RED CELL BAND 41--DEVELOPMENTAL CHANGES IN RNA SPLICING

描述：这份更新建议旨在了解分子 调控选择性前-mRNA剪接的基础，特别是 因为它与正常红系期间基因表达的调节有关 差异化。作为这些分析的一个范例，监管 将探索蛋白4.1前-mRNA的外显子16。外显子16编码 一个位于血影蛋白-肌动蛋白结合区的21个氨基酸的盒 这使蛋白质4.1具有促进 肌动蛋白与膜骨架中的幽灵蛋白。该外显子缺失 (剪接)在早期红系祖细胞的成熟mRNA中，但 在晚期祖细胞的4.1mRNA中存在(拼接)。康博伊博士 假设外显子16剪接的调控取决于两者 4.1 Pre-mRNA中的顺式调控序列元件和反式作用 RNA结合蛋白/剪接因子。生成用于以下目的的RNA底物 这些机制研究表明，一个简单的含有外显子16的3外显子微型基因 在天然内含子序列和构成外显子的两侧， 建造的。这一模型4.1 Pre-mRNA已被证明参与 在跟随完整4.1前-mRNA的选择性剪接程序中， 产生了包含外显子16的两种不同的产物。 建议使用这种微型基因结构来i)表征顺式计算机 可以正向或负向调节外显子16剪接的元件， Ii)SR已知可选剪接因子的测试效果 外显子16上的家族剪接，III)使用RNA亲和层析 分离和克隆4.1 Pre-mRNA相关的新反式因子 剪接，以及iv)表征备选的表达 红系发育过程中的剪接因子。用于分析 4.1在mRNA剪接前，将采用3种模型剪接系统： 显微注射非洲爪哇卵母细胞、体外核提取液和 转基因的MEL细胞。这些研究应该允许详细的 一个关键的拼接开关的调节解释 红系发育，使红细胞稳定 膜骨架。

DESCRIPTION: This renewal proposal seeks to understand the molecular basis of the regulation of alternative pre-MRNA splicing, especially as it pertains to regulation of gene expression during normal erythroid differentiation. As a paradigm for these analyses, regulation of exon 16 of protein 4.1 pre-MRNA will be explored. Exon 16 encodes a 21 amino acid cassette within the spectrin-actin binding domain that endows protein 4.1 with the capacity to promote association of actin with spectrin in the membrane skeleton. This exon is absent (spliced out) in the mature MRNA of early erythroid progenitors but present (spliced in) in 4.1 MRNA of late progenitors. Dr. Conboy hypothesizes that regulation of exon 16 splicing depends on both cis- regulatory sequence elements in 4.1 pre-MRNA and trans-acting RNA binding protein/splicing factors. To generate an RNA substrate for these mechanistic studies, a simple 3 exon minigene containing exon 16 flanked by native intron sequences and constitutive exons, has been constructed. This model 4.1 pre- MRNA has been shown to participate in the alternative splicing program following by intact 4.1 pre-MRNA, yielding two products differing in inclusion of exon 16. Dr. Conboy proposed to employ this minigene construct to i) characterize cis elements that may positively or negatively modulate exon 16 splicing, ii) test effects of known alternative splicing factors of the SR family on exon 16 splicing, iii) employ RNA affinity chromatography to isolate and clone new trans factors involved in 4.1 pre-MRNA splicing, and iv) characterize the expression of the alternative splicing factors during erythroid development. For analyses of 4.1 pre-MRNA splicing, 3 model splicing systems will be employed: microinjected Xenopus oocytes, nuclear extracts in vitro, and transfected MEL cells. These studies should allow a detailed explanation of the regulation of a critical splicing switch during erythroid development that allows stabilization of the erythrocyte membrane skeleton.