Interaction between NANOS2 and the CCR4-NOT Deadenylation Complex Is Essential for Male Germ Cell Development in Mouse

Atsushi Suzuki,Rie Saba,Yoshinori Morita,Kei Miyoshi,Yumiko Saga,Austin John Cooney

doi:10.1371/journal.pone.0033558

Atsushi Suzuki, Rie Saba + Show 4 more

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https://doi.org/10.1371/journal.pone.0033558

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Abstract

Nanos is one of the evolutionarily conserved proteins implicated in germ cell development and we have previously shown that it interacts with the CCR4-NOT deadenylation complex leading to the suppression of specific RNAs. However, the molecular mechanism and physiological significance of this interaction have remained elusive. In our present study, we identify CNOT1, a component of the CCR4-NOT deadenylation complex, as a direct factor mediating the interaction with NANOS2. We find that the first 10 amino acids (AAs) of NANOS2 are required for this binding. We further observe that a NANOS2 mutant lacking these first 10 AAs (NANOS2-ΔN10) fails to rescue defects in the Nanos2-null mouse. Our current data thus indicate that the interaction with the CCR4-NOT deadenylation complex is essential for NANOS2 function. In addition, we further demonstrate that NANOS2-ΔN10 can associate with specific mRNAs as well as wild-type NANOS2, suggesting the existence of other NANOS2-associated factor(s) that determine the specificity of RNA-binding independently of the CCR4-NOT deadenylation complex.

Highlights

The sexual development of mammalian germ cells leading to the generation of eggs and sperm is a critically important biological process
We showed that NANOS2 associates with the CCR4-NOT deadenylation complex in male gonocytes, and that this interaction is responsible for the deadenylation activity of NANOS2 [8]
We further found an upregulated meiotic marker, SCP3 (Figure 2J, K, L) [17], and downregulated male-specific marker, DNMT3L (Figure 2M, N, O) [18]. These phenotypes were almost identical to those observed in Nanos2-null mice [6] and we concluded that the first 10 residues of NANOS2 are essential for almost all of its functions. These results suggest that the interaction of NANOS2 with the CCR4-NOT deadenylation complex is essential for its developmental functions, we cannot exclude the possibility that the association of other factors with the 10 N-terminal amino acids (AAs) of NANOS2 is critical

Summary

Introduction

The sexual development of mammalian germ cells leading to the generation of eggs and sperm is a critically important biological process. The PGCs are potent precursors for both oogonia and spermatogonia, sexual differentiation is induced after the colonization of the embryonic gonads with somatic cells. RA molecules derived from the mesonephros induce the meiotic initiation of germ cells in female embryonic gonads via the induction of the RA responsive gene Stra, which is required for premeiotic replication [4]. At least two somatic factors are required for masculinization of germ cells in male embryonic gonads. Somatically derived fibroblast growth factor 9 (FGF9) promotes the expression of maletype genes including Nanos via its receptors on the surfaces of germ cells [5]. Nanos expression commences by E13.5 after the downregulation of Cyp26b1 and is required for the maintenance of the male germ cell state [6]

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