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

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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.