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

G1/G0 逮捕期间 DND1 介导的小鼠精原细胞转录后调节

基本信息

批准号：
9770539
负责人：
Victor A. Ruthig
金额：
$ 6.16万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2020-12-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9770539
关键词：
Address Adult Alleles Antibodies Cell Cycle Cell Cycle Arrest Cell Differentiation process Cells Chromatin Development Embryo Failure Fertility Fetal Development Foundations G1 Phase Genes Genetic Transcription Genetic Translation Germ Cells Germ cell tumor Goals Immunoprecipitation Label Malignant Neoplasms Mediating Meiosis Messenger RNA Mus Mutation Normal Cell Outcome Pathway interactions Phase Play Population Post-Transcriptional Regulation Pregnancy Process Proteins Proxy RNA RNA Stability RNA immunoprecipitation sequencing RNA-Binding Proteins Regulation Research Resistance Ribosomal Proteins Ribosomes Role Specific qualifier value Spermatogenesis Teratoma Testicular Teratoma Testing Therapeutic Time Transcript Transgenic Organisms Translating Translations Undifferentiated Western Blotting Wild Type Mouse Work base cdc Genes cell type chromatin remodeling experimental study immunocytochemistry in vivo male mutant pluripotency prenatal stem cell fate stem cell population transcriptome transcriptome sequencing tumor tumorigenesis

项目摘要

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.

摘要