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）：该 SHINE II 提案旨在促进对 RNA 加工在红细胞生成中的作用进行更广泛的研究。这项工作的广泛假设是，高度精心策划的 RNA 加工程序对于正常红细胞生成至关重要，其关键推论是 RNA 加工缺陷应对可能导致无法解释的红细胞疾病的分化和/或红细胞功能产生重大不利影响。这些概念被积极利用，为非红细胞生物学和疾病提供令人兴奋的新见解，但直到最近发现骨髓增生异常和其他血液癌症中的剪接机制突变，RNA 加工在红细胞生成中一直未被充分重视。为了研究 RNA 加工对红细胞体内系统的影响，本提案重点关注完全缺乏 MBNL1（Muscleblind-like1）的敲除小鼠模型，MBNL1 是一种调节转录后过程（包括替代性前 mRNA 剪接）的锌指蛋白。通过结合内含子调控元件）和 mRNA 稳定性（通过结合 3'UTR 序列）。三联重复疾病（强直性肌营养不良）中 MBNL1 的功能性缺失会导致与该疾病的特定生理缺陷相关的剪接缺陷。初步数据表明，分化的成红细胞执行强大的选择性剪接程序； MBNL1 是人和小鼠正常成红细胞中最丰富的 RNA 加工因子之一； MBNL1 可以调节机械稳定红细胞膜所需的关键选择性剪接事件（在蛋白质 4.1R 前体 mRNA 中）； MBNL1 基因敲除小鼠存在红细胞缺陷，表现为网织红细胞升高、血细胞比容降低和脾脏极大增大。这些观察结果支持这样的假设：MBNL1 缺陷应该对成红细胞转录组产生重大影响，就像在肌肉中一样。根据 SHINE II 格式，该提案只有一个目标。目标 1A 将使用最先进的 RNA-seq 和计算分析策略对 FACS 纯化的原红细胞、正染色红细胞和网织红细胞进行全局转录组分析。 MBNL1 敲除与正常同窝动物的比较预计会揭示数十到数百个改变的转录本，这些转录本将代表这些动物中疾病的候选介质。目标 1B 将使用允许操纵 MBNL1 表达和 MBNL1 结合基序的小基因方法来验证 1A 中预测的 MBNL1 介导的 RNA 加工事件。这项研究的预期成果是对 MBNL1 调节的 RNA 加工网络的新见解，以及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基因敲除动物的病理学。除了 MBNL1 之外，其他几种具有转录后调节活性并记录在非红细胞组织中的疾病参与的多功能 RBP 在红细胞中也大量表达。这类蛋白质的缺陷可能是红细胞疾病的一个未被认识到的原因。了解对红细胞功能至关重要的剪接事件可以促进未来利用正在开发的用于治疗剪接疾病的反义技术进行治疗干预。