Finch, AJ et al., Supplemental Material Supplemental Figures a. d

20 Pages·2011·0.51 MB·English

by  Alan Warren

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

cSecondary
structure
(N,
Cα,
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