Abstract
SummarySpermatogonial stem cells (SSCs) sustain spermatogenesis and fertility throughout adult male life. The conserved RNA-binding protein NANOS2 is essential for the maintenance of SSCs, but its targets and mechanisms of function are not fully understood. Here, we generated a fully functional epitope-tagged Nanos2 mouse allele and applied the highly stringent cross-linking and analysis of cDNAs to define NANOS2 RNA occupancy in SSC lines. NANOS2 recognizes the AUKAAWU consensus motif, mostly found in the 3′ untranslated region of defined messenger RNAs (mRNAs). We find that NANOS2 is a regulator of key signaling and metabolic pathways whose dosage or activity are known to be critical for SSC maintenance. NANOS2 interacts with components of CCR4-NOT deadenylase complex in SSC lines, and consequently, NANOS2 binding reduces the half-lives of target transcripts. In summary, NANOS2 contributes to SSC maintenance through the regulation of target mRNA stability and key self-renewal pathways.
Highlights
The maintenance of spermatogonial stem cells (SSCs) is essential to sustain life-long spermatogenesis and adult male fertility
We inserted a complex tag that consisted of the V5 tag, the Myc tag, the precision protease site, the His tag, and the enhanced green fluorescent protein (EGFP) that generates an N-terminal NANOS2 fusion protein (Figures 1A and S1)
In the adult testis Nanos2 expression is restricted to undifferentiated spermatogonia, many of which are GFRa1-positive (Sada et al, 2009; Suzuki et al, 2009)
Summary
The maintenance of spermatogonial stem cells (SSCs) is essential to sustain life-long spermatogenesis and adult male fertility. In mouse, this population of stem cells resides within undifferentiated type A spermatogonia (Huckins, 1971; Oakberg, 1971). Upon Gfra downregulation, SSCs give rise to an intermediate population of spermatogenic precursors, marked by Ngn, which are responsive to retinoic acid (RA) signaling and can differentiate into c-Kit-positive cells (Ikami et al, 2015; Nakagawa et al, 2010). Downstream signaling cascades activated by these growth factors, such as phosphatidylinositol 3-kinase (PI3K)-Akt and ERK/MAPK pathways are essential for SSC self-renewal (Hasegawa et al, 2013; Ishii et al, 2012; Lee et al, 2007). Tight control of mTOR complex 1 (mTORC1) activity is critical in regulating the balance between self-renewal and differentiation in SSCs (Busada et al, 2015b; Hobbs et al, 2010; Serra et al, 2017), as shown by the progressive spermatogenic failure associated with the alteration of the mTORC1 component RAPTOR or the regulatory TSC1/2 complex (Hobbs et al, 2015; Serra et al, 2019; Wang et al, 2016)
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