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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：
8251145
负责人：
Clinton C MacDonald
金额：
$ 31.45万
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/8251145
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 public health relevance 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）：聚腺苷化-在mRNA 32末端增加一个聚(a)尾部-是每个组织中基因表达所必需的。然而，聚腺苷化的基本特征——所使用的RNA信号、所选择的位点和所涉及的蛋白质——在男性生殖细胞中与其他任何组织中都是不同的。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腺苷化的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不育，这种情况类似于人类患者的少弱无畸形精子症。我们发现Cstf2t至少控制两类重要基因的多聚腺苷化，长穿插核元件（LINEs）和无内含子小基因（ISGs）。细胞系是导致至少70种遗传疾病的可移动元素，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化来减少LINE mrna，从而减少它们的丰度并抑制它们的增殖。为了确定DCstF-64如何控制LINE表达，我们将在细胞培养实验中测试外源性DCstF-64是否会抑制LINE mRNA表达，DCstF-64是否会在内部聚腺苷化位点与LINE mRNA结合，以及外源性DCstF-64是否会抑制逆转录转位率。isg是一种表达的反转录子，控制着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的多聚腺苷化和终止，我们将测试DCstF-64是否优先与ISG多聚腺苷化位点相关，ISG的正确多聚腺苷化是否需要外源性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试验和转基因小鼠测试DCstF-64结构域的功能。