P-body dynamics revealed by DDX6 protein knockdown via the auxin-inducible degron system.

The transcriptome dynamically changes via several transcriptional and post-transcriptional mechanisms. RNA-binding proteins contribute to such mechanisms to regulate the cellular status. DDX6 is one such protein and a core component of processing bodies (P-bodies), membrane-less cytosolic substructures where RNA and proteins localize and are functionally regulated. Despite the importance of DDX6, owing to the lack of tightly controlled methods for protein knockdown, it was difficult to assess in high time resolution how its depletion exactly affects the P-body assembly structure. Therefore, we adopted an advanced protein degradation method, the auxin-induced degron (AID) system, to degrade DDX6 acutely in ES cells. By introducing AID-tagged DDX6 and the E3 ligase subunit of OsTIR1 into ES cells, we successfully degraded DDX6 following auxin analog (indole-3-acetic acid, IAA) treatment. The degradation rate of DDX6 was slower than that of the cytosolic reporter protein EGFP but was enhanced by increasing the OsTIR1 dosage. Lastly, we confirmed that a substantial portion of P-bodies disappears around the time of 1 hr after IAA addition consistent with DDX6 depletion detected by western blot. In accordance with this, we detected transcriptome changes by 6 hr after IAA treatment. Therefore, we demonstrated the applicability of the AID method to gain insight into the function of P-bodies and their protein components.

A cooperative mechanism of target RNA selection via germ-cell-specific RNA-binding proteins NANOS2 and DND1.

The germ-cell-specific RNA-binding protein (RBP) NANOS2 plays a pivotal role in male gonocyte differentiation and spermatogonial stem cell maintenance. Although NANOS2 interacts with the CNOT deadenylation complex and Dead end 1 (DND1) to repress target RNAs, the molecular mechanisms underlying target mRNA selection remain unclear because of the limited cell resource in vivo. Here, we demonstrate that exogenous NANOS2-DND1 suppresses target mRNAs in somatic cells. Using this somatic cell system, we find that NANOS2 interacts with RNA-bound DND1 and recruits the CNOT complex to the mRNAs. However, a fusion construct composed of the CNOT1-binding site of NANOS2 (NIM) and DND1 fails to repress the target gene expression. Therefore, NANOS2 is required not only for recruitment of the CNOT complex but also for selecting the target mRNA with DND1. This study reveals that NANOS2 functions as a second-layer RBP for the target recognition and functional adaptation of DND1.

Genetic and structural analysis of the in vivo functional redundancy between murine NANOS2 and NANOS3.

NANOS2 and NANOS3 are evolutionarily conserved RNA-binding proteins involved in murine germ cell development. NANOS3 is required for protection from apoptosis during migration and gonadal colonization in both sexes, whereas NANOS2 is male-specific and required for the male-type differentiation of germ cells. Ectopic NANOS2 rescues the functions of NANOS3, but NANOS3 cannot rescue NANOS2 function, even though its expression is upregulated in Nanos2-null conditions. It is unknown why NANOS3 cannot rescue NANOS2 function and it is unclear whether NANOS3 plays any role in male germ cell differentiation. To address these questions, we made conditional Nanos3/Nanos2 knockout mice and chimeric mice expressing chimeric NANOS proteins. Conditional double knockout of Nanos2 and Nanos3 led to the rapid loss of germ cells, and in vivo and in vitro experiments revealed that DND1 and NANOS2 binding is dependent on the specific NANOS2 zinc-finger structure. Moreover, murine NANOS3 failed to bind CNOT1, an interactor of NANOS2 at its N-terminal. Collectively, our study suggests that the inability of NANOS3 to rescue NANOS2 function is due to poor DND1 recruitment and CNOT1 binding.

Oocyte-triggered dimerization of sperm IZUMO1 promotes sperm-egg fusion in mice.

Sperm-egg fusion is indispensable for completing mammalian fertilization. Although the underlying molecular mechanisms are poorly understood, requirement of two spermatozoon factors, IZUMO1 and SPACA6, and two oocyte factors, CD9 and the IZUMO1 counter-receptor JUNO, has been proven by gene disruption, and the binding of cells to an oocyte can be reconstituted by ectopic expression of IZUMO1. Here we demonstrate that robust IZUMO1-dependent adhesion of sperm with an oocyte accompanies the dimerization of IZUMO1. Despite the intrinsic dimeric property of its N-terminal region, IZUMO1 is monomeric in spermatozoa. Interestingly, JUNO associates with monomeric IZUMO1, which is then quickly removed as tight adhesion of the two cells is subsequently established. We therefore propose that global structural rearrangement of IZUMO1 occurs on JUNO recognition and that this rearrangement may then initiate force generation to overcome repulsion between the juxtaposing membranes, through an unidentified receptor on the egg.