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.

项目摘要/摘要 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处理程序对于正常 红细胞生成，关键的推论，RNA处理中的缺陷应对 分化和/或红细胞功能可能是无法解释的红斑疾病的基础。这些概念是 积极利用以提供非恐怖生物学和疾病的令人兴奋的新见解，但直到最近 在骨髓增生和其他血液学癌症中发现剪接机械突变，RNA处理 在红细胞生成中被低估了。研究RNA加工在体内红系中的影响 系统，该提案的重点是完全缺乏MBNL1的敲除小鼠模型（Muscleblind-- 类似1），一种调节转录后过程的锌指蛋白，包括替代前mRNA 剪接（通过与内含子调节元件结合）和mRNA稳定性（通过与3'UTR序列结合）。 MBNL1在三胞胎重复疾病，肌发育症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。初步数据表明差异化 生成细胞执行强大的替代剪接程序；该MBNL1是最丰富的RNA之一 人和小鼠正常的红细胞的加工因子； MBNL1可以调节钥匙 机械稳定的红细胞膜需要的替代剪接事件（在蛋白质4.1R前MRNA中）；和 MBNL1敲除小鼠的红细胞缺陷表现为升高的网状细胞，减少血细胞比容， 大大扩大了脾脏。这些观察结果支持以下假设 像在肌肉中一样，对红细胞转录组的主要影响。根据Shine II格式， 该提议包括一个目标。 AIM 1A将是对FACS纯化的Proerythro-的全球转录组分析 使用最先进的RNA-seq和计算状态 分析策略。 MBNL1敲除与正常同窝仔的比较有望揭示数十个 数百个变化的转录本将代表这些动物中疾病的候选介体。目标1B会 使用允许的微型方法验证1A中预测的MBNL1介导的RNA处理事件 MBNL1表达和MBNL1结合基序的操纵。这项研究的预期结果是新颖的 对MBNL1调节的RNA处理网络的见解，并识别受到影响 这些网络可能有助于淘汰动物的病理学。除了MBNL1，其他几个 具有转录后调节活动的功能性RBP和疾病参与 在红细胞细胞中大量表达非果皮组织。这类蛋白质中的缺陷可能是 未经批准的红斑疾病原因。了解对红细胞功能至关重要的剪接事件可能 通过开发反义技术来治疗剪接疾病，促进未来的治疗干预。