【作者】 倪岚；

【导师】 杨炯； 王桂荣；

【作者基本信息】 武汉大学 ， 内科学， 2013， 博士

【摘要】 研究背景与目的肺表面活性物质(pulmonary surfactant)是主要由肺泡Ⅱ型细胞合成,组装并分泌的一种脂蛋白复合物,主要作用为降低肺泡表面张力,提高肺的顺应性,促进肺泡气体交换并防止肺损伤。其主要功能成分为肺泡表面蛋白(surfactant protein,SP)和脂类。其中SP-B是必需的蛋白质,不正常的SP-B表达与多种肺部疾病相关。SP-B等位基因具有多态性,基因突变除点突变(外显子和内显子),并有包含一段序列的结构区域的插入和缺失突变,这些突变与成熟SP-B的减少有关和/或影响了proSP-B的加工过程进而造成SP-B蛋白合成障碍。SP-B基因内含子4区的前半部分存在11个以不同长度的(CA)。为特征的模体(motif),每个模体由一段20个碱基左右的保守序列和随后的CA重复序列(2到17个)构成。1995年本实验室研究发现此区域序列的基因多态性同ARDS有关。2005年本实验室研究又发现(CA)。模体的缺失变异同SP-B mRNA不完全剪切有关,且此不完全剪切同肺癌相关。显示SP-B基因内含子4区(CA)。模体可能通过影响SP-B mRNA的剪切过程从而导致成熟SP-B mRNA的减少并影响功能性SP-B蛋白的表达。但这11个(CA)。模体的具体作用方式及作用机制并不清楚。RNA剪切(RNA splicing)是真核基因表达中一个重要的转录后调控过程。RNA序列决定了剪切位点,正确的剪切有赖于剪切位点的正确供给。另外剪切调控蛋白可结合到称为外显子／内含子剪切增强子(ESE或者ISE, exonic/intronic splicing enhancer)或者外显子／内含子剪切沉默子(ESS或者ISS, exonic/intronic splicing silencer)的特殊RNA序列,前者增强附近的剪切位点的剪切,后者正好相反。同时,RNA构像即RNA二级结构的变化及稳定性也会影响剪切位点的供给及增强子／沉默子序列的结合能力。基于RNA剪切的发生过程和影响因素,为进一步探讨(CA)。模体对SP-BmRNA剪切过程的影响机制,我们对SP-B基因内含子4区(CA)。模体进行了系统性研究。首先合成野生型SP-B基因内含子4区(WT intron4)基因序列,并以此为基础构建了一系列(CA)。结构域的缺失结构的质粒,然后在细胞中表达这些DNA质粒。利用Northern杂交和实时定量PCR检测(CA)。模体对SP-B mRNA剪切的影响；然后对这一系列缺失结构进行剪切位点预测及RNA二级结构分析；最后通过凝胶阻滞迁移实验(EMSA)研究相关(CA)。模体的RNA序列与其潜在结合蛋白的相互作用,探讨相关(CA)。模体影响SP-B基因剪切的发生机制。方法第一部分：合成含正常(CA)。模体的野生型SP-B基因内含子4区(WT intron4)结构,并以此为基础构建了一系列不同(CA)。模体缺失的内含子4区缺失结构(DC-A, DC-B,...DC-K),并将所有内含子4区结构导入我们构建的已经去除内含子4区的pcDNA3.1SP-B Minigene-del-m (Del-M)中,形成一系列含不同(CA)n模体缺失的SP-B Minigene,将他们转染至CHO细胞中并抽提RNA,利用Northern杂交和实时定量PCR检测mRNA的剪切效率(完全剪切/不完全剪切)分析(CA)n模体对SP-B mRNA剪切的影响。根据结果构建含特定模体的SPB minigene再次进行Northern杂交和RT-PCR以确定特定模体的对剪切的影响。第二部分：利用程序对这一系列缺失结构进行相关剪切位点的预测及RNA二级结构分析(包括分析结构的构型特征和稳定性),对含有特定模体的缺失结构进行比较分析。第三部分：分析特定(CA)。模体可能存在的潜在RNA结合蛋白并合成含有特定蛋白结合位点的RNA序列的探针及突变序列探针,用凝胶阻滞迁移(EMSA)技术研究相关(CA)。模体的RNA序列与其潜在结合蛋白的相互作用。结果第一部分：缺失结构及含特定模体结构的northern blot和RT-PCR分析：1)所有不同(CA)n模体缺失的SP-B Minigene的mRNA剪切效率较之完全(正常)剪切均有下降；2)这些含不同(CA)n模体缺失的SP-B Minigene的剪切效率各不相同,其中第8,9号(CA)。模体显示对剪切产生明显的并且相反的影响作用。第二部分：剪切位点的预测：支点序列与3’端的AG决定区的距离为258bp,共有四组高预测剪切蛋白存在于intron4模体区,HnRNP L顷向与较长(CA)n结合,模体8可能具有SRP20的特异性结合位点。RNA二级结构分析：SP-BMinigene的RNA二级结构呈现出共同的特征：1)所有的(CA)。模体或(CA)。簇团倾向于通过他们自身或和其他(CA)。模体一起形成稳定的环状结构(loop),同时(CA)。模体的保守序列倾向于形成稳定性各不相同的茎状结构(stem)。RNA二级结构稳定性或者茎状结构和/或环状结构的稳定性的变化可能同RNA剪切效率的变化相关。2)当RNA二级结构的影响因素相同时,RNA剪切效率的变化同增强子或沉默子序列的变化有关。第三部分：特定(CA)。模体存在的潜在RNA结合蛋白分析：(CA)n模体可能存在影响RNA剪切的增强子和沉默子,第8号(CA)。模体的保守序列上存在SRP20的特定结合位点,它可能通过发挥增强子的作用从而影响RNA的剪切。结论SP-B内含子4区(CA)。模体区可能存在影响RNA剪切的增强子和沉默子,第8,9号(CA)。模体显示出对剪切产生明显的并且相反(增强和抑制)的影响作用。第8号(CA)。模体的保守序列上存在SRP20的结合位点,它可能通过发挥增强子的作用从而影响RNA的剪切。在对缺失结构进行剪切效率分析时,RNA二级结构也是重要的影响因素。更多还原

【Abstract】 Background and Objectives:Pulmonary surfactants are lipoprotein complexes that are produced, assembled, and synthesized by lung alveolar type II cells. The primary functions of pulmonary surfactants include lowering alveolar surface tension, improving lung compliance, promoting alveolar gas exchange, and preventing lung damage. The major functional components are the surfactant proteins A. B. C and D (e.g., surfactant protein, SP-A, B, C, D) and lipids, of which, SP-B is essential for normal lung function and abnormal expression of SP-B is associated with multiple lung diseases.Human SP-B gene contains a number of polymorphisms. In