Finch, AJ et al., Supplemental Material
Includes 7 Supplemental Figures, 7 Supplemental Tables, (Tables 1 and 2 are
provided as Excel files), Supplemental Materials and Methods, Supplemental
References.
Supplemental Figures
Figure S1. Targeted disruption of the mouse Sbds gene, related to Fig. 1
a. Genomic organization of the mouse Sbds locus (Sbds+); b. targeting vector; c. the
product of homologous recombination (Sbdsfl); d. structure of the Sbds locus
following Cre recombination (Sbds-). Red boxes represent Sbds exons I-V, yellow triangles represent loxP sites. The 5' external probe used for genotyping by filter hybridization is shown as a gray box. B, BamH1.
Figure S2. Sbds deletion does not alter the ratio of 60S to 40S ribosomal subunits,
related to Fig. 2. The ratios of 60S to 40S subunits are compared between Sbdsfl/- mice
treated with 6 x 250 µg poly(I:C) over 2 weeks in the absence (-Cre) or presence
(+Cre) of the pMx1-cre transgene.
Figure S3. A negative genetic interaction network for EFL1 and SDO1, related to
Fig. 3. Genes are represented as nodes, and negative genetic interactions (e.g.
synthetic lethal/sick interactions) are represented as edges that connect the nodes.
Synthetic lethal/sick interactions confirmed by random spore and/or tetrad analysis
are shown as black edges. Network edges representing unconfirmed quantitative
negative interactions that satisfied a stringent confidence threshold (e <-0.12, p <0.05)
1 are shown in grey. Nodes are colored based on a general biological process annotation
described previously (Costanzo et al. 2010).
Figure S4. EFL1 catalytic residues are required for genetic complementation of efl1Δ
cells in vivo, related to Fig. 3.
a. Schematic of the domain structure of yeast EFL1 showing the position of critical
loss of function missense mutations.
b. Sequence alignment of the consensus GTP-binding domain (G domain) for H.
sapiens EFL1 (HsEFL1, NP_078856), S. cerevisiae EFL1 (ScEfl1p, NP_014236) and
S. cerevisiae EF-2 (ScEF-2). The amino sequences were aligned using MacVector
11.1 (http://www.macvector.com). The conserved motifs G1, G2, G3, G4 and G5 of
the G domain are boxed in red. Residues mutated in (c) are shown in red.
c. EFL1 catalytic residues are required for function in vivo. The ability of a yeast
EFL1-expressing plasmid to complement the growth defect of efl1Δ cells is impaired by mutations in the G1 P-loop that spans the α and β phosphates of the bound GTP cofactor (T33A), the G3 switch II region involved in Mg2+ coordination and γ- phosphate binding (D102A, H106A, H106I), the G4 motif involved in binding the
guanine nucleobase of GTP (D159A) or mutation of a residue predicted to contact the
SRL (W240A).
Figure S5. The SBDS protein is highly conserved from archaea to human, related to
Fig. 4. a. Structure-based sequence alignment of representative SBDS protein orthologs.
Numbering corresponds to the amino acid sequence for human SBDS. Shading intensity indicates the degree of amino acid identity. GenBankTM accession numbers:
2 H. sapiens (NP_057122), M. musculus (P70122), S. cerevisiae (NP_013122), D.
melanogaster (NP_648057), D. discoideum (AAO50830), A. fulgidus (NP_069327),
M. thermautotrophicus (NP_275828). Secondary structure elements for the A.
fulgidus and H. sapiens SBDS orthologs are shown above the alignment with domain
I colored in red, domain II in yellow, domain III in green. The alignment was
generated with MacVector 11.1 (http://www.macvector.com).
b-d. The fold of the human and archaeal SBDS proteins is conserved. Superposition
of the structures of human SBDS (pdb code 2L9N) domain I (red) (b), domain II
(yellow) (c) and domain III (green) (d) with the respective domains of A. fulgidus
SBDS (pdb code 1T95) (gray). Rms deviation for domain I (residues A16-T89) is
1.27 Å; domain II (residues D97-K164) is 1.48 Å and domain III (residues H171-
L237) is 1.13 Å. Figures were generated using PyMOL (http://www.pymol.org).
Figure S6. Chemical shift perturbations caused by SDS-associated disease mutants, related to Fig. 4. Overlays of the 1H,15N HSQC spectra for SBDS WT (blue) and 25
SDS-associated mutants (red). a. (I) P6L, (II) N8K, (III) R19Q, (IV) F27L, (V) A30S,
(VI) Y32C, (VII) K33E, (VIII) N34I. b. (IX) E44G, (X) F57L, (XI) K67E, (XII)
K118N, (XIII) C119R, (XIV) C119Y, (XV) N121T, (XVI) T129A. c. (XVII) S143L,
(XVIII) S143W, (XIX) K148R, (XX) K148T, (XXI) Q153R, (XXII) R169C, (XXIII)
R169L, (XXIV) R175W. d. (XXV) R218Q.
