Studies on a Novel RNA that Promotes Heart Development

促进心脏发育的新型RNA的研究

• 批准号: 6729905
• 负责人: LARRY F LEMANSKI
• 金额: $ 24.59万
• 依托单位: FLORIDA ATLANTIC UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-15 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6729905
• 关键词: RNA; RNA binding protein; Urodela; antisense nucleic acid; cardiogenesis; chemical structure function; endoderm; gel mobility shift assay; gene therapy; heart contraction; in situ hybridization; laboratory rabbit; lethal genes; mutant; myofibrils; myogenesis; nucleic acid quantitation /detection; polymerase chain reaction; protein localization; protein purification; site directed mutagenesis; tissue /cell culture; transfection; tropomyosin

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.

描述（由申请人提供）：我们的长期目标是阐明细胞，分子和遗传机制，这些机制调节发育中心的心肌分化早期的心肌细胞分化和肌原纤维化。墨西哥ambystoma墨西哥的Ambystoma Ambystoma中天然存在的隐性致命突变是一种研究早期心脏发育的有趣模型，因为纯合子胚胎（C/C）形成了缺乏Tropomyosin的心脏，缺乏有组织的肌动纤维，并且无法打败。可以通过用正常的内胚层组织培养心脏，在培养基中，由前内胚层调节，或从内胚层或条件培养基中分离出的总RNA来纠正缺陷。此外，我们还从条件培养基RNA构建的cDNA文库中确定了一种新颖的克隆（＃4），该cDNA文库充当了能够纠正心脏缺陷的生物活性RNA的模板。 RNA可以在胚胎中至少结合两个蛋白质。该RNA可能是一种调节分子，可上调突变体心脏中的tropomyosin合成，并直接或与其结合蛋白（S）促进肌原纤维生成。已经开发出以下具体目的是为了解决我们的假设，即这种RNA最有可能与RNA结合蛋白结合使用，可促进心脏细胞中心脏细胞的心肌分化和肌原纤维化发生：1）克隆＃4 RNA在促进胚胎心脏中肌纤维化心脏中的作用； 2）转基因轴突将用于测试克隆＃4在体内拯救突变体心脏的能力； 3）将通过原位杂交和定量RT-PCR来分析生物活性克隆＃4 RNA的表达； 4）生物活性克隆＃4 RNA的结构功能关系将通过体外诱变进行评估； 5）与生物活性RNA结合的蛋白质将在细胞，生化和分子水平上进行纯化和表征； 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。这项研究将提供有关脊椎动物心脏中肌原纤维发生和心肌细胞营救的分子机制的新基本信息。理解和能够将有缺陷的非伴随心脏组织变成收缩肌肉的健康相关性可能是巨大的。如果可以将其应用于人类中，那么损害心脏组织的患者可能会再次将其重新分化为功能性肌肉。