Remodelling of cellular RNA Metabolism during Prader-Willi syndrome

该项目的主要目的是确定遗传疾病期间细胞RNA代谢是如何重塑的，特别是使用Prader-Willi综合征（PWS）作为模型。PWS是一种以早发性贪食为特征的神经发育障碍，发病率约为1 / 10- 30000。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，包括对饥饿的调节。PWS通常是由印迹基因组区域的丢失引起的，该区域包括两个小核仁rna (snoRNAs) snoRD115和snoRD116的多个串联拷贝。这些是从小核仁RNA宿主基因14 （SNHG14）中切除的。产生PWS的已知最小的缺失只删除了snoRD116重复序列。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，snoRD115和snoRD116似乎都不参与核糖体合成，而是被称为“孤儿”snorna，因为它们都没有任何已知的直接靶标。在整个项目中，隆德人类中脑（LUHMES）细胞系将被使用。这些是人类胚胎神经元前体细胞，可以分化成功能性的多巴胺能神经元。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，为什么snorna是神经元特异性的问题将被测试。最初的RNAseq数据表明，宿主基因转录或剪接的变化与snoRD116的积累无关。将进一步分析这些数据以证实这些结论。我们还将验证snorna的稳定是由于纤维蛋白（FBL）同系物纤维蛋白样1 （FBLL1）的神经元表达的假设。FBL是snord结合形成小核仁核糖核蛋白（snoRNPs）的四个核心蛋白之一。然而，初步分析表明，FBL表达在神经元分化过程中丢失，而FBLL1则强烈上调。这表明分化过程中snoRD116的积累可能需要FBLL1的稳定。snoRD115/116与核心蛋白NOP58和FBL的结合将被确定。相反，snoRD115的RNAseq数据表明在分化过程中出现了特定的转录本，因此这些转录本的终点和时间将被确定。其次，为什么snoRD115和snoRD116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。我们将进行小RNA测序，以确认snoRD115、snoRD116和其他神经元特异性snoRNAs不同亚型的相对表达。信息学将用于测试表达最多的snoRNA序列与目标mrna的潜在序列互补性。来自缺乏snoRD116的细胞的RNA也将分析剪接缺陷和潜在目标内含子的互补性测试。此外，还将确定snoRD115和snoRD116的异位表达是否抑制缺失突变体中基因表达的变化，这可能会揭示哪些影响是由成熟snoRNAs的缺失引起的。最后，RNA-RNA相互作用将使用接近连接技术来确定，这些技术要么是基于蛋白质的，如使用标记蛋白的CLASH，要么是使用抗体的hiCLIP，要么是基于rna的，如使用寡核苷酸选择的同志。通过对PWS的研究，可以将见解应用于其他神经元snorna，从而开发出针对rna相关认知障碍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.