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很大一部分的小RNA的功能方面存在着根本的差距。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，这是威胁生命的肥胖的最常见遗传原因。我们的长期目标是阐明小RNA对选择性剪接的调节，据估计小RNA占人类基因表达的40%以上。本应用的目的是确定在Prader-Willi综合征患者中缺失的snoRNAs会影响哪些选择性剪接模式，并了解其中一个snoRNAHBII-52影响选择性剪接模式的分子基础。中心假说是，Prader-Willi综合征患者体内缺失的snoRNAs通过掩盖前mRNAs上的剪接调控元件来调节选择性剪接。因此，这些snoRNAs通过改变它们的选择性剪接模式来调节许多基因的表达。这项拟议研究的基本原理是，受这些snoRNAs调控的基因代表着治疗Prader-Willi综合征的药物靶点。因此，这项建议与美国国立卫生研究院的使命有关，该使命涉及发展基础知识，这可能有助于减轻人类疾病的负担。在我们第一份发表的snoRNA HBII-52影响选择性剪接的报告和额外的强有力的初步数据的指导下，这一假说将通过追求两个具体目标来验证：1)确定在Prader-Willi综合征患者中缺失的snoRNA调控的前mRNAs；以及2)确定HBII-52如何调节备选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，使用生物信息学预测和剪接位点敏感DNA阵列异位表达单个snoRNAs后，将确定前mRNA加工的变化。在第二个目标下，我们将利用建立的体外系统，鉴定与snoRNA HBII-52竞争的抑制物活性，并确定其作用模式。这项拟议的工作是创新的，因为它显示了snoRNAs在调节替代前mRNA剪接中的全新角色。这很可能改变目前snoRNAs只调控非mRNAs的“教科书知识”。这项研究具有重要意义，因为它将揭示snoRNAs的新作用，识别Prader-Willi综合征的分子缺陷，并有助于预测小RNAs对剪接位点选择的影响。 与公共健康相关：拟议的研究深入研究了一个未被充分研究的基因表达领域，并具有潜在的适用性，以了解Prader-Willi综合征和潜在的其他形式的遗传性肥胖的分子原因。这项拟议的研究与公共健康相关，因为它调查了人类基因表达的一种新机制。了解这一机制是改善由前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.