Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development

Abstract

RNA-binding proteins (RBPs) play important roles for generating various cell types in many developmental processes, including eggs and sperms. Nanos is widely known as an evolutionarily conserved RNA-binding protein implicated in germ cell development. Mouse NANOS2 interacts directly with the CCR4-NOT (CNOT) deadenylase complex, resulting in the suppression of specific RNAs. However, the mechanisms involved in target specificity remain elusive. We show that another RBP, Dead end1 (DND1), directly interacts with NANOS2 to load unique RNAs into the CNOT complex. This interaction is mediated by the zinc finger domain of NANOS2, which is essential for its association with target RNAs. In addition, the conditional deletion of DND1 causes the disruption of male germ cell differentiation similar to that observed in Nanos2-KO mice. Thus, DND1 is an essential partner for NANOS2 that leads to the degradation of specific RNAs. We also present the first evidence that the zinc finger domain of Nanos acts as a protein-interacting domain for another RBP, providing a novel insight into Nanos-mediated germ cell development.

Keywords: Dead end; Nanos; RNA; germ cell.

Figure EV1. Identification of NANOS2‐associated proteins

1. Silver staining of proteins co‐precipitated with anti‐FLAG antibody from testis extracts of either wild‐type or transgenic embryos expressing FLAG‐tagged NANOS2 at E15.5. Both wild‐type and transgenic proteins in the gel at the location of numbers (No. 1 to 7) were analyzed by mass spectrometry. Note that the gels at No. 3, 6 and 7 were excised and analyzed despite no apparent band in the picture, because slight signals of several bands could be visualized with a backlight source. *Heavy chain of anti‐FLAG antibody.

2. Proteins identified exclusively in the transgenic proteins of the gels indicated in (A).

3. Scores of DND1 peptides identified by LC–MS/MS and MASCOT.

4. Amino acid sequence of DND1. Residues shown in red correspond to peptide sequence obtained by mass spectrometry, and the underlined residues are the RNA recognition motif.

5. Results of GST pull‐down assay using E. coli extracts expressing GST, GST‐fused NANOS2, or NANOS3 mixed with MBP‐tagged LacZα or DND1. Precipitates were analyzed by CBB staining. An arrowhead indicates MBP‐DND1 co‐precipitated with GST‐tagged NANOS2 or NANOS3. *Non‐specific band.

6. Amino acid sequence alignment of NANOS2 and NANOS3. Grey circles indicate conserved amino acids in NIM, while red and blue circles indicate conserved CCHC residues in the former and latter zinc finger motif. Amino acids enclosed with a square in N‐ and C‐terminus are highly similar sequences deleted in Fig 1D.

Figure EV2. Expression profile of DND1

1. A–L

   Wild‐type whole embryos at E7.5 (A–C), E8.5 (G–I) and E9.5 (J–L), and a Ter/Ter embryo at E7.5 (D–F) were stained with antibodies against DND1 (A, D, G, K), NANOS3 (B, E, H), or OCT3/4 (J). Note that the immunostaining signal of DND1 was not detected in a Ter/Ter embryo, indicating the specificity of the antibody against DND1. Insets in (A–F) and (J–L) show an enlarged version of each picture to better depict PGCs.

2. M–R

   Sections of ovary (M–O) and testes (P–R) from embryos at E14.5 were stained with antibodies against DND1 (green) (M, P) and TRA98 (red) (N, Q).

3. S

   Western blotting analyses of proteins in 1/3 of a male gonad and female gonad per lane at E12.5, E13.5, E14.5, and E15.5 using the antibodies indicated. Note that the amount of loading control protein TUBULIN in male gonads is larger than that in female gonads because of the larger size of male gonads compared to female gonads.

4. T–Y

   NIH3T3 cells were transfected with HA‐tagged Nanos2 (T–V) or Dnd1 (W–Y) and then stained with antibodies against NANOS2 (green) (T) or DND1 (green) (W) and DCP1a (red) (U, X). Note that both HA‐tagged NANOS2 and DND1 do not show clear localization to P‐bodies.

Data information: DNA was labeled with DAPI (C, F, I, L, N, O, Q, R, U, V, X, Y). Scale bars: 100 μm in (A, G, J) for (A–F), (G–I), and (J–L), respectively; 50 μm in (M) for (M–R); 10 μm in (T, W) for (T–V) and (W–Y), respectively.

Figure 1. DND1 interacts directly with NANOS2 via its zinc finger motif

1. A, B

   Western blotting analysis of proteins co‐precipitated with anti‐FLAG antibody from testis extracts of E15.5 wild‐type embryos and transgenic embryos expressing FLAG‐tagged NANOS2 with or without RNase (A) or from extracts of HeLa cells transfected with FLAG‐tagged DND1 and with or without HA‐tagged NANOS2 (B). Precipitates were analyzed with the indicated antibodies.

