1
Figure S1: In vitro validation of home-made rabbit anti-P11, anti-P9 antibodies probed against recombinant
MBNL1 proteins. (a) Position of the MBNL1 sequences corresponding to the two synthesized P9 and P11 peptides,
produced according to the protocol summarised in the Materials and Methods section. Highlighted in dark grey:
exon-5-amino acids making up the P9 peptide sequence, present only in Ex5-MBNL142-43 isoforms. Highlighted in
light grey: exon-6-amino acids making up the P11 peptide sequence, commune to all the MBNL1 isoforms. The
proline-rich motifs are highlighted in the boxes. (b) WB analysis of recombinant MBNL1 isoforms 40-41-42-43
tested for anti-P9 and anti-P11 antibodies. Clear specificity of anti-P9 against only exon-5-included MBNL142-43
isoforms and absence of reaction with ΔEx5-MBNL140-41. As expected, anti-P11 antibody recognizes all the
MBNL1 isoforms. The smear bands are products of degradation, due to the instability of diluted recombinant
proteins. (c) Titre of the novel anti-P9 and -P11 antibodies. Dot blot analysis of different dilutions of recombinant
MBNL141 and MBNL143 isoforms probed with anti-P11, -P9 antibodies and a reference monoclonal antibody
against exon 3 (generous gift of Dr. Holt). Boxes with *: the proteins MBNL1 41 and MBNL143 were mixed with
10µM P11 and P9 peptide, respectively, to test the antibody’s specificity. Anti-P9 antibody has a 10-fold increased
titre toward MBNL143 compared to anti-P11 antibody. Similar results were obtained for MBNL140 and MBNL142
(data not show).
2
Figure S2: In vivo validation of home-made rabbit anti-P11, anti-P9 antibodies. (a) Stable rhabdomyosarcoma
clones transfected with an empty vector (RD EV), or overexpressing MBNL141 (RD41) or MBNL143 (RD43), were
established. The nuclear fraction of rhabdomyosarcoma (RD) cells (20 µg) was tested for anti-P9, -P11 and
reference monoclonal antibody by WB. Anti-P9 cross-reacted only with the Ex5-MBNL142-43 isoforms, which,
when extracted from cells, run at the apparent molecular weight (M r) of 50 kDa, whereas anti-P11 recognised
various bands between 40 and 50 kDa. (b) WB analysis of the nuclear fraction from normal (C) and DM1 muscle,
probed with anti-P9 and anti-P11 antibodies. Only one band, running at an apparent Mr of 50 kDa, was found P9positive, whereas a few bands were P11-positive, most of them running at an apparent Mr of 40 kDa. (c) To exclude
a cross reactivity of anti-P9 antibody with other muscle nuclear proteins, immunoprecipitation (IP) and mass
spectrometry (MS) were performed on the nuclear fraction of human muscle and RD43. Proteins were separated in
10% SDS-PAGE and stained with Coomassie blue. Representative IP images of two independent experiments: lane
1: total lysate of muscle tissue (50 µg); lane 2: immunoprecipitated proteins of muscle with anti-P9 (55 µl), three
bands were cut and analyzed by MS; lane 3: molecular mass markers; lane 4: total lysates of RD cells
overexpressing MBNL143 (RD43) (50 µg); lane 5: immunoprecipitated proteins of RD43 with anti-P9 (55 µl), two
3
bands were cut and analyzed by MS. The MS analysis identified MBNL142-43 in the immunoprecipitate of both
samples. (d). WB of the same samples as in (c), probed with anti-P9 antibodies. The IP proteins from muscle (5 µl)
(lane 2’) and RD43 (5 µl) (lane 5’) both run at apparent MW of 50 kDa. In both 50 kDa IP bands, MBNL142-43 were
identified by MS. The anti-P9 positive band, with apparent Mr of 50 kDa, suggests the occurrence of in vivo posttranscriptional modifications of MBNL142-43, which increase its Mr by 8 kDa.
Figure S3: In trans effect of the MBNL140-41-42-43 protein isoforms on the splicing of IR exon 11 and hc-TNT
exon 5 minigenes. The different MBNL1 isoforms were co-expressed in rhabdomyosarcoma cells in the presence
of minigenes containing IR exon 11 (Minigene B) (a) or hc-TNT exon 5 (RTB300) (b). Cells were lysed 48 h after
transfection, and splicing products were assayed by RT-PCR using primers located on the minigenes sequences
(See Supplemental Table 1). All analyzed MBNL1 isoforms act similarly, promoting inclusion of IR exon 11 and
skipping of hc-TNT exon 5. Results are from at least three independent experiments; data are expressed as indicated
in the Methods section.
4
Figure S4: SPR studies of the interaction of human MBNL143 and the Lyn kinase SH3 domain. (a) The
sensorgram shows the binding of the SH3 domain to MBNL1. Relative units (RU) are plotted as a function of time
(in s). The experiment was conducted as described in the Methods section. (b) A plot of the response as a function
of the SH3 domain concentration was used to estimate the dissociation constant. The diagram is the result of
averaging several analysis. Both the experiments confirm the interaction of the Lyn kinase SH3 domain with human
MBNL1 isoform 43 but not with isoform 41. The dissociation constant for the MBNL1 isoform 43, estimated with
this method, is approximately 120 μM.
