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RMND5 from Xenopus laevis is an E3 ubiquitin-ligase and functions in early embryonic forebrain development.

Pfirrmann T, Villavicencio-Lorini P, Subudhi AK, Menssen R, Wolf DH, Hollemann T - PLoS ONE (2015)

Bottom Line: In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function.Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex.We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development.

View Article: PubMed Central - PubMed

Affiliation: Martin-Luther University Halle-Wittenberg, Institute of Physiological Chemistry, Halle, Germany.

ABSTRACT
In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development.

No MeSH data available.


Related in: MedlinePlus

rmnd5 is expressed during early embryonic development.(A) Temporal RT-PCR analysis of rmnd5 expression (top panel); different developmental stages (NF-stages) indicated at the top. ODC1 functions as RNA input control (bottom). (B) Rmnd5 protein at different developmental stages. Western blot analysis of embryo lysate from indicated stages (top). α-RMND5A (Novus Biological; rabbit, 1:1000); α-Tubulin (AbD Serotec, rat, 1:2500). (C) Spatial analysis of rmnd5 expression. Whole mount in situ hybridisation (Wmish) of wild type Xenopus laevis embryos at different developmental stages. NF stage 3 (panel a, left) and stage 4 (panel c) rmnd5 transcript in the animal pole (top), NF-stage 12 (panel d) rmnd5 transcripts around the prospective head, NF-stage 18; 24 (panel e, f, g, h) neuronal ectoderm (red arrow, panel e) and ciliated cells of the skin (yellow arrow, panel e, g, h), NF-stage 34 (panel j, k, l, m) proencephalon (red arrow) and eyes (green arrow). Negative controls with sense probes (panel b, i).
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pone.0120342.g002: rmnd5 is expressed during early embryonic development.(A) Temporal RT-PCR analysis of rmnd5 expression (top panel); different developmental stages (NF-stages) indicated at the top. ODC1 functions as RNA input control (bottom). (B) Rmnd5 protein at different developmental stages. Western blot analysis of embryo lysate from indicated stages (top). α-RMND5A (Novus Biological; rabbit, 1:1000); α-Tubulin (AbD Serotec, rat, 1:2500). (C) Spatial analysis of rmnd5 expression. Whole mount in situ hybridisation (Wmish) of wild type Xenopus laevis embryos at different developmental stages. NF stage 3 (panel a, left) and stage 4 (panel c) rmnd5 transcript in the animal pole (top), NF-stage 12 (panel d) rmnd5 transcripts around the prospective head, NF-stage 18; 24 (panel e, f, g, h) neuronal ectoderm (red arrow, panel e) and ciliated cells of the skin (yellow arrow, panel e, g, h), NF-stage 34 (panel j, k, l, m) proencephalon (red arrow) and eyes (green arrow). Negative controls with sense probes (panel b, i).

Mentions: To study a potential role of Rmnd5 during development of the neural system, we first analyzed its spatio-temporal expression pattern in embryos of Xenopus laevis. We started out looking at the temporal expression of rmnd5 by semi-quantitative RT-PCR using total RNA from consecutive developmental stages of Xenopus laevis embryos. In Fig. 2A a specific signal at the expected size of 305 bp indicated the existence of rmnd5 transcript (upper panel), while ODC1 primers were used to control the amount of input RNA (lower panel). The expression of rmnd5 receives a strong maternal contribution, as RT-PCR products are detectable at NF (Nieuwkoop and Faber) stage 3 and declines steadily until gastrulation (NF stage 12). Zygotic transcripts of rmnd5 are detectable through all further stages analyzed, though on a much lower level compared to NF stage 3. This finding is confirmed on the protein level, but a comparable decline in Rmnd5 levels is not visible (Fig. 2B). Whole mount in situ hybridisation (WMISH) reveals first expression of rmnd5 at the four-cell stage (Fig. 2C-A, NF stage 3) and the eight-cell stage (Fig. 2C-C, NF stage 4). Here rmnd5 transcript is mostly present in the animal pole (top), an area developing into derivatives of ectoderm and mesoderm. During gastrulation (NF-stage 12) increasing levels of rmnd5 transcripts appear enhanced in the prospective head region (Fig. 2C-D) and during neurulation (Fig. 2C-E, NF stage 18) in the neuronal ectoderm (red arrow) and subpopulations of skin cells, most likely the ciliated cells of the skin (Fig. 2C-E, -H; yellow arrow). In a later stage of development (NF stage 34, Fig. 2C-J, -K), the expression of rmnd5 is mainly restricted to head structures and demarcates neural tissues and derivatives. Strong expression is observed in the eyes (Fig. 2C-J, -K, -L; green arrow) and the prosencephalon (Fig. 2C-J, -K, -L, -M; red arrow).


