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

描述(申请人提供)：这个项目的长期目标是研究人类表面活性蛋白A(SP-A)的mRNA选择性剪接的机制，以及在几种肺部疾病的发病机制中的潜在影响。选择性信使核糖核酸剪接是一种主要的调控机制，它可以产生蛋白质多样性和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。选择性剪接产生的变异约有20%落在非翻译区(5‘非翻译区和3’非翻译区)，这会影响mRNA的稳定性、翻译效率和信使核糖核酸的定位。SP-A是一种重要的天然免疫蛋白，在肺部的天然宿主防御和炎症调节、表面活性物质相关的生理功能和分娩过程中发挥着重要作用。在人类中，有两个功能基因，SP-A1和SP-A2。SP-A1和SP-A2在胎儿和成人肺中有不同的表达，SP-A1和SP-A2基因变异与呼吸窘迫综合征(RDS)、支气管肺发育不良(BPD)等肺部疾病相关。SP-A1和SP-A2的5‘端非编码区产生了多种剪接变异体，这些5’端非编码区变异体对翻译效率和信使核糖核酸的稳定性有不同的影响。此外，SP-A1占总SP-A含量的比例已被证明随着年龄和肺健康状况的变化而变化。在这一建议中，我们假设SP-A1和SP-A2在5‘UTR顺式作用元件和结合这些元件的反式作用因子方面存在差异。其基本原理是基于以下观察结果：A)最常见的SP-A1(A*D‘)和SP-A2(A*BD，A*BD’)剪接变异体之间的关键区别是分别排除和包含外显子B；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/沉默元件(ESE/S#6)在剪接中起作用，特定蛋白(S)可能与ESE/S#6结合。为了验证这一假说，我们提出了两个具体目标：特定目的#1：研究候选顺式作用元件ESE/S在SP-A1和SP-A2 5‘非编码区剪接中的功能影响。具体目的#2：研究SP-A 5‘非编码区剪接调控中的反式作用因子，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。R21项目完成后，可以建立一个新的人SP-A选择性剪接的实验模型系统，并分析和鉴定每个SP-A基因中候选顺式作用元件ESE/S和反式作用因子。这为我们进一步研究(R01应用)人类SP-A1和SP-A2的剪接调控机制，以及更好地理解几种肺部疾病的发病机制提供了基础的方法和见解。 与公共卫生相关：该项目旨在建立一个新的高通量实验系统，用于研究人类表面活性蛋白(SP-A1和SP-A2)的mRNA选择性剪接机制。拟议的实验包括检查候选的ESE/S顺式作用元件的功能影响，以及鉴定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不断增长的mRNA剪接调控领域，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。

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.