RED CELL BAND 41--DEVELOPMENTAL CHANGES IN RNA SPLICING

描述：本更新提案旨在了解分子 选择性前mRNA剪接的调节基础，特别是 因为它涉及正常红细胞系中基因表达的调节 分化 作为这些分析的范例， 将探索蛋白4.1前mRNA的外显子16。 外显子16编码 血影蛋白-肌动蛋白结合结构域内的21个氨基酸盒 赋予蛋白质4.1促进 肌动蛋白与血影蛋白在膜骨架中的结合。 该外显子缺失 在早期红系祖细胞的成熟mRNA中， 在晚期祖细胞的4.1 mRNA中存在（剪接）。 康博医生 假设16号外显子剪接的调控依赖于 4.1前mRNA中的顺式调节序列元件和反式作用元件 RNA结合蛋白/剪接因子。为了产生RNA底物， 这些机制的研究，一个简单的3外显子小基因含有外显子16 两侧是天然内含子序列和组成型外显子， 构建了该模型4.1前mRNA已被证明参与 在选择性剪接程序中， 产生包含外显子16的两种不同产物。 康博医生 建议使用该小基因构建体i）表征顺式 可以正向或负向调节外显子16剪接的元件， ii）测试SR的已知可变剪接因子的作用 iii）使用RNA亲和层析， 4. 1 pre-mRNA中新反式因子的分离和克隆 剪接，和iv）表征所述替代物的表达， 在红细胞发育过程中的剪接因子。 用于分析 4.1前mRNA剪接，将采用3种模型剪接系统： 显微注射非洲爪蟾卵母细胞，核提取物在体外， 转染MEL细胞。这些研究应该允许详细的 解释关键拼接开关的调节， 使红细胞稳定的红细胞发育 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.