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MRNA Processing in Reproduction and Fertilization

生殖和受精中的 mRNA 加工

基本信息

批准号：
8103635
负责人：
Clinton C MacDonald
金额：
$ 31.18万
依托单位：
TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-09-27 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8103635
关键词：
Antibodies Beryllium Binding Binding Proteins Biological Assay C-terminal Cell Culture Techniques Cells Chromosome Segregation Chromosome Structures Complement Complementary DNA Complex Couples Cultured Cells DNA Transposable Elements Data Defect Disease Elements Essential Genes Exons Family Female Fertility Fertilization Gene Expression Genes Genetic Genetic screening method Genomics Germ Cells Grant Hereditary Disease Human Human Genetics Human Genome Immunoprecipitation Individual Infertility Knockout Mice Laboratory Study Learning Length Luciferases Male Infertility Mediating Meiosis Messenger RNA Metabolic Metabolism Molecular Mus Names Nuclear Patients Phenotype Physiological Poly(A) Tail Polyadenylation Polyadenylation Pathway Process Production Proteins RNA RNA Recognition Motif RNA-Binding Proteins RNA-Directed DNA Polymerase Reproduction Retroposon Retrotransposition Retrotransposon Role Running Signal Transduction Site Somatic Cell Spermatids Spermatogenesis Techniques Testing Testis Tissues Transfection Transgenic Mice Transgenic Organisms Variant Wild Type Mouse base crosslink in vivo mRNA Expression mRNA Precursor male mouse genome mutant paralogous gene reproductive sperm cell transcription factor

项目摘要

DESCRIPTION (provided by applicant): Polyadenylation-the addition of a poly(A) tail to an mRNA 32 end-is essential for gene expression in every tissue. However, fundamental features of polyadenylation-the RNA signals used, the sites chosen, and the proteins involved-are different in male germ cells than in any other tissue. These differences are essential for the correct expression of key genes during spermatogenesis, for example altering the protein forms of transcription factors or altering translational efficiency. DCstF-64 (gene name: Cstf2t) is the testis-expressed variant of CstF-64, an RNA-binding protein that regulates polyadenylation in somatic cells. DCstF-64 is essential for normal spermatogenesis: male Cstf2t knockout mice are infertile due to severe defects in meiotic and postmeiotic sperm production, a condition that resembles oligoasthenoteratozoospermia in human patients. We have found that Cstf2t controls polyadenylation of at least two important classes of genes, long interspersed nuclear elements (LINEs) and intronless small genes (ISGs). LINEs are mobile elements that are responsible for at least 70 genetic diseases, thus being a genomic and metabolic burden. DCstF-64 reduces LINE mRNAs by promoting polyadenylation at internal sites in the gene, thus reducing their abundance and suppressing their proliferation. To determine how DCstF-64 controls LINE expression, we will test whether exogenous DCstF-64 will suppress LINE mRNA expression, whether DCstF-64 binds to LINE mRNAs at internal polyadenylation sites, and whether exogenous DCstF-64 will suppress rates of retrotransposition in a cell culture assay. ISGs are expressed retroposons that control major functions in spermatogenesis (metabolism, gene expression, chromosome structure, and more). To determine how DCstF-64 regulates polyadenylation and termination of ISG mRNAs, we will test whether DCstF-64 is associated preferentially with ISG polyadenylation sites, whether exogenous DCstF-64 is required for correct polyadenylation of ISGs, and whether DCstF-64 is required for normal transcriptional termination of ISGs. Finally, to determine germ cell-specific functions of DCstF-64, we will perform a genetic test to determine whether CstF-64 will complement the Cstf2ttm1Ccma infertility phenotype, purify DCstF-64 complexes to look for germ cell-specific components, and test functions of DCstF-64 domains using a luciferase-based cell transfection assay and transgenic mice. PUBLIC HEALTH RELEVANCE: The most heartbreaking of disorders can be infertility, when a couple fails to conceive after at least a year of attempts. We discovered a gene, CSTF2T, that can be a hidden cause of male infertility-hidden, because females missing this gene have normal fertility, while males have severe problems in sperm production. Using a variety of techniques, we want to learn the molecular causes of why sperm production fails in males missing the CSTF2T gene, to better understand how to assist these infertile couples.

描述（由申请人提供）：聚腺苷酸化——在 mRNA 32 末端添加聚腺苷酸尾——对于每个组织中的基因表达都是必需的。然而，雄性生殖细胞中多聚腺苷酸化的基本特征（使用的 RNA 信号、选择的位点以及涉及的蛋白质）与任何其他组织都不同。这些差异对于精子发生过程中关键基因的正确表达至关重要，例如改变转录因子的蛋白质形式或改变翻译效率。 DCstF-64（基因名称：Cstf2t）是 CstF-64 的睾丸表达变体，CstF-64 是一种调节体细胞多聚腺苷酸化的 RNA 结合蛋白。 DCstF-64 对于正常精子发生至关重要：雄性 Cstf2t 基因敲除小鼠由于减数分裂和减数分裂后精子产生的严重缺陷而无法生育，这种情况类似于人类患者的少弱精子症。我们发现 Cstf2t 控制至少两类重要基因的多腺苷酸化，即长散布核元件 (LINE) 和无内含子小基因 (ISG)。 LINE 是导致至少 70 种遗传疾病的移动元件，因此是基因组和代谢负担。 DCstF-64 通过促进基因内部位点的聚腺苷酸化来减少 LINE mRNA，从而降低其丰度并抑制其增殖。为了确定 DCstF-64 如何控制 LINE 表达，我们将测试外源 DCstF-64 是否会抑制 LINE mRNA 表达、DCstF-64 是否在内部多腺苷酸化位点结合 LINE mRNA，以及外源 DCstF-64 是否会在细胞培养测定中抑制逆转录转座率。 ISG 是表达的逆转录子，控制精子发生的主要功能（代谢、基因表达、染色体结构等）。为了确定 DCstF-64 如何调节 ISG mRNA 的多腺苷酸化和终止，我们将测试 DCstF-64 是否优先与 ISG 多腺苷酸化位点相关，ISG 的正确多腺苷酸化是否需要外源 DCstF-64，以及 ISG 的正常转录终止是否需要 DCstF-64。最后，为了确定 DCstF-64 的生殖细胞特异性功能，我们将进行基因测试以确定 CstF-64 是否会补充 Cstf2ttm1Ccma 不育表型，纯化 DCstF-64 复合物以寻找生殖细胞特异性成分，并使用基于荧光素酶的细胞转染测定和转基因小鼠测试 DCstF-64 结构域的功能。公共卫生相关性：最令人心碎的疾病可能是不孕症，即一对夫妇在至少一年的尝试后未能怀孕。我们发现了一个基因，CSTF2T，它可能是男性不育的一个隐藏原因——隐藏，因为缺失这个基因的女性具有正常的生育能力，而男性则在精子生成方面存在严重问题。我们希望利用各种技术来了解缺少 CSTF2T 基因的男性精子生成失败的分子原因，以便更好地了解如何帮助这些不孕夫妇。