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Aberrant RNA processing in MBNL1-deficient mice with erythroid defects

MBNL1 缺陷型红细胞缺陷小鼠的 RNA 加工异常

基本信息

批准号：
9115135
负责人：
JOHN G CONBOY
金额：
$ 27.88万
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：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9115135
关键词：
3&apos Untranslated Regions Adult Affect Alternative Splicing Anemia Animals Antisense Technology Area Binding Biology Bone Marrow Cell membrane Cell physiology Chloride Ion Chlorides Communities Computer Analysis Coupling Data Defect Development Disease Dissection Dysmyelopoietic Syndromes Erythroblasts Erythrocytes Erythroid Erythroid Cells Erythropoiesis Event Exons Foundations Future Gene Expression Genes Health Hematocrit procedure Hematologic Neoplasms Hematology Human Inherited Insulin Resistance Introns KH Domain Knock-out Knockout Mice Mediating Mediator of activation protein Molecular Analysis Mus Muscle Mutation Myotonic Dystrophy NIH Program Announcements Nerve Degeneration Outcomes Research Pathology Physiological Point Mutation Poly A Population Process Pronormoblasts Proteins RNA Processing RNA Recognition Motif RNA Splicing RNA-Binding Proteins Regulation Regulatory Element Reporter Research Reticulocyte count Reticulocytes Role Scientist Sequence Analysis Somatic Mutation Splenomegaly System Testing Therapeutic Intervention Tissues Transcript Trinucleotide Repeats Work Zinc Fingers base disease-causing mutation erythroid differentiation experience fetal human disease in vivo insight interest knockout animal mRNA Precursor mRNA Stability mouse model neglect novel programs protein structure function response transcriptome transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): This SHINE II proposal seeks to promote broader research into the role of RNA processing in erythropoiesis. The broad hypothesis of this work is that a highly orchestrated RNA processing program is essential for normal erythropoiesis, with the critical corollary that defects in RNA processing should have major adverse impacts on differentiation and/or red cell function that may underlie unexplained erythroid disorders. These concepts are actively exploited to provide exciting new insights in nonerythroid biology and disease, but until the recent discovery of splicing machinery mutations in myelodysplasia and other hematologic cancers, RNA processing has been under-appreciated in erythropoiesis. To study the impact of RNA processing in an erythroid in vivo system, this proposal focuses on a knockout mouse model with complete deficiency of MBNL1 (Muscleblind- like1), a zinc finger protein that regulates post-transcriptional processes including alternative pre-mRNA splicing (via binding to intron regulatory elements) and mRNA stability (via binding to 3'UTR sequences). Functional depletion of MBNL1 in the triplet repeat disease, myotonic dystrophy, causes splicing defects that correlate with specific physiological deficits of the disease. Preliminary data show that differentiating erythroblasts execute a robust alternative splicing program; that MBNL1 is one of the most abundant RNA processing factors in normal erythroblasts of both human and mouse; that MBNL1 can regulate a key alternative splicing event (in protein 4.1R pre-mRNA) required for mechanically stable red cell membranes; and that MBNL1 knockout mice have erythroid deficits manifested by elevated reticulocytes, reduced hematocrit, and greatly enlarged spleen. These observations support the hypothesis that MBNL1 deficiency should have a major impact on the erythroblast transcriptome, as it does in muscle. In accordance with the SHINE II format, this proposal consists of one aim. Aim 1A will be a global transcriptome analysis of FACS-purified proerythroblasts, orthochromatic erythroblasts, and reticulocytes using state of the art RNA-seq and computational analytical strategies. Comparison of MBNL1-knockouts with normal littermates is expected to reveal dozens to hundreds of altered transcripts that will represent candidate mediators of disease in these animals. Aim 1B will validate MBNL1-mediated RNA processing events predicted in 1A, using minigene approaches that allow manipulation of MBNL1 expression and MBNL1 binding motifs. Expected outcomes of this research are novel insights into MBNL1-regulated RNA processing networks, and identification of erythroid processes impacted by these networks that may contribute to pathology in knockout animals. Besides MBNL1, several other multi-functional RBPs with post-transcriptional regulatory activities and documented disease involvement in nonerythroid tissues are abundantly expressed in erythroid cells. Defects in this class of proteins may be an unappreciated cause of erythroid disease. Understanding splicing events critical for erythroid function could facilitate future therapeutic intervention with antisense technologies being developed to treat splicing diseases.

描述(由申请人提供)：这项Sare II提案旨在促进对RNA处理在红细胞生成中的作用的更广泛的研究。这项工作的广泛假设是，高度协调的RNA处理程序对于正常的红细胞生成是必不可少的，关键的推论是，RNA处理中的缺陷应该对分化和/或红细胞功能产生重大不利影响，这可能是原因不明的红系疾病的基础。这些概念被积极利用来为非红系生物学和疾病提供令人兴奋的新见解，但直到最近在骨髓发育不良症和其他血液病癌症中发现剪接机制突变之前，RNA加工在红细胞生成中一直被低估。为了研究在体内红系系统中RNA处理的影响，这项建议集中在完全缺乏MBNL1(肌肉盲样1)的敲除小鼠模型上，MBNL1是一种锌指蛋白，调节转录后过程，包括替代的前mRNA剪接(通过与内含子调控元件的结合)和mRNA的稳定性(通过与3‘UTR序列的结合)。在强直性肌营养不良的三重重复疾病中，MBNL1的功能缺失会导致剪接缺陷，这与疾病的特定生理缺陷有关。初步数据显示，分化的红细胞执行强大的选择性剪接程序；MBNL1是人和小鼠正常红细胞中含量最丰富的RNA处理因子之一；MBNL1可以调节红细胞膜机械稳定所需的一个关键的选择性剪接事件(在蛋白质4.1R Pre-mRNA中)；以及MBNL1基因敲除小鼠患有红系缺陷，表现为网织红细胞数量增加，红细胞压积降低，脾显著增大。这些观察结果支持这样一种假设，即MBNL1缺乏应该对红细胞转录组产生重大影响，就像对肌肉一样。根据SIRE II格式，这项提案由一个目标组成。目的使用最先进的RNA-SEQ和计算分析策略，对FACS纯化的前红细胞、正染红细胞和网织红细胞进行整体转录组分析。将MBNL1基因敲除基因与正常胎鼠进行比较，有望发现数十到数百个改变的转录本，这些转录本将代表这些动物疾病的候选介体。目的1B将验证在1A中预测的MBNL1介导的RNA加工事件，使用微基因方法，允许操纵MBNL1表达和MBNL1结合基序。这项研究的预期结果是对MBNL1调节的RNA加工网络的新见解，以及识别受这些网络影响的红系过程，这些过程可能有助于基因敲除动物的病理。除MBNL1外，其他几种具有转录后调控活性的多功能RBPs在红系细胞中大量表达，并被证实参与了非红系组织的疾病。这类蛋白质的缺陷可能是红系疾病的一个未被意识到的原因。了解剪接事件对红系功能至关重要，可以促进未来的治疗干预，反义技术正在开发用于治疗剪接疾病。