Protein 4.1 Gene Expression in Developing Red Cells

描述（由申请人提供）：我们采用蛋白4.1R基因作为模型来研究红细胞生成过程中剪接的分子调控。蛋白4.1R是红细胞骨架的关键元素。在红细胞生成过程中，两种严格调节的剪接事件改变了其表达和功能：1）早期事件决定外显子2'排除，其省略了上游翻译起始位点，仅产生“小”（80 kD）同种型。2)晚期事件诱导包含外显子16，其编码血影蛋白-肌动蛋白结合的关键肽。我们在理解这两个事件方面取得了进展，包括：1）鉴定出一种阶段特异性mFox-2A亚型，该亚型在晚期红细胞中上调，并且对16号外显子剪接开关至关重要; 2）证明晚期红细胞中SF 2/ASF表达增加通过结合外显子剪接增强子刺激16号外显子剪接; 3）在红细胞分化期间一组剪接因子的定量，显示4.1R剪接通过改变所选择的普遍存在的和细胞类型特异性激活剂和抑制剂的量来调节;其中两个，PTB和一个40 kD的新蛋白是所提出的研究的对象：4）鉴定调节外显子2'剪接的偶联转录和剪接途径;和5）检测导致成熟红细胞中产生80 kD同种型的启动子使用的转换。我们现在建议更精确地定义控制这两个剪接事件的关键元件和机制，并建立我们发现的mFox-2A同种型的生理作用，采用以下具体目标：第一章完成PTB和40 kD蛋白在外显子16剪接调控中的功能作用的表征，并表征它们促进外显子16剪接的机制activation.这将在体外和体内系统中采用组合的遗传和生物化学方法。2)确定Fox-2A在指导红细胞特异性选择性外显子选择中的生理影响。Fox-2A的影响将通过使用SpliceArray技术对晚期细胞中发生的红细胞特异性剪接变化进行综合分析来分析。3)研究选择性外显子1A和1C启动子的转录如何调控外显子2 '/2剪接位点的选择。这些研究将提供对特定因素如何发挥作用以编程红系细胞分化的见解。公共卫生相关性：许多不同形式的蛋白质可以通过选择性mRNA剪接从基因中产生;这些蛋白质形式的不当产生可能对人类疾病产生深远影响。我们研究了一个主要的例子（蛋白质4.1R），这种剪接是如何调节的。

DESCRIPTION (provided by applicant): We have employed the protein 4.1R gene as a model to study the molecular regulation of splicing during erythropoiesis. Protein 4.1R is a key element of the erythrocyte cytoskeleton. Two tightly regulated splicing events alter its expression and function during erythropoiesis: 1) An early event dictates exon 2' exclusion, which omits an upstream translation initiation site and produces only the "small" (80 kD) isoform. 2) A late event induces the inclusion of exon 16, which encodes a peptide critical for spectrin-actin binding. We have made progress toward understanding both events, including: 1) Identification of a stage-specific mFox-2A isoform that is up-regulated in late erythroid cells and critical for the exon 16 splicing switch; 2) Demonstration that increased SF2/ASF expression in late erythroid cells stimulates exon 16 splicing by binding an exonic splicing enhancer; 3) Quantification of a panel of splicing factors during erythroid differentiation, showing 4.1R splicing is modulated by varying the amount of selected ubiquitous and cell-type specific activators and inhibitors; two of these, PTB and a 40 kD novel protein are objects of the proposed studies; 4) Identification of a coupled transcription and splicing pathway that regulates exon 2' splicing; and 5) Detection of a switch in promoter usage that results in the production of the 80 kD isoform in mature red cells. We now propose to define more precisely the key elements and mechanisms governing these two splicing events, and to establish the physiological role of the mFox-2A isoform that we discovered, employing the following specific aims: 1) To complete the characterization of the functional roles of PTB and a 40 kD protein in exon 16 splicing regulation and to characterize the mechanisms by which they promote exon 16 splicing activation. This will employ combined genetic and biochemical approaches in both in vitro and in vivo systems. 2) To determine the physiological impact of Fox-2A in directing erythroid specific alternative exon selection. The impact of Fox-2A will be analyzed by comprehensive analysis of erythroid specific splicing changes that occur in late cells, using SpliceArray technologies. 3) To investigate how transcription from the alternative exon 1A and 1C promoters governs exon 2'/2 splice site selection. These studies will provide insights into how particular factors function to program the differentiation of erythroid cells. PUBLIC HEALTH RELEVANCE: Many different forms of a protein can be generated from a gene by alternative mRNA splicing; improper production of these protein forms could have a profound impact on human diseases. We study a major example (Protein 4.1R) of how this splicing is regulated.