DND1 Mediated Posttranscriptional Regulation in Murine Prospermatogonia During G1/G0 Arrest

摘要 生殖细胞特异性RNA结合蛋白（RBP）的表达，即死端1（DND 1），在生殖细胞中启动。 在胚胎第7.5天（E）指定细胞。DND 1在整个胎儿发育和成人中表达 未分化的雄性生殖细胞（MGCs）。在胎儿发育期间，MGCs首先经历快速增殖， 然后进入细胞周期停滞期（G1/G0），占据胎儿发育的最后四分之一。在细胞 周期停滞MGCs过渡到前体细胞。有证据表明RNA的重要性- 结合蛋白和转录后调节在减数分裂，和其他时期的细胞周期暂停或 染色质重塑时，转录可能受到限制。然而，转录后调控过程中， G1/G0期，MGCs向原核细胞的转变尚未被研究。我们假设新的 在此期间，转录是有限的，DND 1在调节转录过程中起着关键作用。 在MGC细胞周期停滞期间mRNA靶标的可用性和翻译。为了解决这个假设， 1)我们将产生一个转录组的时间过程，跨越之前和期间的MGC细胞周期停滞 来追踪记录的稳定状态我们将使用5-乙炔尿苷（EU）或Bru标记来鉴定新的 这段时间的成绩单。2)接下来，我们将使用RNA免疫沉淀测序（RIP-seq），基于 携带GFP标记的DND 1等位基因的新转基因系，以在MGCs中体内鉴定DND 1的靶点 在细胞周期停滞之前和期间。3)然后，我们将使用GFP标记的 核糖体蛋白（L10a）在细胞周期停滞之前和期间的时间点，以确定mRNA的变化 加载到核糖体上。我们将确定是否核糖体加载的转录本翻译使用Western 代表性蛋白质的印迹和荧光免疫细胞化学。这项工作的目的是表征 调节生殖细胞多能性和分化的转录和转录后机制 在产前发育过程中，特别是RBP，DND 1是否调节可用性或 在细胞周期停滞期间，MGCs向原核细胞转变所必需的靶标的翻译。

ABSTRACT Expression of the germ-cell-specific RNA binding protein (RBP), dead end 1 (DND1), is initiated as germ cells are specified at embryonic day (E) 7.5. DND1 is expressed throughout fetal development and in adult undifferentiated male germ cells (MGCs). During fetal development, MGCs first undergo rapid proliferation, then enter a period of cell cycle arrest (G1/G0) occupying the last quarter of fetal development. During cell cycle arrest MGCs transition to prospermatogonia. There is established evidence for the importance of RNA- binding proteins and posttranscriptional regulation during meiosis, and other periods of cell cycle pausing or chromatin remodeling when transcription may be limited. However, posttranscriptional regulation during G1/G0, when MGCs transition to prospermatogonia has not been investigated. We hypothesize that new transcription is limited during this period, and that DND1 plays a critical role in regulating the availability and translation of mRNA targets during MGC cell cycle arrest. To address this hypothesis, 1) We will generate a transcriptome time course spanning the period before and during MGC cell cycle arrest to track the steady state level of transcripts. We will use 5-ethynyluridine (EU) or Bru labeling to identify new transcripts during this period. 2) Next, we will use RNA immunoprecipitation sequencing (RIP-seq), based on a new transgenic line carrying a GFP-tagged allele of DND1, to identify targets of DND1 in vivo in MGCs prior to and during cell cycle arrest. 3) We will then perform immunoprecipitation using a GFP-tagged ribosomal protein (L10a) at time points before and during cell cycle arrest to identify changes in mRNAs loaded onto ribosomes. We will determine whether ribosome-loaded transcripts are translated using western blots and fluorescent immunocytochemistry for representative proteins. This work aims to characterize the transcriptional and posttranscriptional mechanisms that regulate pluripotency and differentiation of germ cells during prenatal development, and specifically whether the RBP, DND1, regulates the availability or translation of targets essential for the transition of MGCs to prospermatogonia during cell cycle arrest.