(9) The Germ Cell-Specific RNA-Binding Protein RBM46 is an Essential Post-Transcriptional Repressor of Meiotic Gene Expression in the Male Germline

Emma A. Gilbert¹; Bryan A. Niedenberger¹; Brett D. Keiper²; Brian P. Hermann³; and Christopher B. Geyer^1,4

1. Anatomy & Cell Biology Department, Brody School of Medicine at East Carolina University, Greenville, North Carolina, USA

2. Biochemistry and Molecular Biology Department, Brody School of Medicine at East Carolina University, Greenville, North Carolina, USA

3. Neuroscience, Developmental and Regenerative Biology Laboratory, University of Texas at San Antonio, San Antonio, Texas, USA

4. East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, North Carolina, USA

Abstract Text:

To produce haploid gametes, mitotic germ cells must enter and progress through the specialized meiotic program. The mechanisms driving meiotic entry are poorly understood in higher eukaryotes, especially mammals. During mammalian spermatogenesis, meiosis initiates in postnatal testes only after mitotic spermatogonia complete a lengthy retinoic acid (RA)-induced differentiation program. It is during this enigmatic differentiation program that epigenetic priming and transcriptional activation of the meiotic gene program occurs. However, these mRNAs remain repressed (inhibited, sequestered, and/or decayed) prior to entry into meiosis. Thus, the meiotic gene expression program is regulated post-transcriptionally, switching from repression in mitotic spermatogonia to efficient translation in meiotic spermatocytes. Little is known about the molecular mechanisms responsible for this post-transcriptional regulatory switch. A candidate component in mice is RBM46, a germ cell-specific and evolutionarily conserved RNA-binding protein that is the mammalian ortholog of the Drosophila tumorous testis (tut) gene. RBM46 is essential for fertility in both female and male mice; in males, RBM46 is expressed in both mitotic spermatogonia and meiotic spermatocytes and exists in a multiprotein complex(es) to bind and regulate transcripts required for both mitosis and meiosis. In the absence of RBM46, male germ cells fail to enter meiosis, resulting in infertility; however, the timing of germ cell dysfunction and the underlying molecular mechanisms of RBM46 remain undefined. To define the molecular mechanisms underpinning post-transcriptional control of meiotic initiation and to understand how RBM46 regulates mRNA utilization, we employed two genetic mouse models: a FLAG-tagged RBM46, with the tag inserted in-frame with the N-terminus of the endogenous gene, as well as an Rbm46 conventional knockout. Using these mice, we synchronized spermatogenesis and found RBM46 binds multiple essential meiotic transcripts. RBM46 is undetectable in spermatogonial stem cells (SSCs), but is upregulated in transit-amplifying undifferentiated and in differentiating spermatogonia and is essential for the final stages of spermatogonial differentiation. In testes of Rbm46 knockout mice, meiotic mRNA targets of RBM46 were aberrantly translated in mitotic spermatogonia. These spermatogonia of Rbm46 knockout mice attempted to precociously enter meiosis and subsequently died by apoptosis. Taken together, our findings position RBM46 as a key post-transcriptional repressor of meiotic gene expression in mitotic spermatogonia, and this repression program regulates the proper timing of meiotic entry in the male germline.