Figure S7. The SDS-associated SBDS variants R126T and K151N bind to 60S
subunits in vitro, related to Fig. 6. Recombinant WT SBDS and variants (R126T,
K151N) were bound together with EFL1 to RRL 60S subunits over the indicated
3 range of KCl concentrations and pelleted through 30% (w/v) sucrose cushions. Bound
SBDS, EFL1 and Rpl28 were visualized by immunoblotting.
Supplemental Tables.
Table S1. Quantitative scoring of EFL1 positive and negative genetic interactions
(Excel file).
Table S2. MS/MS analysis of murine eIF6 tryptic peptides. Observed phosphopeptides are shaded in yellow. The peptide containing S235
(LNEAKPSTIATS235MR) is shaded in blue. Lower case amino acids indicate modifications (Excel file).
4 Table S3. Summary of experimental restraints and structural statistics for the 20 accepted lowest energy structures of the human SBDS protein
Structural constraints Intra‐residue 1490 Sequential 1119 Medium‐range (2 ≤ |i‐j| ≤ 4) 596 Long‐range (|i‐j| > 4) 822 Dihedral TALOS constraints 296 Hydrogen bond constraints 116 Total 4439
Statistics for accepted structures Statistics parameter R.m.s deviation for distance constraints (űSD) 0.0052 ± 0.0010 R.m.s deviation for dihedral constraints (o±SD) 0.0727 ± 0.0486
Mean CNS energy term (kcal.mol‐1±SD) E (overall) 157.7 ± 26.9 E (van der Waals) 40.5 ± 13.1 E (NOE and hydrogen bond constraints) 8.8 ± 3.7 E (chi‐1 dihedral and TALOS constraints) 0.5 ± 1.0
R.m.s deviations from the ideal geometry Bond lengths (űSD) 0.0012 ± 0.0002 Bond angles (o±SD) 0.29 ± 0.01 Improper angles (o±SD) 0.15 ± 0.02
Average atomic r.m.s deviation from the mean structure (űSD) Domain I Residues A16‐T89 (N, Cα, C atoms) 0.44 ± 0.12 aSecondary structure (N, Cα, C atoms) 0.37 ± 0.09 Residues A16‐T89 (all heavy atoms) 0.99 ± 0.12 Domain II Residues D97‐K164 (N, Cα, C atoms) 0.78 ± 0.25 bSecondary structure (N, Cα, C atoms) 0.55 ± 0.14 Residues D97‐K164 (all heavy atoms) 1.42 ± 0.27 Domain III Residues H171‐L237 (N, Cα, C atoms) 0.66 ± 0.11 α cSecondary structure (N, C , C atoms) 0.63 ± 0.10 Residues H171‐L237 (all heavy atoms) 1.30 ± 0.10
Ramachandran Quality parameters (%) Molprobity Residues in favored region of Ramachandran Plot 92.8 Residues in allowed region of Ramachandran Plot 99.1
PROCHECK Most favored regions 90.4 Additionally allowed regions 9.5 Generously allowed regions 0.0 Disallowed regions 0.1
5 aresidues A16-R22, K25-C31, K33-S41, L47-V50, F57-N59, Q64-V65, K68-F75,
Q80-T89. bresidues D97-D117, V130-D139, T150-K164. cresidues H171-L178, E182-
L193, K195-D201, E207-P214, R218-K229, L234-L237.
6 Table S4. Summary of SBDS missense mutations associated with Shwachman-
Diamond syndrome
Mutation Reference N8K (Boocock et al. 2003) E44G K67E I87S R126T R169C
I167M (Nakashima et al. 2004)
R19Q (Shammas et al. 2005) C31W K33E N34I L71P K118N S143L + K148R Q153R R169L
I87T (Makitie et al. 2004)
Y32C (Nicolis et al. 2005)
C84R (Kuijpers et al. 2005)
R175W (Erdos et al. 2006) N121T
P6L C. Bellanné-Chantelot and J. Donadieu, personal communication F57L C119Y, C119R T129A K151N R169L R218Q A154V
A30S M. Schwarz, personal communication
7 Table S5. Impact of SDS-associated mutations
Chemical shift SBDS mutants perturbationa Classb I II III Domain I P6L + – – B N8K + – – B R19Q + – – B F27L * – – A A30S + – – B Y32C * – – A K33E + – – B N34I + – – B E44G + – – A F57L * – – A K67E + – – B C84R * – – A
Domain II K118N – + + B C119R – * * A C119Y – * * A N121T – * * A R126T – + – B T129A – + – B S143W – + – B S143L – + – B K148R – + – B K148T – + – B K151N – + – B Q153R – + – A R169C – + + A R169L – + + A
Domain III R175W – – + B R218Q – – + B
8 a. (–) indicates no chemical shift perturbation compared to WT SBDS; (+), local or global chemical shift perturbation; (*), stability of the domain was compromised leading to unfolding and/or aggregation. b. (A) stability mutants, (B) surface mutants.
