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Shine II提案旨在促进对RNA加工在红细胞生成的作用的更广泛的研究。这项工作的广泛假设是，高度精心策划的RNA加工程序对于正常的红细胞生成是必不可少的，其关键推论是，RNA加工中的缺陷应对分化和/或红细胞功能产生重大不利影响，这可能是无法解释的肾上腺炎疾病的基础。这些概念被积极利用，以提供非炎热生物学和疾病的令人兴奋的新见解，但是直到最近发现骨髓增生和其他血液学癌症的剪接机械突变之前，RNA处理在erythropoiesis中一直被低估。为了研究RNA加工在体内系统中的红细胞系统中的影响，该建议着重于完全缺乏MBNL1（Muscleblind- oke1）的基因敲除小鼠模型，这是一种调节文字后过程的锌指蛋白，包括替代性预MRNA剪接（通过与内含子调节元件的结合）和mRNA稳定性（通过与3'UTR序列结合）。 MBNL1在三胞胎重复疾病，肌动症障碍中的功能耗竭会导致剪接缺陷与疾病的特定生理缺陷相关。初步数据表明，区分红细胞执行强大的替代剪接程序；该MBNL1是人和小鼠正常红细胞中最丰富的RNA加工因子之一。 MBNL1可以调节机械稳定的红细胞膜所需的关键替代剪接事件（在蛋白质4.1R前MRNA中）；并且MBNL1敲除小鼠的红细胞缺陷表现为升高的网状细胞，血细胞比容降低和脾脏肿大。这些观察结果支持以下假设：MBNL1缺乏应像在肌肉中一样对红细胞转录组产生重大影响。根据Shine II格式，该提案包括一个目标。 AIM 1A将是使用最先进的RNA-seq和计算分析策略的FACS纯化的培养基细胞，原色红细胞和网状细胞的全球转录组分析。 MBNL1敲除与正常同窝仔的比较有望揭示数十个转录本改变的转录本，这些转录本将代表这些动物中疾病的候选介体。 AIM 1B将使用允许操纵MBNL1表达和MBNL1结合基序的微型方法来验证1A中预测的MBNL1介导的RNA处理事件。这项研究的预期结果是对MBNL1调节的RNA处理网络的新见解，以及对受这些网络影响的红细胞过程的鉴定，这些过程可能有助于敲除动物的病理学。除MBNL1外，其他几种具有转录后调节活性的多功能RBP和疾病参与非炎症组织的参与在红细胞细胞中大量表达。这类蛋白质中的缺陷可能是红骨疾病的未引起的原因。了解对红细胞功能至关重要的剪接事件可以促进未来的治疗干预措施，以治疗剪接疾病的反义技术。