Protein 4.1 Gene Expression in Developing Red Cells

描述（申请人提供）：我们以蛋白4.1R基因为模型，研究了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。两个严格调控的剪接事件改变了其在红细胞生成过程中的表达和功能：1)一个早期事件决定了外显子2'的排除，它忽略了上游的翻译起始位点，只产生“小”（80 kD）同种异构体。2)一个后期事件诱导了外显子16的包含，该外显子编码了一个对谱蛋白-肌动蛋白结合至关重要的肽。我们已经在理解这两个事件方面取得了进展，包括：1)鉴定了一个阶段特异性的mFox-2A亚型，该亚型在晚期红系细胞中上调，对外显子16剪接开关至关重要；2)证明红细胞晚期SF2/ASF表达增加通过结合外显子剪接增强子刺激外显子16剪接；3)红细胞分化过程中剪接因子的定量分析，表明4.1R剪接是通过改变所选择的普遍存在的和细胞类型特异性的激活因子和抑制剂的数量来调节的；其中两种，PTB和一种40 kD的新蛋白是拟议研究的对象；4)发现调控外显子2'剪接的转录和剪接耦合通路；5)在成熟红细胞中检测到启动子使用的开关，导致80 kD亚型的产生。我们现在建议更精确地定义控制这两个剪接事件的关键因素和机制，并确定我们发现的mFox-2A亚型的生理作用，采用以下具体目标：1)完成PTB和40kd蛋白在外显子16剪接调节中的功能作用的表征，并表征它们促进外显子16剪接激活的机制。这将在体外和体内系统中采用遗传和生化相结合的方法。2)确定Fox-2A在指导红系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技术对发生在晚期细胞中的红系特异性剪接变化进行综合分析。3)研究外显子1A和1C启动子的转录如何影响外显子2'/2剪接位点的选择。这些研究将提供深入了解特定的因素如何发挥作用，以规划红细胞的分化。公共卫生相关性：一个基因可以通过不同的mRNA剪接产生许多不同形式的蛋白质；这些蛋白质形式的不当产生可能对人类疾病产生深远的影响。我们研究了一个主要的例子（蛋白4.1R）是如何调节这种剪接的。

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.