Nazlamova L et al. (2017), A newly identified Rab-GDI paralogue has a role...

XB-ART-52694

Gene 2017 Jan 30;599:78-86. doi: 10.1016/j.gene.2016.11.013.

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A newly identified Rab-GDI paralogue has a role in neural development in amphibia.

Nazlamova L , Noble A , Schubert FR , McGeehan J , Myers F , Guille M , Scarlett G .

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Vesicle shuttling is critical for many cellular and organismal processes, including embryonic development. GDI proteins contribute to vesicle shuttling by regulating the activity of Rab GTPases, controlling their cycling between the inactive cytosol and active membrane bound states. While identifying genes controlled by A-form DNA sequences we discovered a previously unknown member of the GDI family, GDI3. The GDI3 gene is found only in amphibians and fish and is developmentally expressed in Xenopus from neurula stages onwards in the neural plate, and subsequently in both dorsal and anterior structures. Depletion or over-expression of the GDI3 protein in Xenopus embryos gives rise to very similar phenotypes, suggesting that strict control of GDI3 protein levels is required for correct embryonic development. Our analysis suggests the evolutionary origins of GDI3 and that it is functionally distinct from GDI1. Predicted structural analysis of GDI3 suggests that the key difference between GDI1 and GDI3 lies in their lipid binding pockets.

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Species referenced: Xenopus
Genes referenced: arih2 gdi1 gdi2 odc1 prkar2a slc25a20

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	Fig. 1. The Rab GTPase recycling mechanism is controlled by a variety of effectors to ensure a constant Rab soluble pool in the cell cytosol. GDP bound Rab proteins are escorted by Rab escort protein (REP) in order to be prenylated by geranyl-geranyl tranferase (GGT). Once the isoprenoid group is attached, Rab-GDP is solubilised by guanine dissociation inhibitor (GDI) and is ready to regulate vesicle shuttling. Subsequently Rab-GDP is activated by exchange to Rab-GTP, a process assisted by guanine exchange factor (GEF). Once activated Rab- GTP can bind vesicles and export them to the correct destination where Rab-GTP is converted back to Rab-GDP. The conversion from Rab-GTP to Rab-GDP is catalysed by GTPaseactivating protein (GAP).
	Fig. 2. Alignment of the Xenopus tropicalis GDI3 protein sequence against the JGI 7.1 GDI3 predicted protein sequence. The upper rowcorresponds to the observed GDI3 DNA sequence and the lower row the GDI3 predicted protein sequence obtained from Xenbase genome assembly JGI 7.1 of Xenopus tropicalis. Bases highlighted indicate SNPs between the two sequences while the predicted exonic arrangement is indicated by grey arrows.
	Fig. 3. Phylogenetic and syntenic analysis of the GDI genes. (A) Phylogenetic tree of chordate GDI proteins. The tree shows the phylogenetic relationship of GDI protein sequences from representatives of all main vertebrate classes. Nanorana parkeri (XP_018431581 (NCBI)), Latimeria chalumnae (XP_005992613 (NCBI)), Lepisosteus oculatus (XP_015203569 (NCBI)), Callorhinchus milii (XP_007888513 (NCBI)), Scyliorhynchus canicula (SSC-transcript-ctg25260 (SkateBase)), Danio rerio (NP_001307001 (NCBI)), Gasterosteus aculeatus (ENSGACP00000001552 (Ensembl)), Xenopus tropicalis (XP_004914150.1 (NCBI)). The tree was produced using the maximum likelihood method, using the GDI sequence from the basal chordate amphioxus as outgroup. The gnathostome sequences are divided into three distinct groups (GDI1 in blue, GDI2 in green, GDI3 in red), with GDI1 and GDI2 grouped together. The lamprey GDIA and GDIB sequences are placed on their own, unique branches. (B) Organisation of the GDI3 loci in different vertebrates. The GDI3 gene with its four upstream and downstream neighbours is shown for the western clawed frog, coelacanth, spotted gar, stickleback and elephant shark. Teleost fish have undergone a third genome duplication. Both loci are shown, since most genes, including GDI3, have only been retained in one of the two loci. The corresponding locus is also shown for human, chicken and anole lizard, but these species lack the GDI3 gene. For comparison, the GDI1 and GDI2 genes with their neighbours are shown for the western clawed frog. The GDI genes are marked in yellow, while for the neighbouring genes orthologues are shown in the same colour. The GDI3 loci are well conserved between species, notably with Arih2, Slc25A20 and Prkar2a always present close to GDI3. (C) Two substitution sites in GDI3 compared to GDI1 (Y117H and N130D, shown in red) when plotted against the tertiary structure map to the flanking hinge regions of a helix lining the lipid binding pocket. Yeast GDI is shown in blue and bovine GDI1 in yellow and the lipid moiety is highlighted in green. GDI1 helices D and E are part of the lipid binding site of domain II and beta-sheet b2 is contained in domain I.
	Fig. 4. GDI3 expression in X. tropicalis. (A) Xenopus tropicalis embryos were harvested at the Nieuwkoop-Faber stages shown and RNA extracted prior to RT-PCR analysis by odc (as an internal reference) and GDI3 specific primers. GDI3 expression is observed throughout all the sampled developmental stages. (B) In situ hybridisation analysis of Xenopus tropicalis embryos for GDI3. The upper row of each stage set corresponds to probing with an antisense probe and the bottom row is probed with a control sense probe. The red arrow shows the anterior region of the embryos. In Nieukoop and Faber (NF) stages 16, 18 and 22 embryos are shown from lateral, dorsal and anterior views left to right. Embryos at stages 25 and 30 are shown in the lateral and dorsal view only. (C) Analysis of GDI3 expression in adult Xenopus tropicalis tissues show presence of mRNA in the eyes, leg muscle and heart but not in the brain, liver, intestine and kidney.
	Fig. 5. Functional analysis of GDI3 in Xenopus tropicalis. (A) The binding sites ofMO1 andMO2 to the Xenopus GDI3 5â€² UTR. (B) Embryos injected at one cell stagewith either 12 or 24 pg of GDI3 syntheticmRNAwere grown to stage 18 and harvested and tested for translation of the exogenous GDI3. Extraction equivalencewas confirmed by running a parallel gel in the same electrophoresis tank thatwas stainedwith Coomassie Brilliant Blue. (C) Embryos injected with either morpholino for protein level knock-down or GDI3mRNA for protein over-expression demonstrate loss of anterior-posterior axis. In each panel pictures of three different embryos are shown. (D) A variety of anterior marker genes were analysed for their changes as a consequence of GDI3 over-expression by RT-PCR at stage 18.