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Supplementary Methods 1
Saccharomycescerevisiae genomic DNA was extracted from the wild-type strain W303 using standard rapid glass-bead lysis and phenol-chloroform extraction, as described (Trevisson et al., 2009). The coding region of the yeast MSR1 (the ortholog of RARS2) was amplified from genomic DNA using primers: MSR1 -33F (5’-GCTTTGAATGACCTACCTGA-3’) and MSR1 2155R XbaI (5’-agtcatctagaTTGGATCCATCTTCTATTGG-3’), and it was cloned in pCR8/GW/TOPO (Invitrogen, USA) according to the manufacturer’s protocol. The accuracy and the reading frame of the plasmid were rechecked by direct sequencing. MSR1 was then cloned into the pCM189-RfB vector, containing the URA3 selection marker, via homologous recombination using the Gateway LR Clonase II enzyme mix (Invitrogen, USA) according to manufacturer’s protocol. pCM189-RfB was obtained by cloning the RfB cassette (Invitrogen, USA) into the PmeI site of pCM189 (Gari et al., 2007).
Specific mutants were generated by site-directed mutagenesis using the QuickChange kit (Stratagene, Agilent, Italy) according to the manufacturer’s protocol; mismatched-primers were designed with the assistance of the Stratagene Primer Design software. The correctness of each construct was verified by direct sequencing.
The experiments were performed using a W303 strain (Mat a/Mat alpha {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15} [phi+]). Yeast strains were cultured in rich media (YP) or synthetic minimal medium (SM) at 30°C, as described (Burke et al., 2000). YPD (1% yeast extract, 2% peptone, 2% dextrose) was used to routinely maintain wild type and ΔMSR1 strain. Synthetic minimal medium (0.17% yeast nitrogen base without amino acids, ammonium sulphate 0,5%, glucose 2%) contained the necessary auxotrophic supplements was used to select the transformant strains. YPG (1% yeast extract, 2% peptone, 3% glycerol) was used to test the respiratory ability of the mutant strains. SMP (sporulation medium, 1% potassium acetate) was used to induce the sporulation of diploid yeast strains. Two percent agar was added to the solid media. Growth in plates was assayed as described (Trevisson et al., 2009).
Deletion of MSR1 gene was obtained by replacing the MSR1 genomic sequence with the HIS3 gene sequence on a W303 diploid strain. The HIS3MX6 coding sequence was amplified from pFA6a-HIS3MX6 vector using primers: Msr1 Inac His F (5-atgttcgggatagtttatctgaagaatcgcagtttactctgcaagCGTACGCTGCAGGTCGAC-3) and Msr1 Inac His R (5-ttacattctatcaaccgggactagtcccaaaagcgacattgcattATCGATGAATTCGAGCTCG-3) and Expand High Fidelity PCR System (Roche). The primers contained 45 bp of homology with the MSR1 gene (sequence in small caps) to allow homologous recombination between the PCR fragment and the MSR1 genomic region. PCR conditions were as follows: 94°C for 3 min, 25 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 2 min and 30 s, and a final extension step of 7 min at 72°C. The PCR product was transformed into the diploid strain using a high efficiency lithium acetate method as previously described (Gietz and Woods 2006). Colonies with the insertion of the cassette were selected by growth in SM medium lacking histidine, and the correctness of the insertion was proved by PCR after genomic DNA extraction using the primers: MSR1 -385F cagtcagagctcACTGACTACAACGCTGATGA and MSR1 2155R. PCR conditions were as above. Only colonies with heterozygous deletion of MSR1 were selected for further studies.
Transformations of the diploid strain with the pCM189 vectors were performed with the PEG-Lithium acetate method as previously described (Gietz and Woods 2006).
Heterozygous ΔMSR1 mutants were let to sporulate on solid SPM medium for 4 days at 25°C. About 10 tetrads per strain were mechanically dissected as described (Sartori et al., 2000) and the spores were incubated on YPD at 30°C for 3 days. For each spore, the correct 2:2 segregation of the histidine prototrophy and of the different auxotrophic markers was verified by replica-plate on selective media. Haploid cells autotrophic for histidine and uracil were used for subsequent analyses.
References
Burke D, Dawson,D, Stearns, T. Methods in Yeast Genetics 2000, pp. 113–114, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Gari E, Piedrafita L, Aldea M, Herrero E.A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast. 1997; 13: 837-48
Gietz RD, Woods RA. Yeast transformation by the LiAc/SS Carrier DNA/PEG method. Methods Mol Biol 2006; 313:107-20
Sartori G, Mazzotta G, Stocchetto S, Pavanello A, Carignani G. Inactivation of six genes from chromosomes VII and XIV of Saccharomyces cerevisiae and basic phenotypic analysis of the mutant strains. Yeast. 2000; 16: 255-65
Shammas C, Menne TF, Hilcenko C, Michell SR, Goyenechea B, Boocock GR, et al. Structural and mutational analysis of the SBDS protein family. Insight into the leukemia-associated Shwachman-Diamond Syndrome. J Biol Chem. 2005; 280: 19221-98
Trevisson E, Burlina A, Doimo M, Pertegato V, Casarin A, Cesaro L, et al. Functional complementation in yeast allows molecular characterization of missense argininosuccinate lyase mutations.J Biol Chem. 2009; 284: 28926-34
Supplementary Methods 2
Fish breeding and embryo collection. Adult zebrafish were bred by natural crosses in a male-to-female ratio of 2:1 (Westerfield 1995). Immediately after spawning, the bottoms of the aquariums were siphoned. The fertilized eggs were harvested, washed, and placed in 9-cm-diameter Petri dishes in 0.6 mg/l Instant Ocean sea salts (Aquarium Systems, Sarrebourg, France). The developing embryos were incubated at 28.5 °C until use. Developmental stages of zebrafish embryos were expressed as hours or days post-fertilization (hpf/dpf) at 28.5 °C (Dooley and Zon, 2000).
