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Interaction of NANOS2 and NANOS3 with different components of the CNOT complex may contribute to the functional differences in mouse male germ cells.

Suzuki A, Niimi Y, Saga Y - Biol Open (2014)

Bottom Line: Transcription of Nanos2 and Nanos3 was strongly compromised in the presence of this transgene, which resulted in the mimicking of the Nanos2/Nanos3 double- condition in the male gonad.In addition, similar to NANOS2, we found that NANOS3 associated with the CCR4-NOT deadenylation complex but via a direct interaction with CNOT8, unlike CNOT1 in the case of NANOS2.This alternate interaction might account for the molecular basis of the functional redundancy and differences in NANOS2 and NANOS3 functions.

View Article: PubMed Central - PubMed

Affiliation: Division of Materials Science and Chemical Engineering, Faculty of Engineering, Yokohama National University, Yokohama, Kanagawa, Japan Department of Materials Science and Chemical Engineering, Graduate School of Engineering, Yokohama National University, Yokohama, Kanagawa, Japan atsuzuki@ynu.ac.jp ysaga@lab.nig.ac.jp.

No MeSH data available.


Related in: MedlinePlus

Generation of transgenic mice expressing NANOS2-ZM under the control of the Nanos2 enhancer.(A) Flag-tagged NANOS2 or NANOS2-ZM were precipitated with anti-FLAG antibodies from HeLa cell extracts. Precipitates were analyzed by western blotting with the indicated antibodies. (B) Immunoprecipitated Flag-tagged NANOS2 or NANOS2-ZM were incubated with 5′-fluorescein isothiocyanate-labeled poly(A) RNA substrate for 0, 45, 90, and 180 minutes. Samples were then analyzed on a denaturing sequencing gel. (C) Schematic representation of the transgene encoding a NANOS2 variant harboring mutations in zinc finger motifs under the control of the Nanos2 enhancer. (D) Western blot analysis of NANOS2 protein in E14.5 male gonads from the wild-type, Flag-tagged Nanos2-ZM transgenic mouse lines #4 and #5, and Flag-tagged wild-type Nanos2 transgenic mouse line (full). Tubulin was used as a loading control. (E–G) Gross comparison (E) and hematoxylin-eosin-stained sections of testes from 6-week-old wild-type (F) and transgenic (G) mice. Scale bar, 100 µm in F for F–G.
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f01: Generation of transgenic mice expressing NANOS2-ZM under the control of the Nanos2 enhancer.(A) Flag-tagged NANOS2 or NANOS2-ZM were precipitated with anti-FLAG antibodies from HeLa cell extracts. Precipitates were analyzed by western blotting with the indicated antibodies. (B) Immunoprecipitated Flag-tagged NANOS2 or NANOS2-ZM were incubated with 5′-fluorescein isothiocyanate-labeled poly(A) RNA substrate for 0, 45, 90, and 180 minutes. Samples were then analyzed on a denaturing sequencing gel. (C) Schematic representation of the transgene encoding a NANOS2 variant harboring mutations in zinc finger motifs under the control of the Nanos2 enhancer. (D) Western blot analysis of NANOS2 protein in E14.5 male gonads from the wild-type, Flag-tagged Nanos2-ZM transgenic mouse lines #4 and #5, and Flag-tagged wild-type Nanos2 transgenic mouse line (full). Tubulin was used as a loading control. (E–G) Gross comparison (E) and hematoxylin-eosin-stained sections of testes from 6-week-old wild-type (F) and transgenic (G) mice. Scale bar, 100 µm in F for F–G.

Mentions: We have previously shown that the N-terminal region of NANOS2 is required to interact with CNOT1, and this interaction is essential for NANOS2 functions in vivo (Suzuki et al., 2012). In this study, we focused on the evolutionarily conserved zinc finger domain consisting of two consecutive CCHC-type zinc finger motifs, because this domain is indispensable for in vivo functions in Drosophila (Lehmann and Nüsslein-Volhard, 1991). Accordingly, we generated a NANOS2 variant harboring mutations in this domain by substituting the first cysteine residues in the two CCHC motifs of NANOS2 (C61 and C96) with alanine to disrupt the structures. We refer to this NANOS2 variant as NANOS2-ZM hereafter. First, we examined whether the mutations had any effect on the interaction with the CNOT complex using HeLa cells transfected with Flag-tagged Nanos2-ZM. Immunoprecipitation analyses revealed that NANOS2-ZM precipitated endogenous CNOT proteins including CNOT1, which was similar to NANOS2 (Fig. 1A). In addition, and consistent with this finding, the level of deadenylase activity in NANOS2-ZM precipitates was the same as that in wild-type NANOS2 precipitates (Fig. 1B). These data indicate that the NANOS2 zinc finger domain is neither involved in the interaction with CNOT proteins nor the deadenylase activity of the CNOT complex.