2. C

   GST pull‐down assay using E. coli extracts expressing GST, GST‐fused NANOS2, or NANOS3 mixed with MBP‐tagged LacZα or DND1. Precipitates were analyzed by Coomassie Brilliant Blue (CBB) staining or Western blotting with an anti‐MBP antibody. An arrowhead indicates MBP‐DND1 co‐precipitated with GST‐tagged NANOS2 or NANOS3. See also Fig EV1E.

3. D, E

   Western blotting analyses of proteins co‐precipitated with anti‐FLAG antibody from extracts of HeLa cells transfected with FLAG‐tagged NANOS2, NANOS2 (ΔN10), NANOS2 (ΔC10), NANOS2 (C61A, C96A) or NANOS3, and HA‐tagged DND1 (D) or with FLAG‐tagged NANOS2, NANOS2 (C61A), NANOS2 (C96A) or NANOS2 (C61A, C96A), and HA‐tagged DND1 (E). Precipitates were analyzed with the indicated antibodies.

4. F

   GST pull‐down assay using E. coli extracts expressing GST‐fused DND1 mixed with MBP‐tagged LacZα, NANOS2, NANOS2 (C61A, C96A) or NANOS2 (60–114). Arrowheads indicate MBP‐tagged NANOS2 (lane 2) or NANOS2 (60–114) (lane 4).

Figure 2. Relationship between NANOS and PUMILIO

1. A, B

   Western blotting analyses of proteins co‐precipitated with anti‐FLAG antibody from extracts of HeLa cells transfected with HA‐tagged NANOS1, NANOS2, or NANOS3, and FLAG‐tagged DND1 (A) or with FLAG‐tagged PUMILIO1 or PUMILIO2, and HA‐tagged NANOS1, NANOS2, or NANOS3 with or without MYC‐tagged DND1 (B).

2. C

   Characterization of antibodies against PUMILIO1 and PUMILIO2. Western blotting analyses of Flag‐tagged PUMILIO1 and PUMILIO2 in HeLa cells transfected with FLAG‐tagged PUMILIO1 or PUMILIO2 using antibodies against FLAG, PUMILIO1 or PUMILIO2. Note that the antibodies against PUMILIO1 specifically recognized FLAG‐tagged PUMILIO1, while the anti‐PUMILIO2 antibody could detect both Flag‐tagged PUMILIO1 and PUMILIO2.

3. D

   FLAG‐tagged NANOS2 was immunoprecipitated with an anti‐FLAG antibody from E15.5 testicular extracts from transgenic mice expressing FLAG‐tagged NANOS2. Precipitates were analyzed by Western blotting with the indicated antibodies. Note that co‐precipitation of both PUMILIO1 and PUMILIO2 were not detected even though both CNOT1 and DND1 were clearly co‐precipitated.

Figure 3. DND1 co‐localizes with NANOS2 in P‐bodies

1. A–C

   Sections of male gonads from E15.5 embryos were immunostained with antibodies against DND1 (green) and DCP1a (red).

2. D–F

   Squash preparation of a male gonocyte from E16.5 embryo immunostained with antibodies against DND1 (red) and DCP1a (green). Arrowheads indicate co‐localization of DND1 and DCP1a.

3. G–I

   Sections of male gonads from E15.5 embryos were immunostained with antibodies against DND1 (red) and NANOS2 (green). Arrowheads indicate co‐localization of DND1 and NANOS2.

4. J–M

   NIH3T3 cells transfected with HA‐tagged Dnd1 and FLAG‐tagged Nanos2 were then immunostained with antibodies against DND1 (J) (magenta), NANOS2 (K) (red), and DCP1a (L) (green).

5. N–γ

   Biomolecular fluorescence complementation assay. NIH3T3 cells transfected with VENUS‐C‐fused Dnd1 and VENUS‐N‐fused Nanos2 (N–U) or Nanos2 (C61A, C96A) (V–γ) were immunostained with antibodies against DND1 (N, S, V), NANOS2 (O, W, α) or DDX6 (R, Z). Then, the signals of VENUS fusion protein were visualized (P, T, X, β). Arrowheads in (Z) indicate P‐bodies.

Data information: DNA was labeled with DAPI (blue). Scale bars: 50 μm in (A) for (A–C); 10 μm in (D, G, J, N) for (D–F), (G–I), (J–M) and (N–γ), respectively. Insets in (A–C) and (J–γ) show an enlarged version of each picture to better depict localization of each protein. See also Fig EV2T–Y.

Figure EV3. Strategy for conditional knockout of Dnd1

1. A

   Schematic representation of homologous recombination for the generation of a Dnd1‐flox allele.

2. B

   Genomic DNAs from a G418‐resistant and wild‐type TT2 embryonic stem (ES) cell clones were digested with EcoRI and analyzed by southern blotting using the probe indicated in (A).

3. C

   Agarose gel electrophoresis of polymerase chain reaction (PCR) products in the genotyping of Dnd1‐flox allele.

4. D

   Schematic representation of the mating strategy for conditional knockout of Dnd1.