RT-PCR of zebrafish rars2 gene. Total RNA was extracted from a pool of 30 embryos or larvae at different stages of development with the TRIZOL reagent (Roche, Germany). One μg of RNA was reverse transcribed using Quantitect Reverse Transcription Kit (QIAGEN, USA) according to manufacturer’s protocol. The RARS2 ortholog in zebrafish was first identified by tblast option at NCBI ( using the human RARS2 protein against the zebrafish genome which allowed the identification of NM_200617 transcript whose identity was confirmed by direct sequencing. To perform quantitative RT-PCR, primer1 For: 5’-TCACCAGAGACGTGGCAGCGG-3’; Primer 2 Rev: 5’-GCCCTGCACCAGGCCGAACG-3’ were designed using Primer3 tool ( The steady state amount of RARS2 mRNA was evaluated relatively to the adult with respect to 18S rRNA FJ915075.1 amplifying a 107-bp region using primer3 For: 5’-AGCGTGCGGGAAACCACGAG-3’ and primer4 Rev: 5’-AAGCCGCAGGCTCCACTCCT-3’ as a reference gene on the Light Cycler 480 (Roche). Samples were analysed in triplicate for each assay. Relative expression was estimated using the R = 2-ΔΔCt method, where R indicates the relative expression amount. In all real-time experiments, melting curve analysis showed that there were no primer dimers formation.
Whole-mount in situ hybridisation using DIG-labelled RNA probes.A region contained between primer RARS2_For1108 5’-TCACCAGAGACGTGGCAGCGG-3’ and primer RARS2_Rev1829 5’-GGCCAGAACTGAGTGGACGGAGC-3’ was cloned in the pGEM-T easy vector system (Promega, Italy) for antisense RNA probe tagged with dioxygenin-UTP using the SP6/T7 polymearase RNA labelling kit (Roche, Germany). Dechorionated embryos were fixed overnight at 4C° in 4% paraformaldehyde in 0,1% phosphate-buffered saline (PBS), then stored in methanol at –20 °C till further use. Whole mount in situ hybridization was performed as described by others (Thisse and Thisse 2008). The presence of the probe was visualized immunoistochemically using an anti-DIG antibody conjugated to alkaline phosphatase and a chromogenic substrate. After the hybridization procedure, embryos were washed extensively in 0.1% Tween 20 in PBS, re-fixed in 4% paraformaldehyde in PBS, then transferred to 100% glycerol and observed on AZ100 microscope (Nikon, Germany).
References
Dooley K, Zon LI. Zebrafish: a model system for the study of human disease. Curr Opin Genet Dev 2000; 10:252-6
Thisse C, Thisse B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc. 2008; 3:59-69
Westerfield M. The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 2000; 4th ed., Univ. of Oregon Press, Eugene.
Legend to Supplementary Figure
Suppl Figure 1. Schematic representation of the procedure used in yeast to generate haploid mutants. a) One MSR1 allele is replaced by A HIS3 cassette which confers prototrophy for histidine; b) the diploid is transformed with a plasmid carrying wild type or mutant MSR1 and a URA3 cassette which confers prototrophy for uracil; c) diploid yeast is induced to sporulate; d) tetrads are mechanically dissected and individual cells are selected for both uracil and histidine prototrophy. Positive cells are then tested for respiratory growth.
Suppl. Figure 2. Mutations in RARS2 detected by sequencing in patients with early-onset pan-encephalopathy. Electropherograms flanking the mutations in RARS2 in unaffected individuals (Ctrls) and patients in affected kindred (A-II.02 in family A, B-II.02 in family B, C-II.01in family C) are presented. The positions of the heterozygous mutations are arrow-headed and the predicted effect of the mutation is also shown. A. Patient A-II.01 displayed a heterozygous c.25A>G transition, predicting a novel p.I9V variant in compound heterozigosity with c.1586+3A>T. These mutations were also identified in patient A-II.02. The splice-site variant resulted in skipping of exon 18 (see bottom panel). B. (left) Patient B-II.02 harboured the heterozygous c.734G>A (p.R245Q) and c.1406G>A (p.R469H). These mutations were also identified in patient B-II.01. (right) Patient C-II.01 harboured the heterozygous c.354A>G (p.Q12R) and c.721T>A (p.W241R).
Suppl. Figure 3. (top) Relative expression of zf-rars2 gene by quantitative real time PCR during zebrafish development.The value of 2–ΔΔCt represents the expression of the mitochondrial gene in each development stage normalized to 18S rRNA relative to the normalized expression of genes in adult sample. (bottom) Whole-mount in situ hybridization during different stages of embryogenesis. A1: early stages, 10hpf (magnification 40X); B1: 24hpf (80X); C1: 48hpf (80X); C2: 48hpf (300X); D1: 3dpf (80X); D2: 3dpf (200X); E1: 4dpf (240X); E2: 4dpf (80X); F1: 5dpf (80X); F2: 5dpf (400X). F3: 5dpf (100X). hpf, hours post fertilization; dpf, days post fertilization.