Nuclear small RNA control of the germ to soma transition

核小RNA控制细菌到体细胞的转变

• 批准号: 9003797
• 负责人: Brian Matthew Farley
• 金额: $ 5.8万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9003797
• 关键词: Antibodies; Base Pairing; Base Sequence; Biochemical; Biological; Biological Process; Cell Nucleus; Cell division; Cells; Chromatin; Chromosomes; Complex; DNA Transposable Elements; Defect; Development; Diploidy; Embryonic Development; Employee Strikes; Epitopes; Event; Exhibits; Fertilization; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Germ; Germ Cells; Human; Immunoprecipitation; Individual; Invertebrates; Link; Location; Macronucleus; Mass Spectrum Analysis; Mediating; Meiosis; Mitosis; Modification; Molecular; Molecular Biology; Molecular Mechanisms of Action; Nuclear; Nucleic acid sequencing; Organism; Pathway interactions; Play; Polyploidy; Population; Process; Protein Family; Proteins; RNA Interference; Recruitment Activity; Regulation; Repression; Reproduction; Role; Site; Small Nuclear RNA; Small RNA; Somatic Cell; Study models; System; Techniques; Tetrahymena; Tetrahymena thermophila; Transcript; Untranslated RNA; Vertebrates; Work; chromatin immunoprecipitation; crosslink; egg; gel electrophoresis; genetic information; histone modification; member; micronucleus; neuronal cell body; programs; public health relevance; sperm cell; tool

 DESCRIPTION (provided by applicant): The chromosomes contained in gametes are very different from the chromosomes contained in somatic cells just a few cell divisions following fertilization. Gametic chromosomes are transcriptionally inaccessible, while somatic chromosomes exhibit accessible chromatin in specific locations linked to actively transcribed genes. Establishment of these specific regions of transcriptionally permissive chromatin following fertilization is essential for early embryogenesis and beyond, but the neither the identities of the regulatory factors that directly participate in this process nor their molecular mechanisms of action are well understood. A candidate class of factors that directly mediate the process of sequence-specific establishment of somatic chromosome modifications are nuclear localized members of the Argonaute/Piwi family of proteins. These proteins associate with small RNAs, which enable them to specifically recognize and regulate complementary nucleic acid sequences by base pairing. Nuclear localized Piwi proteins are essential for repressing the mobilization of transposable DNA elements in the nuclei of germ cells of a wide variety of invertebrate and vertebrate species, but it is likely that they have additional function in gametes both before and after fertilization. Disentangling these functions from transposon repression is challenging in most experimental systems, which further complicates attaining a detailed understanding of their function. The ciliate Tetrahymena thermophila is a useful system for the study of nuclear small RNA functions during the germ to soma transition, as multiple, separate nuclear Piwi-guided pathways regulate transposon repression and other functions during sexual reproduction. The Tetrahymena Piwi protein Twi1p represses transposons by establishing repressive histone modifications, while the biological role and function of Twi8p is largely unknown. My work has demonstrated that Twi8p is essential for events that follow fertilization and that it is loaded with small RNAs derived from genomic loci that give rise to non coding RNAs, termed Twi8p-associated small RNA loci (TASLs). I propose that Twi8p guides the establishment of somatic chromosome modifications through TASLs during sexual reproduction. I will determine what these modifications are and what proteins establish them using the powerful molecular biology tools developed in Tetrahymena. I will also determine the biological role of the TASLs during sexual reproduction by specifically disrupting them in either the parental or zygotic genome and characterizing the molecular defects. By leveraging the unique tools and techniques established in Tetrahymena for small RNA gene regulation, I will contribute to our understanding of these conserved processes that are essential in invertebrates and vertebrates alike.

 描述（由申请人提供）：配子中包含的染色体与受精后仅进行几次细胞分裂的体细胞中包含的染色体非常不同。配子染色体在转录上是不可接近的，而体细胞染色体在与活跃转录基因相关的特定位置上表现出可接近的染色质。受精后转录允许染色质的这些特定区域的建立对于早期胚胎发生及以后至关重要，但直接参与这一过程的调节因子的身份及其作用的分子机制尚不清楚。 直接介导体细胞染色体修饰序列特异性建立过程的一类候选因子是 Argonaute/Piwi 蛋白家族的核定位成员。这些蛋白质与小 RNA 结合，使它们能够通过碱基配对特异性识别和调节互补核酸序列。核定位的 Piwi 蛋白对于抑制多种无脊椎动物和脊椎动物生殖细胞核中转座 DNA 元件的动员至关重要，但它们很可能在受精前后的配子中具有其他功能。在大多数实验系统中，将这些功能与转座子抑制分开是具有挑战性的，这使得详细了解它们的功能变得更加复杂。 纤毛虫嗜热四膜虫是研究胚芽到体细胞转变过程中核小 RNA 功能的有用系统，因为多个独立的核 Piwi 引导通路调节有性生殖过程中的转座子抑制和其他功能。四膜虫 Piwi 蛋白 Twi1p 通过建立抑制性组蛋白修饰来抑制转座子，而 Twi8p 的生物学作用和功能很大程度上未知。我的工作已经证明 Twi8p 对于受精后的事件至关重要，并且它装载有来自基因组位点的小 RNA，这些小 RNA 产生非 编码 RNA，称为 Twi8p 相关小 RNA 位点 (TASL)。我认为 Twi8p 在有性生殖过程中通过 TASL 指导体细胞染色体修饰的建立。我将使用四膜虫开发的强大分子生物学工具来确定这些修饰是什么以及哪些蛋白质建立它们。我还将通过在亲本或合子基因组中专门破坏 TASL 并表征分子缺陷，来确定 TASL 在有性生殖过程中的生物学作用。通过利用四膜虫中建立的用于小 RNA 基因调控的独特工具和技术，我将有助于我们理解这些对无脊椎动物和脊椎动物都至关重要的保守过程。