Regulation of alternative pre-mRNA processing by small nucleolar RNAs

描述（由申请人提供）：在理解构成人类基因表达的大部分的小RNA的功能方面存在根本性的差距。例如，我们不知道小核仁RNA（snoRNA）表达的缺失如何导致Prader-Willi综合征，这是危及生命的肥胖症最常见的遗传原因。我们的长期目标是阐明小RNA对可变剪接的调控，据估计，小RNA占人类基因表达的40%以上。本申请的目的是确定Prader-Willi综合征个体中缺失的snoRNA影响哪些选择性剪接模式，并了解其中一种snoRNA HBII-52影响选择性剪接模式的分子基础。中心假设是Prader-Willi综合征患者中缺失的snoRNA通过掩蔽前mRNA上的剪接调控元件来调节选择性剪接.因此，这些snoRNA通过改变它们的选择性剪接模式来调节许多基因的表达。这项研究的基本原理是，由这些snoRNA调控的基因代表了治疗Prader-Willi综合征的药物靶点。因此，该建议与NIH的使命相关，该使命涉及开发可能有助于减轻人类疾病负担的基础知识。在我们首次发表的snoRNA HBII-52影响选择性剪接的报告和其他强有力的初步数据的指导下，这一假设将通过追求两个具体目标进行测试：1）确定Prader-Willi综合征患者中缺失的snoRNA所调控的前mRNA; 2）确定HBII-52如何调节选择性剪接位点的使用并测试其表达的替代方案。在第一个目标下，将使用生物信息学预测和剪接位点敏感的DNA阵列确定异位表达单个snoRNA后前mRNA加工的变化。在第二个目标下，我们将使用已建立的体外系统鉴定与snoRNA HBII-52竞争的阻遏物活性并确定其作用模式。这项工作是创新的，因为它显示了snoRNA在调节选择性前mRNA剪接中的全新作用。它很可能改变目前的“教科书知识”，snoRNA只调节非mRNA。这项研究具有重要意义，因为它将显示snoRNA的新作用，确定Prader-Willi综合征的分子缺陷，并有助于预测小RNA对剪接位点选择的影响。 公共卫生相关性：拟议的研究深入研究了基因表达的一个未充分研究的领域，并具有潜在的适用性，以了解普拉德-威利综合征的分子原因和潜在的其他形式的遗传性肥胖。这项研究与公共卫生有关，因为它研究了人类基因表达的新机制。了解这种机制是改善由前mRNA加工缺陷引起的人类疾病的诊断和治疗选择的基础。

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding the function of small RNAs that constitute a large part of human gene expression. For example, we do not know how the loss of small nucleolar RNA (snoRNA) expression contributes to the Prader-Willi syndrome, the most frequent genetic cause for life-threatening obesity. Our long-term goal is to elucidate the regulation of alternative splicing by small RNAs that are estimated to comprise more than 40% of human gene expression. The objective of this application is to determine which alternative splicing patterns are influenced by the snoRNAs missing in individuals with Prader-Willi syndrome and to understand the molecular basis by which one of these snoRNAs, HBII-52, influences alternative splicing patterns. The central hypothesis is that snoRNAs missing in individuals with Prader- Willi syndrome regulate alternative splicing by masking splicing regulatory elements on pre- mRNAs. Thus these snoRNAs regulate expression of numerous genes by changing their alternative splicing patterns. The rationale for the proposed research is that the genes regulated by these snoRNAs represent drug targets for treatment of the Prader-Willi syndrome. This proposal is therefore relevant to the NIH's mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human disease. Guided by our first published report that the snoRNA HBII-52 influences alternative splicing and additional strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Identify the pre-mRNAs that are regulated by the snoRNAs missing in people with Prader-Willi syndrome; and 2) Determine how HBII-52 regulates alternative splice site usage and test alternatives for its expression. Under the first aim, changes in pre-mRNA processing will be determined after ectopically expressing individual snoRNAs using bioinformatic predictions and splice-site sensitive DNA arrays. Under the second aim, we will identify the repressor activity that competes with the snoRNA HBII-52 and determine its mode of action, using an established in vitro system. The proposed work is innovative, because it shows a complete new role for snoRNAs in the regulation of alternative pre-mRNA splicing. It may well change the current `textbook knowledge' that snoRNAs only regulate non-mRNAs. The proposed research is significant because it would show a novel role of snoRNAs, identify the molecular defects in Prader-Willi syndrome and help to predict the influence of small RNAs on splice site selection. PUBLIC HEALTH RELEVANCE: The proposed studies delve into an under-investigated area of gene expression and have the potential applicability to understand the molecular cause of the Prader-Willi Syndrome and potentially other forms of inherited obesity. The proposed research is relevant for public health, because it investigates a new mechanism of human gene expression. Understanding this mechanism is the basis to improve diagnostics and therapeutic options for human diseases caused by defects in pre-mRNA processing.