addition to point mutations (exons and introns), there are sequence insertion and deletion mutations that directly affect the structural region of the SP-B protein; therefore, these mutations may be related to the decreased expression of mature SP-B, and/or affect proSP-B post translational processing and secretion, which cause SP-B protein synthesis disorder.There are11different (CA)n motifs in the first half of intron4of the human SP-B gene. Each motif is composed of a conserved sequence (-20bp) and2-17subsequent CA repeats. In1995, the work in our laboratory demonstarted that gene polymorphisms in this region were associated with acute respiratory distress syndrome (ARDS). In2005, the work further found that (CA)n motif deletions were related to the incomplete splicing of SP-B mRNA, which was associated with lung cancer. These studies indicated that the intron4(CA)n motifs in the SP-B gene may-by influencing SP-B mRNA splicing processes-lead to the reduction of mature SP-B mRNA and influence the expression of functional SP-B protein. However, the detailed mechanisms of SP-B mRNA splicing regulation by the11(CA)n motifs are unknown.RNA splicing is an important post-transcriptional regulation process in eukaryotic gene expression. RNA sequences can determine RNA splice sites, and correct RNA splicing requires the availability of the correct splicing site. In addition, splice-regulating proteins can bind to specific RNA sequences called exon/intron splicing enhancers (ESE or ISE, exonic/intronic splicing enhancer) or exon/intron splicing silencers (ESS or ISS, exonic/intronic splicing silencer); the former enhances splicing near the splicing site, while the latter inhibits splicing. Additionally, RNA conformation, namely the alteration of RNA secondary structure and stability, also affects the availability of the splicing site and the binding ability of the enhancer/silencer sequence.To further investigate the role of the (CA)n motif on the SP-B mRNA splicing process, we conducted a systematic study of the intron4(CA)n motifs in the SP-B gene. We synthesized the intron4gene sequence from the wild-type SP-B gene (WT intron4) by PCR method. Based on this sequence, we constructed a series of (CA)n deletion mutant plasmids. These DNA plasmids were transfected into CHO cells and then Northern blot and real-time quantitative PCR analyses were used to determine the effects of the (CA)n motif on SP-B mRNA splicing. Additionally, through electrophoretic mobility shift assay (EMSA) experiments, we studied the (CA)n motif-related RNA sequences and the potential interactions with binding proteins and discussed the mechanism of (CA)n motif-regulated SP-B gene splicing.Methods Part Ⅰ:pcDNA3.1SP-B minigene and WT intron4fragment were synthesized from the SP-B gene by PCR method. By using the WT intron4sequence, we constructed a series of (CA)n motif mutated fragments (DC-A, DC-B,... DC-K) that characterized by deletion of the different motif one by one. Each of intron4(CA)n motif fragment was inserted into the pcDNA3.1SP-B minigene, so that a series of mutanted SP-B minigene plasmids contained different (CA)n motif deletion