RED CELL BAND 4.1:DEVELOPMENTAL CHANGES IN RNA SPLICING

作为理解结构和功能的长期承诺的一部分 在哺乳动物的红细胞中，这个实验室正在研究 调节独特膜簇合成的机制 在红细胞中表达的骨骼蛋白。其中一具骷髅 组分，蛋白质4.1，是一种多功能结构蛋白， 与其他几种骨骼元素相互作用，并表现出广泛的 红系和非红系细胞的相对分子质量谱。 这种结构蛋白的多种异构体是从一个家族翻译而来的 来自单个大的4.1的差异剪接的mRNA 基因，但具有不同的组织特异性表达模式。这项建议 旨在研究替代RNA剪接的作用和机制 蛋白质4.1组织特异性表达的相关事件 “拼接形状。”红系特异性剪接事件在两个区域的 基因将被调查：一个监管替代方案的使用 翻译起始点位于该基因的两个不同的5‘外显子， 而第二个控制结构不同的几个的表达 幽灵蛋白-肌动蛋白结合域。复制的细胞模型 蛋白质4.1中的发育剪接开关，如小鼠 将培养红白血病(MEL)细胞；体外核剪接 提取物将通过标准技术制备；以及拼接底物 以转录自蛋白质4.1基因组“迷你基因”的前mRNAs的形式 将被合成。对这些关键问题的实验操作 组件将允许对调节核苷酸序列进行表征 在前信使核糖核酸中，以及推测的反式作用核剪接 与这些序列相互作用的因素。最终，假定的剪接 将使用特定的探针(抗体或 寡核苷酸)或真核表达克隆技术。特定的 以前用于表征和克隆DNA的方法学 结合蛋白，如凝胶迁移率分析、DNA亲和力 层析，用寡核苷酸探针克隆原核表达， 和使用生物活性分析的真核表达克隆，将 适用于研究假定的RNA结合蛋白，这些蛋白 另一种拼接。 红细胞生成是一个显著的分化过程，在这个过程中 有核的红系前体细胞被广泛重塑，直到它们 实现成熟红细胞独特的形态特征。这个 这里提出的研究应该阐明以下机制(S) 选择性剪接影响红细胞的重要结构变化 红细胞生成过程中的膜蛋白。然而，在更广泛的意义上，这 这一提议代表了对血液学一个重大问题的探索 研究，即利用什么遗传策略来影响 基因表达的实质性变化，这是戏剧性表型的基础 红细胞生成的分化？这些研究最终将揭示 选择性RNA剪接是否是红细胞生成的主要效应因子，如 在果蝇体细胞性分化途径中，或简单地说是一个 选育基因结构改变的经济遗传机制 膜蛋白。

As part of a long term commitment to understand the structure and function of the mammalian erythrocyte, this laboratory is studying the genetic mechanisms that regulate synthesis of the unique constellation of membrane skeletal proteins expressed in red cells. One of those skeletal components, protein 4.1, is a multi-functional structural protein that interacts with several other skeletal elements and exhibits a broad spectrum of molecular weights in erythroid and nonerythroid cells. Multiple isoforms of this structural protein are translated from a family of differentially-spliced mRNAs that are derived from a single large 4.1 gene but have distinct tissue-specific expression patterns. This proposal seeks to investigate the role and mechanisms of alternative RNA splicing events responsible for tissue-specific expression of protein 4.1 "spliceoforms." Erythroid-specific splicing events in two regions of the gene will be investigated: one regulates utilization of alternative translation initiation sites located in two different 5' exons of the gene, while a second controls expression of several structurally different spectrin-actin binding domains. Cellular models that reproduce developmental splicing switches in protein 4.1, such as mouse erythroleukemia (MEL) cells, will be developed; in vitro nuclear splicing extracts will be prepared by standard techniques; and splicing substrates in the form of pre-mRNAs transcribed from protein 4.1 genomic "minigenes" will be synthesized. Experimental manipulation of these critical components will allow characterization of regulatory nucleotide sequences within the pre-mRNA, as well as putative trans-acting nuclear splicing factors that interact with these sequences. Ultimately, putative splicing factor cDNAs will be cloned using either specific probes (antibodies or oligonucleotides) or eukaryotic expression cloning techniques. Specific methodologies employed previously in characterization and cloning of DNA binding proteins, such as gel shift mobility assay, DNA-affinity chromatography, prokaryotic expression cloning with oligonucleotide probes, and eukaryotic expression cloning using biological activity assays, will be adapted for use in studying putative RNA binding proteins that mediate alternative splicing. Erythropoiesis constitutes a remarkable differentiation process in which nucleated erythroid precursor cells are extensively remodeled until they achieve the unique morphology characteristic of mature erythrocytes. The research proposed here should elucidate the mechanism(s) whereby alternative splicing effects important structural changes in red cell membrane proteins during erythropoiesis. In a broader sense, however, this proposal represents an exploration of a major question in hematology research, namely, what genetic strategies are utilized to effect the substantial changes in gene expression that underly the dramatic phenotypic differentiation in erythropoiesis? these studies will ultimately reveal whether alternative RNA splicing is a major effector of erythropoiesis, as in the Drosophila somatic sexual differentiation pathway, or simply an economical genetic mechanisms for changing the structure of selected membrane proteins.