Noncoding RNAs at the heart of the Prader-Willi locus

描述（由申请人提供）：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基础。PWS是一种印迹疾病，这意味着只在父亲而不是母亲染色体15 q11 -13区域表达的基因是负责的。此外，与影响蛋白质编码基因的基因突变不同，导致PWS的最小遗传缺失只影响RNA的非编码转录本。在PWS中最小缺失区域的中心是两种类型的非编码RNA。首先，HBII-85/SNORD 116小核仁RNA（snoRNA）定位于成熟神经元中的核仁并影响rRNA和核仁成熟。第二，HBII-85/SNORD 116 snoRNA周围的宿主基因外显子被剪接，并作为长的非编码RNA（lncRNA）保留在核中，形成大的RNA云状结构，其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理解snoRNA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中潜在的神经发育缺陷可能具有同等重要性，如果不是更重要的话。在这个建议中，我们试图回答三个主要的悬而未决的问题，PWS的分子发病机制。1)在成熟神经元中，染色质去凝聚的机制基础是什么？2)哪种ncRNA负责PWS表型，snoRNA还是宿主lncRNA？3)与PWS基因座缺陷和重复相关的小鼠/人表型差异的遗传和机制基础是什么？这些方法包括用于组合检测小鼠和人脑中的R-环、ncRNA和染色质去凝聚的新型荧光原位方法，以及新型小鼠-人杂交神经元细胞系和人PWS诱导的多能细胞衍生的神经元。这些实验的结果有望提高对lncRNA在PWS基因座中心的功能作用的理解，并可能使未来的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.