Pfirrmann T , Villavicencio-Lorini P , Subudhi AK , Menssen R , Wolf DH , Hollemann T .

Xla Wt + rmnd5a MO (fig.3.A.f,g,j)
Xla Wt + rmnd5a MO (fig.3.A.i)
Xla Wt + rmnd5a MO (fig.3.A.j)
Xla Wt + rmnd5a MO (fig.3.A.n,o)
Xla Wt + rmnd5a MO (fig.3.A.n,o,q)
Xla Wt + rmnd5a MO (fig.3.A.q)
Xla Wt + rmnd5a MO (fig.3.A.u,v)
Xla Wt + rmnd5a MO (fig.3.A.x^1)
Xla Wt + rmnd5a MO (fig.3.A.x^1)
Xla Wt + rmnd5a MO (fig.3.B.b,c,d,e)
Xla Wt + rmnd5a MO (fig.3.B.b,e)
Xla Wt + rmnd5a MO (fig.3.B.d)

	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. 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. 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. 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.
	Sup. F1 Catabolite degradation of fructose-1,6-bisphosphatase (FBPase) in yeast. Δgid2 (YWO0906) was transformed with plasmid pRM41 harbouring V5-tagged rmnd5. YWO0906 and YWO2023 (containing GID2-V5) were transformed with empty plasmid pRS426 as controls. Cells were grown for 12h in synthetic complete medium without uracil containing 2% glucose. After addition of 2% glucose 1.5 OD600 of cells were taken at the indicated time points. Total protein was extracted and precipitated with trichloroacetic acid, resuspended in urea buffer and subjected to Western blot analysis with polyclonal FBPase antiserum, Pgk antibody (Molecular probes) and V5 antibody (Thermo Scientific), respectively. Digital data were quantified using TotalLab Quant and Excel; FBPase signals were normalised with 3-phosphoglycerate kinase (Pgk) (A) Representative Western blot of a complementation experiment. (B) Quantification of FBPase signal after glucose addition. Graphs include data from n = 10 (Rmnd5, blue), n = 7 (gid2 = Δgid2, red) and n = 5 (GID2, purple) experiments, respectively.
	S2 Fig. The CTLH complex functions during early embryonic neurogenesis. As Fig. 3 with standard morpholino injected embryos
	rmnd5a (required for meiotic nuclear division 5 homolog A ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, lateral view,.
	rmnd5a (required for meiotic nuclear division 5 homolog A ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 4, lateral view.
	rmnd5a (required for meiotic nuclear division 5 homolog A ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, antereo-lateral view
	rmnd5a (required for meiotic nuclear division 5 homolog A ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.

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