5
Wb: Lyn
e
at
s
Ly
i
le
c
Nu
M
c
ito
dr
n
ho
ia
s
ro
c
i
M
om
es
s
to
y
C
ol
293T cells
293T cells
+ 7.5µg pCMV6-XL4/Lyn
293T cells
+ 20 µg pCMV6-XL4/Lyn
Hela cells
Figure S5: Standardization of the amount of Lyn-encoding plasmid transfected into 293T cells. Western blot
analysis of total lysate and various cellular fractions of not transfected 293T cells, which express a low basal level
of SFKs, and transfected with 7,5 µg and 20 µg of Lyn-encoding plasmid (pCMV6-XL4/Lyn), and HeLa cells,
expressing a normal basal level of SFKs. As shown, the 293T cells, transfected with a lower amount of Lynencoding plasmid, expressed Lyn at the comparable level to HeLa cells.
6
Supplemental Table 1: List of oligo (primers and siRNAs) names and sequences used in this study.
Sequence (5’-3’)
Oligo name
MBNL1-2F
Oligo name
MBNL1-10R
MBNL1-2F
MBNL1-F
MBNL1-10R
MBNL1-R
MBNL1-F
MBNL1-Ex4-6
MBNL1-R
MBNL1-Ex8
MBNL1-Ex4-6
MBNL1-Ex3-4
MBNL1-Ex8
MBNL1-Ex4-5
MBNL1-Ex3-4
βactinSBR-F
MBNL1-Ex4-5
βactinSBR-R
βactinSBR-F
PSG5-F
βactinSBR-R
PSG5-R
PSG5-F
TB300-F
PSG5-R
RTB300-R
TB300-F
siMBNL1-Ex5 sense
RTB300-R
siMBNL1-Ex5 antisense
siMBNL1-Ex5 sense
siMBNL1-Ex5 antisense
7
Supplemental Experimental Procedures
MBNL1 recombinant protein
The clones of MBNL1 full-length isoforms 40, 41, 42 and 43 were amplified and inserted into the
expression vectors pET28a (MBNL140 and MBNL143) or pQE50 (MBNL141 and MBNL142) using
standard techniques. The various ORFs were inserted in order to obtain a recombinant protein with a
hexa-histidine tag at both N- and C-terminus (plasmid pET28a), or with a single hexa-histidine tag at the
C-terminus (pQE50). The obtained constructs were sequenced and used to transform E. coli expression
strains BL21(DE3) (MBNL140 and MBNL143) or SG (MBNL141 and MBNL142). Protein expression was
induced at an OD600 of about 0.8 by the addition of 0.25 mM IPTG and allowed to proceed for 16 h at
20°C. All the isoforms were expressed as inclusion bodies, and purified under denaturing conditions in 7
M urea using a HisTrap FF column (GE Healthcare, Little Chalfont, UK) according to the manufacturer’s
instructions. Fractions containing the target recombinant proteins were checked by SDS-PAGE, pooled
and quantified. His-tagged MBNL1 was then diluted to about 1 mg/ml in 7 M urea and refolded by a twostep protocol involving a 1:10 dilution in 20 mM Tris/HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM 2mercaptoethanol and 50 μM ZnCl2 and a successive overnight dialysis against the same buffer without
ZnCl2. Refolded protein samples were centrifuged at 15,000g for 30 min to remove aggregates and
precipitated proteins at 4°C.
Morphological analysis
Immunofluorescence analysis. Primary myoblasts from DM1 patients and control subjects were grown
as monolayers on cover glasses in a humidified 5% (v/v) CO2 atmosphere at 37°C. Then, 48 h after
transfection with plasmids expressing different MBNL1 protein isoforms, cells were fixed with 4%
paraformaldehyde for 20 min at room temperature, permeabilized with 0.3% Triton X-100 in PBS. After
blocking with 3% BSA in PBS, the cells were incubated overnight at 4°C in presence of the primary
antibodies. The MBNL1 isoforms cloned into the pEF1-Myc/HisA expression vector were detected using
an anti-cMyc antibody (sc789, Santa Cruz Biotechnology, Santa Cruz, CA) while those cloned into the
pEF4-V5/His A were detected using an anti-V5 antibody (R960-25, Invitrogen). After washing, the
coverslips with pEF1-Myc/HisA were exposed to DyLight 549 labelled secondary antibodies
(ThermoFisher Scientific, Waltham, MA), while the cells transfected with pEF4-V5/HisA were exposed
to AlexaFluor 488-labelled secondary antibodies (Molecular Probes, Eugene, OR) for 1 h. Similar
processing was carried out on 293T cells co-transfected with plasmids expressing the various MBNL1
protein isoforms and Lyn tyrosine kinase. MBNL1 isoforms and Lyn localization was visualized by using
P9 and P11 antibodies and anti-Lyn antibody (sc-15, Santa Cruz Biotechnology). The samples, mounted
in Vectashield medium containing 0.1 mg/ml DAPI (Vector Laboratories, Burlingame, CA, USA), were
8
observed using an Axioplan 2 fluorescence microscope (Zeiss, Thornwood, NY) or a TCSP5 confocal
microscope (Leica, Weltzar, Germany).