9 Table S6. Yeast strains
Name Genotype Source SE1 S288c; MATa/α, lyp1Δ/+, mfα1Δ::MFα1prLEU2/+; AJW can1Δ::MFA1prHIS3/+, his3Δ1/his3Δ1, leu2Δ0/leu2Δ0, ura3Δ0/ura3Δ0, met15Δ0/met15Δ0, sdo1Δ::NatMX4/SDO1, efl1Δ::KanMX4/EFL1 C375 S288c, MATα, can1Δ::MFA1prHIS3, mfα1Δ::MFα1pr AJW LEU2, lyp1Δ, his3Δ1, leu2Δ0, ura3Δ0, met15Δ0, sdo1Δ::NatMX4, TIF6I58T NS0 S288c, MATα, can1Δ::MFA1prHIS3, lyp1Δ, his3Δ1, This study leu2Δ0, ura3Δ0, met15Δ0, efl1Δ::NatMX4, TIF6R61G Y5538 S288c, MATa/α, lyp1Δ/+, mfα1Δ::MFα1prLEU2/+; C. Boone can1Δ::MFA1prHIS3/+, his3Δ1/his3Δ1, leu2Δ0/leu2Δ0, ura3Δ0/ura3Δ0, met15Δ0/met15Δ0 MATa, CAN1, ade2, trp1, ura3-52, BCY123 + + A. Newman, his3, leu2-3, 112, pep4::his+, prb1::leu2 , bar1::HisG , LMB lys2::pGAL1/10-GAL4+
10 Table S7. Primers
Primer Sequence
Hs eIF61-225 5' GGAATTCCATATGGCGGTCCGAGCTTCG
Hs eIF61-225 3' CGGAATTCTCAATTCAGCTTGAAGACACTCTC
Hs EFL1 5' GCGGATCCATGGTGCTCAACAGTTTG
Hs EFL1 3' GCCTCGAGTTAATGGTGATGGTGGTGATGCTTATTTTTGCTGAGTGT
Hs SBDS 5' GGGGGATCCATGTCGATCTTCACCCCCACCAACCAGATCCGCC
Hs SBDS 3' GCGGATCTGGTTGGTGAGGGTGAAGATCGAC
Sbdsfl/+ geno 5' TGAGTAATCCTGTGCCCAGAGTTC
Sbdsfl/+ geno 3' TGGAAATCGCAGCAATCCTC
Sbds- geno 5' AAAACAAGGTCGTCGGCTGG
Sbds- geno 3' CCCCTCAAACAGGACAACCAAC
11 Supplemental Materials and Methods
Mouse gene targeting
The mouse genomic Sbds locus was PCR-amplified from mouse 129 DNA. Targeting vector pMCTV1 was generated by inserting the positive selection FRT/Neo/FRT cassette into intron 2 of Sbds, with one loxP site placed in intron 1 and a second in intron 2, 3’ to the selection cassette. Exposure to Cre recombinase results in deletion of exon 2 and the selection cassette. G418-resistant ES cell clones were screened by filter hydridization using two distinct mouse genomic probes 5’ and 3’ to the targeting vector. Targeted clones were verified as having a single integration using a neomycin resistance gene fragment. Male chimeras were generated by injection of an ES cell clone into C57BL/6J blastocysts and germline transmission verified by filter hybridization.
Activation of the pMx1-cre transgene was achieved by intraperitoneal injection of 250
µg poly(I:C) (Invivogen) in saline three times per week. Following euthanasia, livers were dissected, frozen on dry ice and stored at -80°C. Samples of liver were fixed in
10% neutral buffered formalin, embedded in paraffin, sectioned and stained with haematoxylin and eosin. Images were acquired with a Leica DMR microscope and
JVC 3CCD KY-F75U digital camera.
