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: 6576496
• 负责人: LARRY F LEMANSKI
• 金额: $ 24.24万
• 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/6576496
• 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(申请人提供)：我们的长期目标是阐明调控发育中心脏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。一种自然产生的隐性致死突变--墨西哥钝顶藻，是研究早期心脏发育的一个有趣的模型，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。通过用正常的前内胚层组织培养心脏，在前内胚层的条件培养液中培养，或者从内胚层或条件培养液中提取总RNA，可以纠正这种缺陷。此外，我们还从条件培养基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可以与胚胎中的至少两种蛋白质结合。这种核糖核酸可能是一种调节分子，它直接或与其结合蛋白(S)结合，上调突变心脏中原肌球蛋白的合成，促进肌原纤维的形成。为了解决我们的假设，这种RNA很可能与RNA结合蛋白一起促进发育中的心脏细胞的心肌细胞分化和肌纤维形成：1)克隆#4 RNA在促进胚胎心脏肌原纤维形成中的作用将被确定；2)转基因轴突将被用于测试克隆#4在体内拯救突变心脏的能力；3)通过原位杂交和定量RT-PCR将分析具有生物活性的克隆#4 RNA在胚胎发育中的表达；4)通过体外诱变来评估具有生物活性的克隆#4 RNA的结构与功能的关系；5)与生物活性核糖核酸结合的蛋白质将被纯化，并在细胞、生化和分子水平上进行鉴定；6)生物活性克隆#4RNA的潜在同源物(S)将在其他动物系统中进行检测。这项研究将为脊椎动物心脏肌原纤维形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肌肉的健康相关性可能是巨大的；如果这可以应用于人类，受损心脏组织的患者可能能够将该组织重新分化为功能肌肉。