Red Cell Band 4.1 - Developmental Changes in RNA Splicing

该更新提案探讨了在分化红系细胞中调节替代性前mRNA剪接的机制，重点关注调节外显子剪接中红系阶段特异性开关的顺式调节序列和反式作用剪接因子蛋白。研究将调查激活蛋白4.1R外显子16（E16）的晚期成红细胞中的原型剪接开关，已知其对红细胞膜的机械稳定具有生理学重要性，以及其他红细胞前体mRNA中的几种新认识和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的高度保守内含子下游中拮抗或协同Fox 2增强子功能的新因子;（2）测试四个新的红细胞剪接开关与E16通过Fox 2或其他E16调节因子协同调节的假设，以提供对更广泛的红细胞剪接程序的新见解;和（3）开始一项新的努力，以表征顺式调节元件和反式剪接因子的功能， 体内与动物模型。除了其对红细胞功能的生物学重要性外，E16剪接开关是分析任何细胞系统中组织特异性剪接的最佳模型之一。拟议研究的创新特征包括：详细分析内含子16 Fox 2位点侧翼的保守内含子，这可能会提供对Fox-2剪接活性调节的深入了解;对其他几种红细胞前体mRNA中puereco-regulated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导致更好地理解的阶段特异性开关在4.1R前mRNA剪接，并可能提供初步证据，福克斯-2在介导红细胞分化阶段特异性选择性剪接程序的潜在更大的作用。这些研究还应该提供对异常剪接引起的疾病机制的见解，最终导致剪接疗法来纠正这些缺陷。

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.