5. E–J

   Sections of E16.5 testes from Dnd1 ^flox/flox (control) and Dnd1 ^flox/flox embryos with the Oct4ΔPE‐CreER^{T 2} transgene (Dnd1‐cKO), both of which were administered with tamoxifen at E13.5, were immunostained with the antibodies against DND1 (green) (E, H) and TRA98 (red) (F, I). DNA was labeled with DAPI (blue) (F, G, I, J). Scale bar: 50 μm in (E) for (E–J).

Figure 4. Conditional deletion of DND1 causes similar but not identical phenotypes to those of Nanos2‐KO male gonocytes

1. A

   Western blotting analyses of proteins in testes from E13.5 to E16.5 embryos of Dnd1 ^flox/flox or Dnd1 ^flox/flox _Tg(Oct4ΔPE‐Cre ER^{T 2} ) each administered with tamoxifen at E13.5 and E15.5 embryos of Nanos2 ^+/− or Nanos2 ^−/− with the indicated antibodies.

2. B–P

   Sections of testes from Dnd1 ^flox/flox (B, E, H, K, N), Dnd1 ^flox/flox _Tg(Oct4ΔPE‐Cre ER^{T 2} ) (C, F, I, L, O) or Nanos2 ^−/− (D, G, J, M, P) embryos were prepared at E16.5 and then immunostained with antibodies against pH3 (B–D), STRA8 (E–G), SYCP3 (H–J), activated caspase‐3 (K–M) or LAMININ (N–P) (green). Germ cells were immunostained with TRA98 (B–D) or DAZL (N–P) (red), and DNA was labeled with DAPI (blue). Tamoxifen was administered at E13.5. Arrowheads indicate apoptotic cells (L, M) or cells outside the tubules (O, P). Scale bars: 50 μm in (B) for (B–D) and (K–P), 50 μm in (E) for (E–J). See also Fig EV3.

Figure 5. DND1 is required for loading of the target RNAs to NANOS2–CNOT complex

1. A–R

   Sections of testes from Dnd1 ^flox/flox (A–C, G–I, M–O) and Dnd1 ^flox/flox _Tg(Oct4ΔPE‐CreERT2) (D–F, J–L, P–R) embryos were prepared at E16.5 and then immunostained with antibodies against NANOS2 (A, D) (green), DCP1a (G, J) (green), DDX6 (M, P) (green) and TRA98 (B, E, H, K, N, Q). DNA was labeled with DAPI (blue). Tamoxifen was administered at E13.5. Scale bars: 50 μm in (A) for (A–F); 50 μm in (G) for (G–L); 50 μm in (M) for (M–R). Insets show an enlarged version of each picture to better depict localization of NANOS2, DCP1a and DDX6.

2. S–V

   Immunoprecipitation with an anti‐FLAG antibody from E15.5 male gonadal extracts of wild‐type and the transgenic mouse line expressing FLAG‐tagged DND1 (S, T), or with an anti‐NANOS2 antibody from E15.5 male gonadal extracts of Dnd1 ^flox/flox mice with or without Oct4ΔPE‐CreERT2 (U, V). Precipitates were analyzed by Western blotting (S, U) or by RT–qPCR (T, V). The RT–qPCR data in (T) and (V) are shown as average relative mRNA levels ± SE (n = 3).

3. W, X

   Western blotting analyses of proteins in NANOS2‐depleted (W) or DND1‐depleted (X) testis extracts from E15.5 embryos. Proteins were analyzed with the indicated antibodies.

Figure EV4. Characterization of the transgenic mouse line expressing FLAG‐tagged DND1

1. A

   Schematic representation of the bacterial artificial chromosome (BAC) transgene. The DNA sequence encoding 3×FLAG‐tag is inserted at the C‐terminus of Dnd1, resulting in the expression of DND1‐3×FLAG fusion protein under the direct control of the Dnd1 enhancer.

2. B

   Western blotting analyses of proteins in E15.5 testes from wild‐type and two independent transgenic embryos (line #1 and #2) with the antibodies indicated. Line #1 was used for further analysis because it produced larger amounts of DND1‐3×FLAG.

3. C

   Western blotting analyses of proteins in E15.5 testes from wild‐type, Ter‐homozygous, and from Ter‐homozygous embryos expressing the FLAG‐tagged Dnd1 transgene with the antibodies indicated.

4. D–F

   Sections of testes from E16.5 Ter‐heterozygous, Ter‐homozygous, and from Ter‐homozygous embryos with the FLAG‐tagged Dnd1 transgene were immunostained with anti‐MVH antibodies. DNA was labeled with DAPI (blue). Scale bar: 50 μm in (D) for (D–F).

5. G–L

   Sections of adult testes (G–I) or ovaries (J–L) from Ter‐heterozygous, Ter‐homozygous, and from Ter‐homozygous mice with the FLAG‐tagged Dnd1 transgene were stained with hematoxylin and eosin.