Remodelling of cellular RNA Metabolism during Prader-Willi syndrome

该项目的主要目的是确定细胞RNA代谢在遗传性遗传疾病中是如何重塑的，特别是使用Prader-Willi综合征（PWS）作为模型。PWS是一种神经发育障碍，其特征是早期发作的摄食过多，发生率约为1/10- 30，000。这种疾病是由下丘脑的基因表达变化引起的，下丘脑是大脑的一个小而中心的区域，参与代谢稳态，这涉及到饥饿的调节。PWS通常由印迹基因组区域的丢失引起，该印迹基因组区域包括两个小核仁RNA（snoRNA）的多个串联拷贝，snoRD 115和snoRD 116。这些是从小核仁RNA宿主基因14（SNHG 14）切除的。产生PWS的最小已知缺失仅去除snoRD 116重复。大多数snoRNA在所有细胞类型中表达，并且是核糖体合成的组成部分。然而，snoRD 115和snoRD 116似乎都不参与核糖体合成，而是被称为“孤儿”snoRNA，因为没有任何已知的直接靶点。在整个项目中，将使用隆德人中脑（LUHMES）细胞系。这些是人胚胎神经元前体细胞，可以分化为功能性多巴胺能神经元。为了确定PWS背后的RNA代谢，该项目目前将集中在两个主要问题上。首先，将测试为什么snoRNA是神经元特异性的问题。最初的RNAseq数据表明，宿主基因转录或剪接的变化并不是snoRD 116积累的原因。将进一步分析这些数据，以证实这些结论。我们还将检验snoRNA的稳定化是由于原纤蛋白（FBL）同系物，原纤蛋白样1（FBLL 1）的神经元表达的假设。FBL是与snoRDs结合形成小核仁核糖核蛋白（snoRNP）的四种核心蛋白之一。然而，初步分析表明，FBL表达在神经元分化过程中丢失，而FBLL 1强烈上调。这表明分化过程中snoRD 116的积累可能需要FBLL 1的稳定。将测定snoRD 115/116与核心蛋白N 0 P58和FBL的结合。相比之下，snoRD 115的RNAseq数据表明分化期间出现了特定转录物，因此将确定这些转录物的终点和时间。其次，为什么snoRD 115和snoRD 116的缺失会改变神经发育后期许多ncRNA和mRNA的丰度，这个问题将通过确定snoRNA的主要靶点和机制来回答。将进行几项测试来确定这一点。将进行小RNA测序以确认snoRD 115、snoRD 116和其他神经元特异性snoRNA的不同亚型的相对表达。信息学将用于测试表达最多的snoRNA序列与靶mRNA的潜在序列互补性。还将分析来自缺乏snoRD 116的细胞的RNA的剪接缺陷，并测试潜在靶内含子的互补性。此外，将确定snoRD 115和snoRD 116的异位表达是否抑制缺失突变体中基因表达的变化，这将潜在地揭示哪些效应是由成熟snoRNA的缺失引起的。最后，RNA-RNA相互作用将使用邻近连接技术来确定，这些技术可以是基于蛋白质的，如CLASH，使用标记的蛋白质，或hiCLIP，使用抗体，或基于RNA的，如COMRADES，使用寡核苷酸selection.With对PWS的这项研究的执行，可以将见解应用于其他神经元snoRNA，并因此可以开发针对RNA相关认知障碍疾病的潜在靶向干预措施。此外，早期诊断和促进未来的先发制人的治疗也可能得到改善。

The major aim of this project is to determine how cellular RNA metabolism is remodelled during inherited genetic disorders, specifically using Prader-Willi syndrome (PWS) as a model. PWS is a neurodevelopmental disorder characterised by early onset of hyperphagia and occurs in ~1 in 10-30,000 people. This disorder is caused by gene expression changes in the hypothalamus, a small and central region of the brain involved in metabolic homeostasis, which involves the regulation of hunger. PWS is typically caused by loss of an imprinted genomic region that includes multiple, tandem copies of two small nucleolar RNAs (snoRNAs), snoRD115 and snoRD116. These are excised from the Small Nucleolar RNA Host Gene 14 (SNHG14). The smallest known deletions generating PWS remove only the snoRD116 repeats. Most snoRNAs are expressed in all cell types and are integral to ribosome synthesis. However, both snoRD115 and snoRD116 appear not to be involved in ribosome synthesis and are instead known as 'orphan' snoRNAs, since there aren't any known direct targets for either. Throughout this project, the Lund human mesencephalic (LUHMES) cell line will be employed. These are human embryonic neuronal precursor cells that can be differentiated into functional, dopaminergic neurons. To determine the RNA metabolism underlying PWS, this project will currently focus on two main questions. First, the question of why snoRNAs are neuronal specific will be tested. Initial RNAseq data indicated that changes in transcription or splicing of the host gene aren't responsible for snoRD116 accumulation. This data will be analysed further to confirm these conclusions. We will also test the hypothesis that stabilisation of snoRNAs is due to the neuronal expression of Fibrillarin (FBL) homolog, Fibrillarin-like 1 (FBLL1). FBL is one of the four core proteins that snoRDs associate with to form small nucleolar ribonucleoproteins (snoRNPs). However, preliminary analyses showed that FBL expression is lost during neuronal differentiation, whereas FBLL1 is strongly upregulated. This suggested that snoRD116 accumulation during differentiation may require stabilisation by FBLL1. Binding of snoRD115/116 to core proteins NOP58 and FBL will be determined. In contrast, RNAseq data for snoRD115 indicates the appearance of specific transcripts during differentiation, so the endpoints and timing of these transcripts will be determined. Secondly, the question of why snoRD115 and snoRD116 loss alters the abundance of many ncRNAs and mRNAs in late neurodevelopment will be answered by determining the primary targets and mechanisms of snoRNAs. Several tests will be performed to determine this. Small RNA sequencing will be performed to confirm the relative expression of different isoforms of snoRD115, snoRD116 and other neuronal-specific snoRNAs. Informatics will be used to test potential sequence complementarity to target mRNAs for the most expressed snoRNA sequences. RNA from cells lacking snoRD116 will also be analysed for splicing defects and potential target introns tested for complementarity. Moreover, whether ectopic expression of snoRD115 and snoRD116 suppress changes in gene expression in deletion mutants will be determined, which would potentially reveal which effects are caused by the loss of mature snoRNAs. Finally, RNA-RNA interactions will be determined using proximity ligation techniques that are either protein-based, such as CLASH, using tagged proteins, or hiCLIP, using antibodies, or RNA-based, such as COMRADES, using oligonucleotide selection.With execution of this research on PWS, insights could be applied to other neuronal snoRNAs and potential targeted interventions for RNA-linked cognitive-impairment disorders could be consequently developed. Furthermore, the early diagnosis and facilitation of future, pre-emptive treatments could also be potentially improved.