were generated. These plasmids were transfected into CHO cells and the RNA was extracted. Northern blot and real-time quantitative PCR analyses were used to determine the mRNA-splicing efficiency (completed splicing/incompleted splicing), and then the effects of the intron4(CA)n motifs on SP-B mRNA splicing were evaluated.Part Ⅱ:Online programs were used to predict the splicing site and to analyze the RNA secondary structure of the deletion mutants, which included the analysis of structural characteristics and stability. Additionally, comparative analysis among the specific motifs relative to their RNA secondary structure was performed. Part Ⅲ:Specific (CA)n motifs that potentially bind RNA-binding proteins were analyzed. To confirm RNA-binding protein interaction with specific (CA)n motif, RNA probes that contained potentially specific binding sites as well as mutated-sequence probe were synthesized. EMSA technology was used to study the correlation and interactions between (CA)n motif and potential binding proteins.Results Part Ⅰ:1) The splicing efficiencies of RNA in all SP-B minigenes that deleted different (CA)n motifs were lower than that of WT minigene.2) All minigene constructs, including WT, expressed visible variable amount of incompletely spliced RNA in addition to completely spliced RNA (or normal mRNA). The Motif8and Motif9were able to enhance and attenuate, respectively, RNA splicing.Part Ⅱ:Prediction of splice sites:The distance between the branch point site and the3’-end of the possible AG-determining regions was258bp, and4groups of potential splicing proteins have a possibility to bind in the intron4motif region. HnRNP L was found to bind the longer (CA)n repeat, while the motif8may have SRp20-specific binding site. We observed that:1) All RNA secondary structures of the minigene and mutants shared similar properties:CA-repeat regions or CA clusters tended to form stable loops by themselves or with other CA rich regions. On the other hand, the conservative sequences (GC rich) tended to form stems with varying stability.2) The stability of the secondary structures of the various constructs, and/or "microstability" of stem/loop elements may differ within constructs and these differences correlated with the observed splice ratios.3) When the stability of the secondary structures was the same in different minigene constructs, the missing motif sequence(s) with potential splicing site may be the reason of different splicing efficiency.Part III:The analysis of specific (CA)n motif demonstrated potential RNA-binding proteins in the(CA)n motifs.:(CA)n motifs may exist in the enhancers and silencers that affect RNA splicing. A specific binding site of SRp20exists in the conserved sequence of the motif8, which may enhance RNA splicing in this study.Conclusions:The (CA)n Motif8and Motif9of SP-B intron4are able to enhance and attenuate, respectively, RNA splicing. A specific RNA-binding site of SRp20exists in the conserved sequence of the motif8, which may have a role of enhancing RNA splicing. The RNA secondary structures may be the considerable element in the analysis of different splicing efficiency.更多还原