Red Cell Band 4.1 - Developmental Changes in RNA Splicing

红细胞带 4.1 - RNA 剪接的发育变化

基本信息

批准号：
7533943
负责人：
JOHN G CONBOY
金额：
$ 71.06万
依托单位：
UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7533943
关键词：
Accounting Actins Affinity Affinity Chromatography Alleles Alternative Splicing Animal Model Binding Binding Proteins Binding Sites Biological Biological Assay Biological Models Brain Cell membrane Cell model Cells Cultured Cells Data Defect Development Disease Elements Engineering Enhancers Erythroblasts Erythrocytes Erythroid Erythroid Cells Erythropoiesis Event Exons Fishes Foxes Funding Genes Goals Hemolytic Anemia Human Individual Introns Lead Longitudinal Studies Mammals Mass Spectrum Analysis Measurement Mechanics Mediating Membrane Methods Modeling Molecular Mus Muscle Nuclear Extract Pathway interactions Play Price Protein Isoforms Proteins RNA Binding RNA Splicing Regulation Regulatory Element Repression Role Site Specificity Spectrin Staging System Systems Analysis Technology Testing Therapeutic Time Tissue-Specific Splicing Tissues Trans-Splicing Vertebrates cell type cofactor combinatorial erythroid differentiation gene function genetic regulatory protein hnRNP A1 human disease in vivo innovation insight interest mRNA Precursor mouse model mutant novel precursor cell programs protein activation research study

项目摘要

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.

该更新建议探讨了调节区分红细胞细胞中替代性mRNA剪接的机制，重点关注顺式调节序列和跨作用剪接因子蛋白，以调节外显子拼接中的eryythroid阶段特异性开关。研究将研究激活蛋白质4.1R外显子16（E16）的蛋白质细胞晚期典型剪接开关，已知对于红细胞膜的机械稳定在生理上很重要，并且在其他红细胞中的一些新的和进化上保存的旋转开关中的其他红细胞前MRNA中很重要。该提案的主要目的包括（1）在E16下游高度保守的内部中与FOX2增强子功能拮抗或协同作用的新因素的亲和力净化；（2）检验以下假设：FOX2或其他E16调节因素对四个新的红细胞剪接开关与E16进行协调调节，以提供对更广泛的细红色剪接程序的新见解；（3）启动新的努力，以表征顺式调节元素的功能和跨性因子的功能带有动物模型的体内。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。拟议的研究的创新特征包括：对内含子16 FOX2位点的保守内含子的详细分析，这些内含子可能会深入了解FOX-2剪接活性的调节；对其他几个红细胞前MRNA中推定共同调节的剪接开关的初步研究； E16调节元件的体内分析。这些目标的成功实现将使人们更好地理解4.1R Premrna剪接中特定阶段的转换，并可能提供有关FOX-2在介导红细胞分化阶段特定替代剪接程序中潜在较大作用的初步证据。这些研究还应提供对异常剪接引起的疾病机制的见解，最终导致剪接治疗剂纠正此类缺陷。