Aberrant RNA processing in MBNL1-deficient mice with erythroid defects

描述（由申请人提供）：本SHINE II提案旨在促进RNA加工在红细胞生成中的作用的更广泛研究。这项工作的广泛假设是，高度协调的RNA加工程序对正常的红细胞生成至关重要，关键的推论是，RNA加工缺陷应该对分化和/或红细胞功能产生重大不利影响，这可能是无法解释的红细胞疾病的基础。这些概念被积极地利用来为非红细胞生物学和疾病提供令人兴奋的新见解，但直到最近在骨髓异常增生和其他血液学癌症中发现剪接机制突变，RNA加工在红细胞生成中的作用一直被低估。为了研究RNA加工对红系体内系统的影响，本研究将重点放在一个完全缺乏MBNL1 （Muscleblind- like1）的敲除小鼠模型上，MBNL1是一种锌指蛋白，可调节转录后过程，包括mRNA前剪接（通过结合内含子调节元件）和mRNA稳定性（通过结合3'UTR序列）。在三联体重复疾病（肌强直性营养不良）中，MBNL1的功能缺失导致剪接缺陷，而剪接缺陷与该疾病的特定生理缺陷相关。初步数据显示，分化的红母细胞执行一个强大的选择性剪接程序；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一个关键的选择性剪接事件（在蛋白4.1R pre-mRNA中）；MBNL1敲除小鼠存在红细胞缺陷，表现为网织红细胞升高、红细胞压积降低和脾脏肿大。这些观察结果支持了MBNL1缺乏应该对红母细胞转录组产生重大影响的假设，就像在肌肉中一样。根据SHINE II的格式，该建议包括一个目标。Aim 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、正色红细胞和网织红细胞进行全局转录组分析。mbnl1基因敲除与正常幼崽的比较有望揭示数十到数百个转录本的改变，这些转录本将代表这些动物疾病的候选介质。Aim 1B将验证1A中预测的MBNL1介导的RNA加工事件，使用允许操纵MBNL1表达和MBNL1结合基序的minigene方法。本研究的预期结果是对mbnl1调控的RNA加工网络的新见解，以及鉴定受这些网络影响的红系过程，这些过程可能有助于敲除动物的病理。除MBNL1外，其他几种具有转录后调控活性的多功能rbp也在红系细胞中大量表达，这些rbp在非红系组织中具有文献记载的疾病参与。这类蛋白的缺陷可能是红系疾病未被认识到的原因。了解剪接事件对红细胞功能至关重要，可以促进未来利用反义技术治疗剪接疾病。

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.