Molecular function of the RNAbinding protein NANOS2 in mouse spermatogonial stem cells

Spermatogonial stem cells (SSCs) are adult unipotent stem cells which are able to both self-renew and differentiate to support spermatogenesis throughout adult life. NANOS2 is a conserved RNA-binding protein, and its genetic deletion in adult mice leads to male infertility due to loss of SSCs. However, the precise mechanism underlying NANOS2 function during SSCs maintenance remains largely unexplored at the transcriptomic level. To accurately identify NANOS2 RNA-occupancy, we generated a Nanos2TAG mouse model, I derived and expanded SSCs in vitro and performed CRAC (UV-Cross-linking and Analysis of cDNA). CRAC revealed that NANOS2 strongly and specifically interacts with RNA, despite its low abundance in cells. It also showed that NANOS2 mainly binds mRNAs within their 3' UTRs, which are enriched in the novel ‘AUNAANU’ motif. These mRNAs are involved in cellular metabolism, and they also include positive regulators of the mTORC pathway, which is known to control SSCs self-renewal. Additionally, we analysed the expression profile of these mRNAs throughout adult spermatogonia development. This revealed that one-third of NANOS2 targets are upregulated during the transition from self-renewing SSCs to transitamplifying spermatogonia. To further explore NANOS2 function, we performed immunoprecipitation followed by mass spectrometry and identified components of the CCR4-NOT complex amongst the strongest interactors. Since the CCR4-NOT promotes RNA decay, I investigated the mRNA stability of NANOS2 targets by using SLAM-seq, a novel technique that enables the determination of mRNA half-life transcriptome-wide. This analysis suggested that NANOS2 mRNA targets have a shorter half-life, compared to the average mRNA stability in SSCs. In summary, I demonstrated that NANOS2 shortens the half-life of hundreds of mRNAs involved in metabolism, through the recruitment of the CCR4-NOT RNA deadenylation complex, in SSC lines. Therefore, we speculate that NANOS2 promotes SSCs self-renewal by repressing active metabolism and cellular growth. This would preserve cellular quiescence and ensures the presence of a long-term SSCs pool in adult mice. Overall, these findings provide new insight into NANOS2 mechanism of action in mouse SSC