Mechanisms of 5'UTR splicing of human surfactant protein (SP)-A1 and SP-A2

人表面活性蛋白(SP)-A1和SP-A2 5UTR剪接机制

• 批准号: 7776643
• 负责人: GUIRONG WANG
• 金额: $ 23.27万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7776643
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Acute; Address; Adult; Affect; Age; Alternative Splicing; Binding; Binding Proteins; Biological Assay; Biological Models; Birth; Bronchopulmonary Dysplasia; Chronic lung disease; DNA; Elements; Enhancers; Exclusion; Exons; Experimental Models; Fetal Lung; Future; Gene Expression; Generations; Genes; Genomics; Goals; Health Status; Host Defense; Human; Immune; Indium; Infant; Inflammatory; Introns; Knowledge; Lung; Lung diseases; Malignant Neoplasms; Mass Spectrum Analysis; Messenger RNA; Methodology; Mutate; Mutation; Pathogenesis; Pattern; Physiological; Play; Premature Birth; Process; Protein Binding; Proteins; Proteome; Pulmonary Surfactant-Associated Protein A; RNA Splicing; Regulation; Reporter; Role; System; Testing; Trans-Activators; Translations; Untranslated Regions; Variant; Western Blotting; activator 1 protein; base; cis acting element; falls; fetal; genetic variant; human disease; insight; lung development; mRNA Precursor; mRNA Stability; novel; public health relevance; research study; respiratory distress syndrome; surfactant

DESCRIPTION (provided by applicant): The long-term goal of this project is to investigate mechanisms of alternative mRNA splicing of human surfactant protein A (SP-A) and potential implications/effects in the pathogenesis of several pulmonary diseases. Alternative mRNA splicing is a major regulatory mechanism that can generate protein diversity and increase proteome complexity. More than 70% of human genes express multiple mRNA variants through alternative splicing. About 20% of the variability from alternative splicing falls within the untranslated regions (5'UTR and 3'UTR) that affects mRNA stability, translation efficiency, and mRNA localization. SP-A, an important innate immune protein, plays a critical role in the innate host defense and the inflammatory regulation of lung, in surfactant-related physiological functions, and in parturition. In humans, there are two functional genes, SP-A1 and SP-A2. Differential expression of SP-A1 and SP-A2 has been observed in fetal and adult lung, and SP-A1 and SP-A2 genetic variants have been associated with pulmonary diseases including respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), and other lung diseases. The 5'UTR of SP-A1 and SP-A2 generates a number of splice variants, and these 5'UTR variants differentially influence translation efficiency and mRNA stability. Moreover, the ratio of SP-A1 to total SP-A content has been shown to vary as a function of age and lung health status. In this proposal, we hypothesize that differences exist between SP-A1 and SP-A2 in 5'UTR cis-acting elements and in trans-acting factors binding these elements. The rationale is based on the observation that: a) the key difference between the most frequently found SP-A1 (A*D') and SP-A2 (A*BD, A*BD') splice variants is the exclusion and inclusion, respectively, of exon B; b) our preliminary findings have shown that SP-A1 cis-acting element #6, i.e. exon splicing enhancer/silencer (ESE/S # 6), plays a role in splicing, and that specific protein(s) may bind to ESE/S #6. To test this hypothesis, we have proposed two specific aims: Specific Aim #1: to examine the functional impact of candidate cis-acting ESE/S elements in the SP-A1 and SP-A2 5'UTR splicing. Specific Aim #2: to study trans-acting factors involved in the regulation of SP-A 5'UTR splicing, with a focus on binding proteins associated with the SP-A1-specific cis-acting element ESE/S # 6. Upon completion of this R21 project, a novel experimental model system for alternative splicing of human SP-A can be established, and aspects of candidate cis-acting ESE/S elements within each SP-A gene and of trans-acting factors be analyzed and identified. These could provide foundational methodologies and insight for our further studies (R01 application) of the mechanisms involved in the splicing regulation of human SP-A1 and SP-A2, and of better understanding of the pathogenesis of several lung diseases. PUBLIC HEALTH RELEVANCE: This project is to establish a novel high-throughout experimental system for the study of mechanisms of alternative mRNA splicing of human surfactant proteins (SP-A1 and SP-A2). The proposed experiments include examination of the functional impact of candidate cis-acting ESE/S elements and identification of trans-acting factors binding these elements. New methodologies used and knowledge gained will contribute to the growing field of mRNA splicing regulation and to future studies that can help us to better understand the pathogenesis of several lung diseases.

描述（由申请人提供）：该项目的长期目标是研究人表面活性剂蛋白 A (SP-A) 的选择性 mRNA 剪接机制以及对几种肺部疾病发病机制的潜在影响/影响。 mRNA 选择性剪接是一种主要的调控机制，可以产生蛋白质多样性并增加蛋白质组的复杂性。超过 70% 的人类基因通过选择性剪接表达多种 mRNA 变体。大约 20% 的可变剪接变异位于非翻译区域（5'UTR 和 3'UTR），影响 mRNA 稳定性、翻译效率和 mRNA 定位。 SP-A是一种重要的先天免疫蛋白，在先天宿主防御和肺部炎症调节、表面活性剂相关生理功能和分娩中发挥着关键作用。在人类中，有两个功能基因：SP-A1 和 SP-A2。在胎儿和成人肺部中观察到 SP-A1 和 SP-A2 的差异表达，SP-A1 和 SP-A2 基因变异与肺部疾病相关，包括呼吸窘迫综合征 (RDS)、支气管肺发育不良 (BPD) 和其他肺部疾病。 SP-A1和SP-A2的5'UTR产生许多剪接变体，这些5'UTR变体对翻译效率和mRNA稳定性有不同的影响。此外，SP-A1 与总 SP-A 含量的比率已被证明随着年龄和肺部健康状况的变化而变化。在此提议中，我们假设 SP-A1 和 SP-A2 之间在 5'UTR 顺式作用元件和结合这些元件的反式作用因子中存在差异。基本原理基于以下观察：a) 最常见的 SP-A1 (A*D') 和 SP-A2 (A*BD, A*BD') 剪接变体之间的主要区别分别是外显子 B 的排除和包含； b) 我们的初步研究结果表明，SP-A1 顺式作用元件 #6，即外显子剪接增强子/沉默子 (ESE/S # 6)，在剪接中发挥作用，并且特定蛋白质可能与 ESE/S #6 结合。为了检验这一假设，我们提出了两个具体目标： 具体目标#1：检查 SP-A1 和 SP-A2 5'UTR 剪接中候选顺式作用 ESE/S 元件的功能影响。具体目标#2：研究参与 SP-A 5'UTR 剪接调节的反式作用因子，重点是与 SP-A1 特异性顺式作用元件 ESE/S #6 相关的结合蛋白。完成此 R21 项目后，可以建立一个用于人类 SP-A 选择性剪接的新实验模型系统，以及每个 SP-A 基因内候选顺式作用 ESE/S 元件的各个方面，以及 的反式作用因子进行分析和鉴定。这些可以为我们进一步研究（R01应用）人类SP-A1和SP-A2剪接调节机制以及更好地了解几种肺部疾病的发病机制提供基础方法和见解。 公共健康相关性：该项目旨在建立一种新型高通量实验系统，用于研究人类表面活性蛋白（SP-A1和SP-A2）的选择性mRNA剪接机制。拟议的实验包括检查候选顺式作用 ESE/S 元件的功能影响以及识别结合这些元件的反式作用因子。使用的新方法和获得的知识将有助于 mRNA 剪接调控领域的不断发展，并有助于未来的研究，帮助我们更好地了解几种肺部疾病的发病机制。