NANOS2 interacts with the CCR4-NOT deadenylation complex and leads to suppression of specific RNAs

Abstract

Nanos is one of the evolutionarily conserved proteins implicated in germ cell development. We have previously shown that NANOS2 plays an important role in both the maintenance and sexual development of germ cells. However, the molecular mechanisms underlying these events have remained elusive. In our present study, we found that NANOS2 localizes to the P-bodies, known centers of RNA degradation that are abundantly accumulated in male gonocytes. We further identified by immunoprecipitation that the components of the CCR4-NOT deadenylation complex are NANOS2-interacting proteins and found that NANOS2 promotes the localization of CNOT proteins to P-bodies in vivo. We also elucidated that the NANOS2/CCR4-NOT complex has deadenylase activity in vitro, and that some of the RNAs implicated in meiosis interact with NANOS2 and are accumulated in its absence. Our current data thus indicate that the expression of these RNA molecules is normally suppressed via a NANOS2-mediated mechanism. We propose from our current findings that NANOS2-interacting RNAs may be recruited to P-bodies and degraded by the enzymes contained therein through NANOS2-mediated deadenylation.

Fig. 1.

NANOS2 localizes to the P-bodies in male mouse gonocytes. (A–L) Sections prepared from wild-type E15.5 male gonads were double-stained with mouse anti-NANOS2 (green) (A and D) and either hDCP2 (B) or mXRN1 (E) antibodies (red staining in each case). Arrowheads indicate colocalization of NANOS2 and hDCP2 (C) or XRN1 (F). DNA was counterstained with DAPI (blue). (Scale bar in A, 20 μm for all panels.)

Fig. 2.

Functional role of NANOS2 during the formation of the P-bodies. (A–E) Male gonadal sections from Nanos2 ^+/− (A and C), Nanos2 ^−/− (B and D), and Nanos2 ^−/− Bax ^−/− (E) embryos at stages E13.5 (A and B), and E16.5 (C, D, and E) were immunostained with p54/RCK (green) and TRA98 (red) antibodies. (F) Average number of p54/RCK foci per male gonocyte at E16.5 was quantified in each picture using ImageJ software (National Institutes of Health) and a cell counter, with the foci of less than a 20 permission value excluded using Photoshop (Adobe). The data shown correspond to two to three pictures. (G–I) A female gonadal section from a NANOS2-expressing embryo at E16.5 was immunostained with anti-FLAG (green) (G) and anti-p54/RCK (red) (H) antibodies. DNA was counterstained using DAPI (blue). (Scale bar in A, 20μm for A–E and G–I.)

Fig. 3.

Interaction between NANOS2 and the CCR4-NOT deadenylation complex. (A) Proteins coimmunoprecipitated with NANOS2 from E14.5 wild-type (lane 1) and Nanos2 ^−/− (lane 2) male gonadal extracts using rabbit anti-NANOS2 antibodies. Arrowheads indicate CNOT1. *1, nonspecific band; *2, IgG polypeptide. (B) Immunoprecipitation–Western blot analyses of proteins from male gonadal extracts of wild-type and transgenic embryos expressing 3×FLAG-NANOS2. *3, IgG polypeptide from the anti-FLAG antibody. (C–E) Male gonadal sections from E15.5 embryos were immunostained with mouse NANOS2 (green) (C) and CNOT3 (red) (D) antibodies. Arrowheads in C–E indicate colocalization between NANOS2 and CNOT3. (F–K) Male gonadal sections from Nanos2 ^+/− (F–H) and Nanos2 ^−/− (I–K) embryos at E15.5 were immunostained with DCP1A (red) (G and J) and CNOT3 (green) (F and I) antibodies. DNA was labeled via DAPI counterstaining (blue). (L) Western blot analyses of proteins from the male gonads of Nanos2 ^+/− and Nanos2 ^−/− embryos at E15.5.

Fig. 4.

The protein complex of NANOS2 and CCR4-NOT complex has in vitro deadenylase activity. (A) Schematic representation of the in vitro deadenylase assay method using NANOS2 over-expressing (O/E) testes. (B) FLAG-tagged NANOS2 was precipitated with anti-FLAG antibodies from the testis extracts of a 6-week-old NANOS2 O/E mouse and incubated with 5′-fluorescein isothiocyanate-labeled poly(A) RNA substrate for 0, 45, 90, and 180 min. Samples were then analyzed on a denaturing sequencing gel, as previously described (21) (G). (C) Western blot analyses reveaing that CNOT6L and CNOT7 are coprecipitated with FLAG-tagged NANOS2.

Fig. 5.

NANOS2 interacts with specific mRNAs and may promote their degradation. (A) Male gonadal extracts from wild-type (wt) and transgenic (tg) mice expressing FLAG-NANOS2 at E15.5 were subjected to immunoprecipitation (IP) with FLAG antibodies. RNA precipitates were analyzed by semi-quantitative RT-PCR. (B) Quantification of each mRNA enrichment from a FLAG IP of tg extracts using real-time RT-PCR. Fold enrichment of each mRNA coprecipitated from tg compared with those from wt is indicated. Mean value of three independent QRT-PCR results is shown. (C–G) Expression profiling of the Sycp3 (C), Stra8 (D), Taf7l (E), Dazl (F), and Meisetz (G) genes in male gonads from Nanos2 ^+/− and Nanos2 ^−/− embryos at E13.5-E15.5 using the Affymetrix GeneChip System as previously described (43) (X-axis; embryonic stage, Y-axis; expression level, black bars; Nanos2 ^+/− embryos, white bars; Nanos2 ^−/− embryos).