Protein expression and purification
The coding sequence for human eIF6 (NP_852133), residues 1-225, was PCR- amplified (from clone IMAGE:2823211) and cloned into pET-28a (Novagen) to generate plasmid peIF6. N-terminal 6xHis-tagged eIF6 was purified by Ni-NTA (GE
Healthcare) affinity chromatography. The coding sequence for human EFL1
(NP_078856) was PCR-amplified (from clone IMAGE:100016131) and cloned into
12 pYES2 to generate plasmid pEFL1.1 that encodes a C-terminal 6xHis-tagged EFL1.
Plasmid pEFL1.1 was transformed into yeast strain BCY123 (provided by A.
Newman) and EFL1 protein expression induced with 2% (w/v) galactose. EFL1 protein was purified using Ni-NTA (GE Healthcare) affinity chromatography and a
Hiload 26/60 Superdex 200 column (GE Healthcare). The coding sequence for human
SBDS (NP_057122) was amplified by RT-PCR from human skeletal muscle RNA
(Clontech) and cloned into a modified pRSETA vector (Invitrogen) to generate plasmid pSBDS that encodes SBDS fused at the N-terminus to a 6xHis/lipoamyl domain/thrombin cleavage sequence. SBDS mutants were generated using the
QuickChange kit (Stratagene). pSBDS was transformed into E. coli C41(DE3) cells and SBDS protein purified by Ni-NTA affinity (GE Healthcare) and a Hiload 26/60
Superdex 75 column (GE Healthcare). For all protein preparations, purity was assessed by SDS-PAGE and the identity of each sample confirmed by mass spectrometry.
NMR sample preparation
Isotopically labeled (15N,13C) samples were grown in M9 minimal medium supplemented with vitamin mix, trace elements and the appropriate nitrogen
15 13 13 ( NH4Cl) and carbon ( C-glucose, U- C6, 99%) sources and purified as described
above. The production of perdeurated samples followed a similar protocol using
2 13 13 H2O-based M9 medium and deuterated C-glucose (U- C6, 99%; 1,2,3,4,5,6,6-D7,
98%). NMR samples at a concentration of 500 µM were prepared in 25 mM
2 phosphate buffer, pH 6.5, 150 mM NaCl, 10% H2O, 1 mM deuterated DTT (d11),
0.05% (w/v) NaN3 and 0.1 mg/ml Pefabloc (Roche). Analytical ultracentrifugation analysis confirmed the monomeric state of the protein under NMR conditions.
13
Spectroscopic measurements
Spectra were recorded at 25°C on Bruker Avance or DRX spectrometers equipped
with 5 mm triple resonance inverse cryoprobes. Backbone resonance assignments
were obtained with TROSY-based three-dimensional experiments (HNCA, HNCO,
HN(CO)CA, HBHA(CO)NH, HNCACB, HNCACO) (Wüthrich 1986; Bax 1994) on
[13C,15N,2H]-labeled samples at 700 MHz 1H frequency for the human SBDS protein and at 750 MHz (European NMR Large-Scale Facility, Utrecht, Holland) for the yeast
SBDS protein. Backbone resonance assignments were confirmed by a HN(CAN)NH spectra (Weisemann et al. 1993). Side-chain resonance assignments were completed
1 13 1 2 using two-dimensional H, C-HSQC in H2O-based buffer and 100% H2O and three- dimensional HCCH-TOCSY, HCCH-COSY, (H)CC(CO)NH and H(CCCO)NH.
NOESY experiments (two-dimensional 1H, 1H-NOESY, three-dimensional 1H, 1H,15N-
1 1 1 13 1 13 13 NOESY in H2O-based buffer and three-dimensional H, H, C- and H, C, C-
2 NOESY in 100% H2O) were recorded at 900 MHz (European NMR Large-Scale
Facility, Utrecht, Holland). The mixing time in all NOESY spectra was 90 ms. 14% of the Hβ resonances were assigned stereospecifically using an HNHB spectrum. 95% of valine Hγ and 84% of leucine Hδ resonances were assigned stereospecifically using a 10% fractionally 13C-labeled sample of human SBDS protein. Spectra were
1 internally referenced to the H2O signal at 4.70 ppm, processed with TopSpin version
2.1 (Bruker), and analyzed with SPARKY (Goddard TD & Kneller DG, SPARKY 3,
University of California, San Francisco).
14 Structure calculation
Structures were calculated using a standard torsion angle dynamics simulated
annealing protocol with the program CNS (Brunger et al. 1998). Intensities of cross- peaks from within regions of known secondary structures were used for calibration
α purposes. Backbone φ and ϕ angular constraints were determined from the HN, H ,
13Cα, 13Cβ, 13CO and 15N chemical shifts using the program TALOS (Cornilescu et al.