Interaction of NANOS2 and NANOS3 with different components of the CNOT complex may contribute to the functional differences in mouse male germ cells.

Suzuki A, Niimi Y, Saga Y - Biol Open (2014)

Generation of transgenic mice expressing NANOS2-ZM under the control of the Nanos2 enhancer.(A) Flag-tagged NANOS2 or NANOS2-ZM were precipitated with anti-FLAG antibodies from HeLa cell extracts. Precipitates were analyzed by western blotting with the indicated antibodies. (B) Immunoprecipitated Flag-tagged NANOS2 or NANOS2-ZM were incubated with 5′-fluorescein isothiocyanate-labeled poly(A) RNA substrate for 0, 45, 90, and 180 minutes. Samples were then analyzed on a denaturing sequencing gel. (C) Schematic representation of the transgene encoding a NANOS2 variant harboring mutations in zinc finger motifs under the control of the Nanos2 enhancer. (D) Western blot analysis of NANOS2 protein in E14.5 male gonads from the wild-type, Flag-tagged Nanos2-ZM transgenic mouse lines #4 and #5, and Flag-tagged wild-type Nanos2 transgenic mouse line (full). Tubulin was used as a loading control. (E–G) Gross comparison (E) and hematoxylin-eosin-stained sections of testes from 6-week-old wild-type (F) and transgenic (G) mice. Scale bar, 100 µm in F for F–G.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4265758&req=5

f01: Generation of transgenic mice expressing NANOS2-ZM under the control of the Nanos2 enhancer.(A) Flag-tagged NANOS2 or NANOS2-ZM were precipitated with anti-FLAG antibodies from HeLa cell extracts. Precipitates were analyzed by western blotting with the indicated antibodies. (B) Immunoprecipitated Flag-tagged NANOS2 or NANOS2-ZM were incubated with 5′-fluorescein isothiocyanate-labeled poly(A) RNA substrate for 0, 45, 90, and 180 minutes. Samples were then analyzed on a denaturing sequencing gel. (C) Schematic representation of the transgene encoding a NANOS2 variant harboring mutations in zinc finger motifs under the control of the Nanos2 enhancer. (D) Western blot analysis of NANOS2 protein in E14.5 male gonads from the wild-type, Flag-tagged Nanos2-ZM transgenic mouse lines #4 and #5, and Flag-tagged wild-type Nanos2 transgenic mouse line (full). Tubulin was used as a loading control. (E–G) Gross comparison (E) and hematoxylin-eosin-stained sections of testes from 6-week-old wild-type (F) and transgenic (G) mice. Scale bar, 100 µm in F for F–G.
Mentions: We have previously shown that the N-terminal region of NANOS2 is required to interact with CNOT1, and this interaction is essential for NANOS2 functions in vivo (Suzuki et al., 2012). In this study, we focused on the evolutionarily conserved zinc finger domain consisting of two consecutive CCHC-type zinc finger motifs, because this domain is indispensable for in vivo functions in Drosophila (Lehmann and Nüsslein-Volhard, 1991). Accordingly, we generated a NANOS2 variant harboring mutations in this domain by substituting the first cysteine residues in the two CCHC motifs of NANOS2 (C61 and C96) with alanine to disrupt the structures. We refer to this NANOS2 variant as NANOS2-ZM hereafter. First, we examined whether the mutations had any effect on the interaction with the CNOT complex using HeLa cells transfected with Flag-tagged Nanos2-ZM. Immunoprecipitation analyses revealed that NANOS2-ZM precipitated endogenous CNOT proteins including CNOT1, which was similar to NANOS2 (Fig. 1A). In addition, and consistent with this finding, the level of deadenylase activity in NANOS2-ZM precipitates was the same as that in wild-type NANOS2 precipitates (Fig. 1B). These data indicate that the NANOS2 zinc finger domain is neither involved in the interaction with CNOT proteins nor the deadenylase activity of the CNOT complex.

Bottom Line: Transcription of Nanos2 and Nanos3 was strongly compromised in the presence of this transgene, which resulted in the mimicking of the Nanos2/Nanos3 double- condition in the male gonad.In addition, similar to NANOS2, we found that NANOS3 associated with the CCR4-NOT deadenylation complex but via a direct interaction with CNOT8, unlike CNOT1 in the case of NANOS2.This alternate interaction might account for the molecular basis of the functional redundancy and differences in NANOS2 and NANOS3 functions.

View Article: PubMed Central - PubMed

Affiliation: Division of Materials Science and Chemical Engineering, Faculty of Engineering, Yokohama National University, Yokohama, Kanagawa, Japan Department of Materials Science and Chemical Engineering, Graduate School of Engineering, Yokohama National University, Yokohama, Kanagawa, Japan atsuzuki@ynu.ac.jp ysaga@lab.nig.ac.jp.

No MeSH data available.


Related in: MedlinePlus