Red Cell Band 4.1 - Developmental Changes in RNA Splicing

这一更新建议探索了在红系细胞分化过程中调节替代前mRNA剪接的机制，重点是顺式调节序列和反式剪接因子蛋白，这些蛋白质调节外显子剪接中红系阶段特定的开关。研究将研究晚期红细胞中激活蛋白4.1R外显子16(E16)的典型剪接开关，已知E16对红细胞膜的机械稳定具有重要的生理意义，以及其他红系前体mRNAs中几个新发现的、进化上保守的剪接开关。该提案的主要目的包括：(1)亲和纯化在E16下游高度保守的内含子中与Fox2增强子功能拮抗或协同的新因子；(2)检验四个新的红系剪接开关与E16协调调节的假设，由Fox2或其他E16调节因子，为更广泛的红系剪接计划提供新的见解；以及(3)启动一项新的努力，以表征E16中顺式调节元件和反式剪接因子的功能 活体动物模型。除了对红系功能的生物学重要性外，E16剪接开关也是分析任何细胞系统中组织特异性剪接的最佳模型之一。建议研究的创新特征包括：详细分析内含子16 Fox2位点两侧的保守内含子，这可能有助于深入了解Fox-2剪接活性的调节；对其他几个红系前体mRNAs中推测共同调控的剪接开关的初步研究；以及对E16调控元件的体内分析。这些目标的成功实现将有助于更好地理解4.1R premRNA剪接中的阶段特异性开关，并可能为Fox-2在介导红系分化阶段特异性选择性剪接程序中发挥更大的潜在作用提供初步证据。这些研究还应该为异常剪接引起的疾病机制提供洞察力，最终导致剪接疗法来纠正这种缺陷。

This renewal proposal explores mechanisms that regulate alternative pre-mRNA splicing in differentiating erythroid cells, focusing on cis-regulatory sequences and trans-acting splicing factor proteins that regulate erythroid stage-specific switches in exon splicing. Studies will investigate the prototypical splicing switch in late erythroblasts that activates protein 4.1R exon 16 (E16), known to be physiologically important for mechanical stabilization of the red cell membrane, and several newly appreciated and evolutionarily conserved splicing switches in other erythroid pre-mRNAs. Major aims of the proposal include (1) affinity purification of novel factors that antagonize or synergize with Fox2 enhancer function in the highly conserved intron downstream of E16; (2) testing the hypothesis that four new erythroid splicing switches are coordinately regulated with E16, by Fox2 or other E16-regulatory factors, to provide new insights into the broader erythroid splicing program; and (3) initiation of a new effort to characterize functionality of cis-regulatory elements and trans-splicing factors in vivo with animal models. In addition to its biological importance for erythroid function, the E16 splicing switch is one of the best models for analysis of tissue-specific splicing in any cell system. Innovative features of the proposed studies include: detailed analysis of the conserved intron flanking the intron 16 Fox2 sites, that may provide insight into modulation of Fox-2 splicing activity; initial studies of the putatively co-regulated splicing switches in several other erythroid pre-mRNAs; and in vivo analysis of E16 regulatory elements. Successful accomplishment of these objectives will lead to a better understanding of the stage-specific switch in 4.1R premRNA splicing, and may provide preliminary evidence regarding a potential larger role for Fox-2 in mediating the erythroid differentiation stage-specific alternative splicing program. These studies should also provide insights into disease mechanisms caused by aberrant splicing, ultimately leading to splicing therapeutics to correct such defects.