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

• 批准号: 8613315
• 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
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8613315
• 关键词: 3' 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; Gene Expression Profile; Genes; 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; public health relevance; response; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT 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 proerythro- blasts, 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.

项目总结/摘要 SHINE II的提案旨在促进对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，一个高度协调的RNA加工程序对正常的细胞生长至关重要。 红细胞生成，与关键的推论，在RNA加工的缺陷应该有重大的不利影响， 红细胞分化和/或红细胞功能可能是原因不明的红细胞疾病的基础。这些概念是 积极利用，以提供令人兴奋的新见解，在非红细胞生物学和疾病，但直到最近， 在骨髓增生异常和其他血液癌症中发现剪接机制突变，RNA加工 在红细胞生成中被低估了研究RNA加工在体内红细胞中的影响 系统，该建议集中于具有MBNL1完全缺陷的敲除小鼠模型（肌盲- Like1），一种锌指蛋白，调节转录后过程，包括替代前mRNA 剪接（通过与内含子调控元件结合）和mRNA稳定性（通过与3'UTR序列结合）。 在三联体重复疾病，强直性肌营养不良中MBNL1的功能缺失，导致剪接缺陷， 与疾病的特定生理缺陷相关。初步数据显示， 成红细胞执行强有力的选择性剪接程序; MBNL1是最丰富的RNA之一 在人类和小鼠的正常成红细胞的加工因子; MBNL1可以调节一个关键的 机械稳定红细胞膜所需的可变剪接事件（在蛋白4.1R前mRNA中）;和 MBNL1敲除小鼠具有红细胞缺陷，表现为网织红细胞升高，血细胞比容降低， 脾脏肿大这些观察结果支持了MBNL1缺陷应该具有 对成红细胞转录组的主要影响，就像在肌肉中一样。根据SHINE II格式， 这一建议只有一个目的。目的1A将是一个全球性的转录组分析的FACS纯化的proglycoprotein， 原始细胞、正染性成红细胞和网织红细胞，使用现有技术水平的RNA-seq和计算 分析战略。将MBNL1基因敲除与正常同窝出生的小鼠进行比较，预计将发现数十种 数以百计的改变的转录本将代表这些动物疾病的候选介质。目标1B将 使用微基因方法验证1A中预测的MBNL1介导的RNA加工事件， MBNL1表达和MBNL1结合基序的操纵。本研究的预期结果是新颖的 深入了解MBNL1调控的RNA加工网络，并鉴定受MBNL1影响的红细胞过程。 这些网络可能会导致基因敲除动物的病理学。除了MBNL1，其他几个多- 具有转录后调节活性的功能性RBP和记录的疾病参与， 非红系组织在红系细胞中大量表达。这类蛋白质中的缺陷可能是一种 红细胞疾病的不明原因。了解对红细胞功能至关重要的剪接事件， 促进未来的治疗干预与反义技术正在开发治疗剪接疾病。