RMND5 from Xenopus laevis is an E3 ubiquitin-ligase and functions in early embryonic forebrain development.

Pfirrmann T, Villavicencio-Lorini P, Subudhi AK, Menssen R, Wolf DH, Hollemann T - PLoS ONE (2015)

rmnd5 is expressed during early embryonic development.(A) Temporal RT-PCR analysis of rmnd5 expression (top panel); different developmental stages (NF-stages) indicated at the top. ODC1 functions as RNA input control (bottom). (B) Rmnd5 protein at different developmental stages. Western blot analysis of embryo lysate from indicated stages (top). α-RMND5A (Novus Biological; rabbit, 1:1000); α-Tubulin (AbD Serotec, rat, 1:2500). (C) Spatial analysis of rmnd5 expression. Whole mount in situ hybridisation (Wmish) of wild type Xenopus laevis embryos at different developmental stages. NF stage 3 (panel a, left) and stage 4 (panel c) rmnd5 transcript in the animal pole (top), NF-stage 12 (panel d) rmnd5 transcripts around the prospective head, NF-stage 18; 24 (panel e, f, g, h) neuronal ectoderm (red arrow, panel e) and ciliated cells of the skin (yellow arrow, panel e, g, h), NF-stage 34 (panel j, k, l, m) proencephalon (red arrow) and eyes (green arrow). Negative controls with sense probes (panel b, i).
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pone.0120342.g002: rmnd5 is expressed during early embryonic development.(A) Temporal RT-PCR analysis of rmnd5 expression (top panel); different developmental stages (NF-stages) indicated at the top. ODC1 functions as RNA input control (bottom). (B) Rmnd5 protein at different developmental stages. Western blot analysis of embryo lysate from indicated stages (top). α-RMND5A (Novus Biological; rabbit, 1:1000); α-Tubulin (AbD Serotec, rat, 1:2500). (C) Spatial analysis of rmnd5 expression. Whole mount in situ hybridisation (Wmish) of wild type Xenopus laevis embryos at different developmental stages. NF stage 3 (panel a, left) and stage 4 (panel c) rmnd5 transcript in the animal pole (top), NF-stage 12 (panel d) rmnd5 transcripts around the prospective head, NF-stage 18; 24 (panel e, f, g, h) neuronal ectoderm (red arrow, panel e) and ciliated cells of the skin (yellow arrow, panel e, g, h), NF-stage 34 (panel j, k, l, m) proencephalon (red arrow) and eyes (green arrow). Negative controls with sense probes (panel b, i).
Mentions: To study a potential role of Rmnd5 during development of the neural system, we first analyzed its spatio-temporal expression pattern in embryos of Xenopus laevis. We started out looking at the temporal expression of rmnd5 by semi-quantitative RT-PCR using total RNA from consecutive developmental stages of Xenopus laevis embryos. In Fig. 2A a specific signal at the expected size of 305 bp indicated the existence of rmnd5 transcript (upper panel), while ODC1 primers were used to control the amount of input RNA (lower panel). The expression of rmnd5 receives a strong maternal contribution, as RT-PCR products are detectable at NF (Nieuwkoop and Faber) stage 3 and declines steadily until gastrulation (NF stage 12). Zygotic transcripts of rmnd5 are detectable through all further stages analyzed, though on a much lower level compared to NF stage 3. This finding is confirmed on the protein level, but a comparable decline in Rmnd5 levels is not visible (Fig. 2B). Whole mount in situ hybridisation (WMISH) reveals first expression of rmnd5 at the four-cell stage (Fig. 2C-A, NF stage 3) and the eight-cell stage (Fig. 2C-C, NF stage 4). Here rmnd5 transcript is mostly present in the animal pole (top), an area developing into derivatives of ectoderm and mesoderm. During gastrulation (NF-stage 12) increasing levels of rmnd5 transcripts appear enhanced in the prospective head region (Fig. 2C-D) and during neurulation (Fig. 2C-E, NF stage 18) in the neuronal ectoderm (red arrow) and subpopulations of skin cells, most likely the ciliated cells of the skin (Fig. 2C-E, -H; yellow arrow). In a later stage of development (NF stage 34, Fig. 2C-J, -K), the expression of rmnd5 is mainly restricted to head structures and demarcates neural tissues and derivatives. Strong expression is observed in the eyes (Fig. 2C-J, -K, -L; green arrow) and the prosencephalon (Fig. 2C-J, -K, -L, -M; red arrow).

Bottom Line: In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function.Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex.We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development.

View Article: PubMed Central - PubMed

Affiliation: Martin-Luther University Halle-Wittenberg, Institute of Physiological Chemistry, Halle, Germany.

ABSTRACT
In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development.

No MeSH data available.


Related in: MedlinePlus