Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Mar 20;10(3):e0120342.
doi: 10.1371/journal.pone.0120342. eCollection 2015.

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

Thorsten Pfirrmann  1 , Pablo Villavicencio-Lorini  2 , Abinash K Subudhi  1 , Ruth Menssen  3 , Dieter H Wolf  3 , Thomas Hollemann  1
Affiliations

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

Thorsten Pfirrmann et al. PLoS One. .

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.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist. Funding by "Fonds der Chemischen Industrie" does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Xenopus laevis Rmnd5 protein is structurally and functionally related to human RMND5A.
(A) Phylogenetic tree of Rmnd5 orthologs. The taxonomic tree of representative eukaryotic species rendered by Phylogeny.fr software [20]. Respective Gid2/Rmnd5 sequences obtained from NCBI with indicated accession numbers (Saccharomyces cerevisiae [NP_010541.3], Candida albicans [XP_712238.1], Aspergillus niger [XP_001388791.2], Caenorhabditis elegans [NP_508444.1], Arabidopsis thaliana [NP_196525.1], Drosophila melanogaster [NP_611536.3], Xenopus laevis [NP_001086276.1], Falco peregrinus [XP_005229906.1], Gallus gallus [XP_004936301.1] Homo sapiens [NP_073617.1; NP_073599.2], Ornithorhynchus anatinus [XP_007670084.1; XP_001515875.2], Sarcophilus harrisii [XP_003758697.1; XP_003756956.1], Canis lupus familiaris [XP_852129.1; XP_531873.2], Mus musculus [NP_077250.2; NP_079622.1], Rattus norvegicus [XP_232051.4; NP_001017473.1]); homolog A (blue), homolog B (red). (B) Sequence alignment of Xenopus laevis Rmnd5 (top), Homo sapiens RMND5A (middle) and RMND5B (bottom). Identical residues (red), similar residues (blue), others (black). Identities (%): Xenopus laevis Rmnd5 to human RMND5A (94%), to human RMND5B (70%). (C) Localization of Homo sapiens RMND5A (RMND5a, middle panel), RMND5B (RMND5b, bottom panel) and Xenopus laevis Rmnd5 (Rmnd5, top panel) in HEK293 cells. GFP signal (left column), DAPI signal (middle column), merged signals (right column).
Fig 2
Fig 2. 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).
Fig 3
Fig 3. The CTLH complex functions during early embryonic neurogenesis.
(A) rmnd5-mo injected embryos were used for in situ hybridisation with indicated marker probes; pax6 (upper lane), n-tubulin (middle lane), k20/en22/rx1/c-actin, emx1.2, nkx2.1 (bottom lanes). Abbreviations: IS, injected side; NIS, non-injected side. Quantitative representation of phenotypes are presented as a bar graph (percent embryos with phenotype to total amount (%); black, phenotype; grey, no phenotype); n = number of independent experiments, N = number of injected embryos analysed for respective marker, *P ≤0.05, ***≤0.001 (Chi Square test). (B) As (A) with sox2 as probe. (C) Xenopus embryos co-injected with rmnd5 morpholino (2.5 pmol/embryo) and synthetic capped RNA (100 pg/embryo) were used for in situ hybridisation and quantified as shown in A.
Fig 4
Fig 4. Rmnd5 is part of an ubiquitin ligase complex.
(A) Glycerol step gradient of Xenopus laevis NF stage 36 embryo lysates. Molecular mass (MW) standard: albumin (67 kDa), fraction 1, 2; LDH (140 kDa), fraction 4; catalase (232 kDa), fraction 6,7. Western blot analysis with α-RMND5A (Rmnd5; upper panel) (1:1000) and α-ARMC8 (lower panel) (1:1000). (B) In vitro polyubiquitination assay with recombinant Xenopus Rmnd5 and Rmnd5-C354S (lane 3, 4). Reactions are performed in the presence (+) or absence (-) of E1 (lane 1), E2 (lane 2) and purified Rmnd5 protein. HDM2 is used as a positive control (lane 5). Polyubiquitination (Poly-Ub) is detected with α-HA and α-RMND5A as control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Regelmann J, Schule T, Josupeit FS, Horak J, Rose M, Entian KD, et al. Catabolite degradation of fructose-1,6-bisphosphatase in the yeast Saccharomyces cerevisiae: a genome-wide screen identifies eight novel GID genes and indicates the existence of two degradation pathways. Mol Biol Cell. 2003;14(4):1652–63. Epub 2003/04/11. 10.1091/mbc.E02-08-0456 PubMed PMID: . - DOI - PMC - PubMed
    1. Menssen R, Schweiggert J, Schreiner J, Kusevic D, Reuther J, Braun B, et al. Exploring the Topology of the Gid Complex, the E3 Ubiquitin Ligase Involved in Catabolite-induced Degradation of Gluconeogenic Enzymes. J Biol Chem. 2012;287(30):25602–14. Epub 2012/05/31. 10.1074/jbc.M112.363762 PubMed PMID: - DOI - PMC - PubMed
    1. Braun B, Pfirrmann T, Menssen R, Hofmann K, Scheel H, Wolf DH. Gid9, a second RING finger protein contributes to the ubiquitin ligase activity of the Gid complex required for catabolite degradation. FEBS Lett. 2011. Epub 2011/11/03. S0014-5793(11)00789-7 [pii]10.1016/j.febslet.2011.10.038 PubMed PMID: . - DOI - PubMed
    1. Santt O, Pfirrmann T, Braun B, Juretschke J, Kimmig P, Scheel H, et al. The yeast GID complex, a novel ubiquitin ligase (E3) involved in the regulation of carbohydrate metabolism. Mol Biol Cell. 2008;19(8):3323–33. Epub 2008/05/30. E08-03-0328 [pii]10.1091/mbc.E08-03-0328 PubMed PMID: . - DOI - PMC - PubMed
    1. Sommer T, Wolf DH. The ubiquitin-proteasome-system. Biochim Biophys Acta. 2014;1843(1):1 Epub 2013/09/24. 10.1016/j.bbamcr.2013.09.009 PubMed PMID: . - DOI - PubMed

Publication types

Grants and funding

The project was supported by funding from the Fonds der Chemischen Industrie, Frankfurt and the Wilhelm Roux program (FKZ 26/22), Halle (Saale). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.