Imprinted SnoRNA Genes in the PWS deletion Region

PWS 缺失区域中的印记 SnoRNA 基因

• 批准号: 6640307
• 负责人: UTA FRANCKE
• 金额: $ 28.64万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6640307
• 关键词: Prader Willi syndrome; SDS polyacrylamide gel electrophoresis; Xenopus oocyte; cell line; chromosome aberrations; clinical research; fluorescent in situ hybridization; gene deletion mutation; gene expression; gene targeting; genetic transcription; genomic imprinting; human tissue; immunoprecipitation; laboratory mouse; messenger RNA; methylation; microarray technology; northern blottings; nucleic acid quantitation /detection; phenotype; polymerase chain reaction; protein localization; small nuclear RNA; tissue /cell culture; transfection

A human disease model for the study of genomic imprinting. Prader- Willi syndrome (PWS) is a complex neurodevelopmental disorder caused by the inactivation of deletion of imprinted, paternally expressed genes within 4 Mbp of chromosome band 15q11.2. The overall goals of this project are to determine the mechanisms of imprinting in the PWS region and to elucidate the genetic and pathophysiologic pathways that lead to the PWS phenotype that involves metabolic and behavioral changes such as growth deficiency and lack of appetite control. A recently discovered novel paternally expressed imprinted gene cluster in the PWS deletion region (PWCR1 in human and Pwcr1 in mouse) encodes small nucleolar RNAs (snoRNAs) with short conserved sequence elements, called C and D boxes (de Los Santos et al. Amer. J. Hum. Genet. 67:1067, 2000). PWCR1 and Pwcr1 are the first mammalian snoRNA genes that show imprinted expression. C/D box snoRNAs are conserved in all organisms and usually serve to direct the site-specific methylation of the ribose 2-hydroxyl group of specific nucleotides in rRNA., PWCR1/Pwcr1, belong to a novel class of snoRNAs, found mostly in brain, in which the sequences that would basepair with the target RNA are not complementary to any known rRNA or snRNA sequences. It is hypothesized that the modification target(s) of PWCR1snoRNAs are brain-specific mRNAs whose processes, such as alternative splicing, methylation or other modification, or intranuclear trafficking is controlled by binding to snoRNA-containing particles; and that lack of PWCR1 snoRNAs contributes to the hypothalamic dysfunction that causes the physical and behavioral manifestation of the PWS phenotype. The proposed work will characterize the organization, transcription, processing, localization and function of this novel kind of snoRNAs. Experimental strategies include the mining of the human genome for organization of the PWCR1snoRNA gene cluster, for a putative "host" gene for the PWCR1snoRNA genes and for candidate RNA modification targets. The expression patterns of putative target RNAs will be studied in the brain from PWS patients and in a PWS mouse model in which the Pwcr1 snoRNA cluster is deleted. PWCR1 snoRNAs will be tested for function by primer extension studies to detect 2'-O-methylation of synthetic complementary target RNAs. The work has the potential for discovery of novel RNA molecules that are modified as a result of a complex formation between PCR1snboRNPs and target RNA, and thus, for major breakthroughs in understanding the role of mRNA modification, and for innovation of new diagnostic and treatment modalities.

用于基因组印记研究的人类疾病模型。Prader-Willi综合征（PWS）是由染色体15q11.2带4 Mbp内的印迹、父系表达基因缺失失活引起的复杂神经发育障碍。该项目的总体目标是确定PWS区域的印记机制，并阐明导致PWS表型的遗传和病理生理途径，该表型涉及代谢和行为变化，如生长缺陷和食欲控制不足。最近发现的PWS缺失区中的新的父系表达的印记基因簇（人的PWCR 1和小鼠的Pwcr 1）编码具有短保守序列元件的小核仁RNA（snoRNA），称为C盒和D盒（de洛斯桑托斯等人，Amer.J.Immunol.2005）。Genet. 67：1067，2000）。PWCR 1和Pwcr 1是第一个显示印迹表达的哺乳动物snoRNA基因。C/D盒snoRNA在所有生物体中是保守的，通常用于指导rRNA中特定核苷酸的核糖2-羟基的位点特异性甲基化。PWCR 1/Pwcr 1属于一类新的snoRNA，主要存在于大脑中，其中与靶RNA碱基配对的序列与任何已知的rRNA或snRNA序列都不互补。据推测，PWCR 1 snoRNA的修饰靶点是脑特异性mRNA，其过程（如选择性剪接、甲基化或其他修饰或核内运输）是通过与含snoRNA的颗粒结合来控制的;并且PWCR 1 snoRNA的缺乏有助于下丘脑功能障碍，从而导致PWS表型的物理和行为表现。这项工作将对这种新型snoRNA的组织、转录、加工、定位和功能进行研究。实验策略包括挖掘人类基因组的PWCR 1 snoRNA基因簇的组织，为PWCR 1 snoRNA基因和候选RNA修饰靶的推定的“宿主”基因。将在PWS患者的脑中以及在缺失Pwcr 1 snoRNA簇的PWS小鼠模型中研究推定靶RNA的表达模式。将通过引物延伸研究检测PWCR 1 snoRNA的功能，以检测合成互补靶RNA的2 '-O-甲基化。这项工作有可能发现由于PCR 1 snboRNP和靶RNA之间形成复合物而被修饰的新型RNA分子，从而在理解mRNA修饰的作用方面取得重大突破，并创新新的诊断和治疗方式。