Studies on a Novel RNA that Promotes Heart Development

描述（由申请人提供）：我们的长期目标是阐明在心脏发育过程中调控早期心肌细胞分化和肌原纤维形成的细胞、分子和遗传机制。在蝾螈（蝾螈）中自然发生的隐性致死突变Ambystoma mexicanum是研究早期心脏发育的一个有趣模型，因为纯合子胚胎（c/c）形成的心脏缺乏原肌球蛋白，缺乏有组织的肌原纤维，并且不能跳动。这种缺陷可以通过将心脏与正常的前内胚层组织、前内胚层调节的培养基、或从内胚层或条件培养基中分离的总RNA进行培养来纠正。此外，我们从条件培养基RNA构建的cDNA文库中鉴定了一个新的克隆（#4），该文库作为能够纠正心脏缺陷的生物活性RNA的模板。这种RNA可以在胚胎中结合至少两种蛋白质。这种RNA可能是一种调节分子，可以上调突变心脏中原肌球蛋白的合成，并直接或与其结合蛋白复合促进肌纤维形成。我们的假设是，这种RNA极有可能与RNA结合蛋白一起促进心肌细胞分化和心肌纤维形成：1)将确定克隆4号RNA在胚胎心脏中促进心肌纤维形成的作用；2)转基因蝾螈将用于测试克隆4号在体内拯救突变心脏的能力；3)通过原位杂交和定量RT-PCR分析生物活性克隆4号RNA在发育胚胎中的表达；4)通过体外诱变评价生物活性克隆4号RNA的结构-功能关系；5)结合生物活性RNA的蛋白质将被纯化并在细胞、生化和分子水平上进行表征；6)生物活性克隆4号RNA的潜在同源物将在其他动物系统中进行检测。本研究将为研究脊椎动物心脏肌原纤维形成和心肌细胞抢救的分子机制提供新的基础信息。理解并能够将有缺陷的、不跳动的心脏组织转化为收缩肌肉对健康的意义是巨大的；如果这项技术能应用于人类，心脏组织受损的患者或许能将该组织重新分化为功能性肌肉。

DESCRIPTION (provided by applicant): Our long-term objectives are to elucidate the cellular, molecular and genetic mechanisms that regulate early myocardial cell differentiation and myofibrillogenesis in developing hearts. A naturally occurring recessive lethal mutation in axolotls (salamanders), Ambystoma mexicanum, is an intriguing model for studying early heart development, because homozygous embryos (c/c) form hearts that are deficient in tropomyosin, lack organized myofibrils, and fail to beat. The defect can be corrected by culturing hearts with normal anterior endoderm tissue, in medium conditioned by the anterior endoderm, or in total RNA isolated from endoderm or conditioned medium. In addition, we have identified a novel Clone (#4) from a cDNA library constructed from conditioned medium RNA which acts as a template for a bioactive RNA capable of correcting the heart defect. The RNA can bind at least two proteins in the embryos. It is possible that this RNA is a regulatory molecule which upregulates tropomyosin synthesis in mutant hearts and promotes myofibrillogenesis either directly or in complex with its binding protein(s). The following specific aims have been developed to address our hypothesis that this RNA, most likely in conjunction with RNA binding proteins, promotes myocardial cell differentiation and myofibrillogenesis in developing heart cells: 1) The role of the Clone #4 RNA in promoting myofibrillogenesis in embryonic hearts will be determined; 2) Transgenic axolotls will be used to test the ability of Clone #4 to rescue mutant hearts in vivo; 3) Expression of the bioactive Clone #4 RNA will be analyzed in developing embryos by in situ hybridization and quantitative RT-PCR; 4) The structural-functional relationships of the bioactive Clone #4 RNA will be evaluated by in vitro mutagenesis; 5) The proteins that bind to the bioactive RNA will be purified and characterized at the cellular, biochemical and molecular levels; 6) Potential homolog(s) of the bioactive Clone #4 RNA will be examined in other animal systems. This research will provide new basic information on the molecular mechanisms Of myofibrillogenesis and myocardial cell rescue in vertebrate hearts. The health relevance of understanding and being able to turn defective, non-beating cardiac tissue into contracting muscle could be tremendous; if this can be applied in humans, patients who have damaged heart tissue might be able to have that tissue redifferentiate into functional muscle again.