1999). A total of 36 structures out of 50 were accepted where no distance violation was > 0.3 Å and no angle violations were > 5º. The quality of the structure was analyzed with Molprobity (Lovell et al. 2003) and PROCHECK (Laskowski et al.
1996).
Backbone relaxation measurements
All relaxation measurements were performed on [2H,15N]-labeled human and yeast
SBDS at 700 MHz and 25°C, using standard Bruker pulse programs. Ten experiments
15 were recorded to measure N R1, with delays of 32, 56, 88, 136, 248, 376, 488, 776,
1384 and 2000 ms. The time courses were acquired in randomized order, and decaying peak intensities were fit using CurveFit (Arthur G. Palmer, III, 1998). For
{1H}15N-heteronuclear NOE measurements, two pairs of interleaved spectra were
recorded with and without 1H saturation, achieved by applying 120˚ pulse every 5 ms
15 for 7 s. Back-calculated values for N R1 were obtained at 700 MHz and 25°C using
the program HYDRONMR (Garcia de la Torre et al. 2000) and the human SBDS
coordinate file.
Antibodies
15 Rabbit polyclonal antibodies were raised against human SBDS (amino acids 236-250) and human EFL1 (residues 1106-1120) and affinity purified. Other antibodies used for immunoblotting were anti-Rps14 (Aviva Systems Biology, #ARP40321), anti-
Rpl28 (ProteinTech Group, #16649), anti-eIF6 (Sigma, #GW22375), anti-Nmd3
(ProteinTech Group, #16060), anti-Ebp1 (ProteinTech Group, #ab33613), anti-Nog1
(ProteinTech Group, #13897), anti-Npm (Abcam, #ab15440), anti-Gapdh (Sigma,
#G9545), anti-Histone H3 (Abcam, #ab1791). Antibody to human Lsg1 was a kind gift from Emmanuel Reynaud, University College Dublin, Ireland. Antibody to human P0 was a kind gift of Marek Tchórzewski, Maria Curie-Skodowska University,
Lublin, Poland. Secondary HRP-conjugated antibodies were obtained from Sigma.
Peptide separation, mass spectrometry and database analysis
Digested peptide mixtures were subjected to LC-MS/MS using an LTQ Velos-
Orbitrap MS (Thermo) coupled to a Ultimate RSLCnano-LC system (Dionex) fitted with an Acclaim PepMap 100 column (C18, 3 µm, 100 Å) (Dionex) with an internal diameter of 75 µm and capillary length of 25 cm. A flow rate of 350 nL/min was used with a solvent gradient of 5% B to 50% B in 57 min. Solvent A was 0.1% (v/v) formic acid and aqueous 80% (v/v) acetonitrile in 0.1% (v/v) formic acid was used as solvent B. The mass spectrometer was operated using an Nth order double play method to automatically switch between Orbitrap-MS and LTQ Velos-MS/MS acquisition. Survey full scan MS spectra (from m/z 400 to 1600) were acquired in the
Orbitrap with resolution R= 60,000 at m/z 400 (after accumulation to a target of
1,000,000 charges in the LTQ). The method used allowed sequential isolation of the
20 most intense ions for fragmentation in the linear ion trap, depending on signal intensity, using CID at a target value of 5,000 charges. For accurate mass
16 measurements the lock mass option was enabled in MS mode and the
polydimethylcyclosiloxane (PCM) ions generated in the electrospray process from
ambient air were used for internal recalibration during the analysis. Target ions
already selected for MS/MS were dynamically excluded for 60 s. General mass
spectrometry conditions were: electrospray voltage, 1.76 kV with no sheath or
auxiliary gas flow, an ion selection threshold of 1000 counts for MS/MS, an
activation Q-value of 0.25, activation time of 30 ms, capillary temperature of 250°C and an S-Lens RF level of 60% were also applied for MS/MS. Raw files were processed using Proteome Discoverer v.1.1 (Thermo). Processed files were searched against the SwissProt mouse database using the Mascot search engine version 2.3.0.
Possible structure modifications allowed were carbamidomethyl cysteine as a fixed
modification. Oxidized methionine, deamidation of asparagine and glutamine,
phosphorylated serine, threonine and tyrosine were searched as variable
modifications. Searches were done with tryptic specificity allowing up to 2 mis- cleavages and a tolerance on mass measurement of 7 ppm in MS mode and 0.6 Da for
MS/MS ions. Phosphorylation sites assigned by Mascot were cross-checked against
the fragmentation data in Proteome Discoverer.
17 Supplemental References
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20