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调节发育中心脏中早期心肌细胞分化和肌原纤维生成的细胞、分子和遗传机制。墨西哥蝾螈中自然发生的隐性致死突变是研究早期心脏发育的一个有趣模型，因为纯合胚胎 (c/c) 形成的心脏缺乏原肌球蛋白，缺乏有组织的肌原纤维，并且无法跳动。该缺陷可以通过将心脏与正常前内胚层组织一起在前内胚层条件培养基中培养，或者在从内胚层或条件培养基中分离的总RNA中培养来纠正。此外，我们还从条件培养基 RNA 构建的 cDNA 文库中鉴定出一种新型克隆 (#4)，该文库可作为能够纠正心脏缺陷的生物活性 RNA 的模板。 RNA可以结合胚胎中的至少两种蛋白质。该RNA可能是一种调节分子，它上调突变心脏中的原肌球蛋白合成，并直接或与其结合蛋白复合地促进肌原纤维生成。我们制定了以下具体目标来解决我们的假设，即这种 RNA 最有可能与 RNA 结合蛋白结合，促进发育中心脏细胞中的心肌细胞分化和肌原纤维生成：1) 将确定克隆 #4 RNA 在促进胚胎心脏中肌原纤维生成中的作用； 2) 转基因蝾螈将用于测试克隆#4在体内拯救突变心脏的能力； 3) 将通过原位杂交和定量 RT-PCR 分析发育中胚胎中生物活性克隆 #4 RNA 的表达； 4) 将通过体外诱变评估生物活性克隆#4 RNA的结构-功能关系； 5) 与生物活性RNA结合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) 将在其他动物系统中检查生物活性克隆 #4 RNA 的潜在同源物。该研究将为脊椎动物心脏中肌原纤维生成和心肌细胞拯救的分子机制提供新的基础信息。了解并能够将有缺陷的、不跳动的心脏组织转化为收缩的肌肉，对健康的意义可能是巨大的。如果这可以应用于人类，心脏组织受损的患者可能能够使该组织再次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.