亲染色体15q11-13区域的基因负责。此外，与影响蛋白质编码基因的基因突变不同，导致PWS的最小基因缺失只影响RNA的非编码转录本。在PWS中最小缺失区域的核心是两种类型的非编码RNA。首先，HBII-85/SNORD116小核仁RNA(SnoRNAs)定位于成熟神经元的核仁，影响rRNA和核仁的成熟。其次，HBII-85/SNORD116 snoRNAs周围的宿主基因外显子被剪接，核保留为一个长的非编码RNA(LncRNA)，形成一个大的RNA云状结构，其大小随着神经元的成熟而增大。虽然PWS领域的大部分焦点都集中在了解snoRNAs和蛋白质编码基因的功能上，但宿主snoRNA-lncRNA对于理解和治疗PWS潜在的神经发育缺陷可能具有同等甚至更重要的作用。在这项提案中，我们试图回答关于PWS分子发病机制的三个主要未解答的问题。1)成熟神经元中染色质解缩的机制基础是什么？2)哪个ncRNA负责PWS表型，snoRNAs还是宿主lncRNA？3)与PWS基因座缺失和复制相关的人/鼠表型差异的遗传和机制基础是什么？这些方法包括在小鼠和人脑中联合检测R环、ncRNAs和染色质解缩的新的荧光原位方法，以及新的鼠-人杂交神经元细胞系和人PWS诱导的多能细胞来源的神经元。这些实验的结果有望提高对位于PWS基因中心的lncRNAs功能作用的理解，并有可能使未来的PWS表观遗传疗法成为可能。

DESCRIPTION (provided by applicant): Prader-Willi syndrome (PWS) is a neurodevelopmental disorder with a known genetic etiology, but a complex epigenetic basis. PWS is an imprinted disorder, meaning that genes expressed only on the paternal but not the maternal chromosome 15q11-13 region are responsible. Furthermore, unlike genetic mutations that affect protein-coding genes, the smallest genetic deletions causing PWS only affect noncoding transcripts of RNA. At the heart of the minimally deleted region in PWS are two types of noncoding RNAs. First, the HBII-85/SNORD116 small nucleolar RNAs (snoRNAs) localize to the nucleolus in maturing neurons and impact rRNA and nucleolar maturation. Second, the host gene exons surrounding the HBII-85/SNORD116 snoRNAs are spliced and nuclear retained as a long noncoding RNA (lncRNA), forming a large RNA cloud-like structure that increases in size with neuronal maturity. While most of the focus in the PWS field has been on understanding the function of the snoRNAs and protein coding genes, the host snoRNA-lncRNA may be of equal if not greater importance to understanding and treating the underlying neurodevelopmental defect in PWS. In this proposal, we seek to answer three major unanswered questions regarding the molecular pathogenesis of PWS. 1) What is the mechanistic basis for chromatin decondensation specifically in mature neurons? 2) Which ncRNA is responsible for the PWS phenotype, the snoRNAs or the host lncRNA? 3) What are the genetic and mechanistic bases of the mouse/human phenotypic differences associated with the PWS locus deficiency and duplication? The approaches include novel fluorescence in situ methods for combined detection of R-loops, ncRNAs, and chromatin decondensation in mouse and human brain and novel mouse-human hybrid neuronal cell lines and human PWS induced pluripotent cell derived neurons. The results of these experiments are expected to improve understanding of functional role of the lncRNAs at the heart of the PWS locus and potentially enable future epigenetic therapies for PWS.