RNA fluorescence in situ hybridisation (RNA-FISH). Muscle sections and myoblasts, grown on
coverslips, were fixed in 4% paraformaldehyde, 10% acetic acid in PBS for 15 min at 4°C, permeabilized
in 0.2% Triton X-100 in PBS for 5 min at room temperature. The samples were then pre-hybridised with
2X SSC (300 mM NaCl, 30 mM sodium citrate, pH7) plus 50% formamide and hybridized at 37°C for 4 h
in a moist dark chamber with 30 ng/ml Cy3-(CAG)10 oligonucleotide probe in 40% formamide, 10%
dextran sulphate in 2X SSC. Stained tissue and myoblasts were washed twice in 2XSSC for 10 min at
room temperature, then mounted in Vectashield (Vector Laboratories) with 0.1 mg/ml DAPI and observed
with a TCSP5 confocal microscope (Leica) collecting 10 µm z-stacks.
P9 and P11 antibody production and validation
Peptide
synthesis.
We synthesised two peptides, corresponding to amino acids 270-288
(TQSAVKSLKRPLEATFDL; this epitope is encoded by exon 5 of MBNL1 gene), hereafter defined as P9,
and 294-308 (LPPLPKRPALEKTNG, encoded by exon 6 of MBNL1 gene), hereafter defined as P11, of
the human MBNL143 protein (Figure S1A) with an extra cysteine at the C-terminus, in dark in the Figure
S1A. This was performed using Fmoc-based solid-phase procedures (Fields and Noble, 1990) with a fully
automatic parallel peptide synthesizer (Model Syro II, MultiSynTech GmbH, Witten, Germany) on preloaded Wang resins (Merck Chemicals, Nottingham, UK). Coupling reagent was 2-(7-Aza-1Hbenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU). Peptide cleavage was
performed
by
reacting
for
2.5
h
the
peptidyl-resins
with
a
mixture
containing
TFA/H2O/thioanisole/ethanedithiol/phenol (10 ml/0.5 ml/0.5 ml/0.25 ml/750 mg). The crude peptides
were purified by preparative reverse-phase HPLC. Molecular masses of the peptides were confirmed by
mass spectrometry on a MALDI TOF-TOF mass spectrometer (model 4800-Applied Biosystems, Foster
City, CA). The purity of the peptides was in the range 95-98% as evaluated by analytical reverse-phase
HPLC.
Rabbit immunizations and purification of anti-MBNL1 peptide antisera. Antibodies were raised in
New Zealand rabbits against the synthetic peptides P9 and P11. The animals were injected intradermally
with 500 μg of peptide-keyhole limpet haemocyanin (KLH) conjugates emulsified in Freund’s complete
adjuvant, followed every two weeks by three booster doses diluted 1:1 in Freund’s incomplete adjuvant.
Antibodies were purified by affinity chromatography using a column consisting of 2 mg of synthetic
peptide coupled to 2 ml of iodoacetyl-activated agarose resin (Sulfo Link Coupling Gel, Pierce,
Northumberland, UK) according to the manufacturer’s instructions. Antisera were diluted 1:1 in PBS and
passed through a 0.25-μm pore-size syringe filter prior to loading onto the affinity resin. Anti-P9 and -P11
9
antibodies were eluted with 0.1 M glycine (pH 2.2), neutralized with Tris/HCl buffer, and dialyzed against
PBS.
Antibody validation. Anti-P9 and anti-P11 antibodies were validated for their specificity and titre
towards different MBNL1 isoforms by immunoblot and dot blot against recombinant MBNL1 proteins.
As shown in Supplemental Figure S1B, anti-P11 recognised isoforms 40, 41, 42 and 43, whereas anti-P9
antibody recognised only the longer isoforms 42 and 43 that contain the epitope encoded by exon 5. Dotblot analysis (Figure S1C) confirmed the specificity of anti-P9 MBNL143 and demonstrated a higher titre
of anti-P11 for MBNL141 and MBNL140 (not shown) than for MBNL143 and MBNL142 (not shown).
Immunoprecipitation and protein identification. MBNL142-43 immunoprecipitation was carried out on
samples lysed in RIPA buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA
pH 8.0) supplemented with protease and phosphatase inhibitors, followed by incubation on ice for 30 min,
then sonicated and centrifuged at 16,000 g for 10 min. An aliquot of the supernatant, corresponding to 1
mg of total protein, at the concentration of 2 µg/µl, was incubated overnight at 4°C with 100 µl of
Dynabeads (Invitrogen) coated with 2 µg of anti-P9 [by mixing the antibody for 1 h at 4°C on a spinning
wheel and washing 2 times with PBS:RIPA (10:1)]. The Dynabeads-antigen-antibody complexes, washed
twice with PBS, were added with 25 µl of glycine buffer (0.2 M glycine pH 2.5) for 2 min on ice. The
immunoprecipitated proteins were recovered in 50 µl final volume after removal of the beads by magnetic
force (twice for 2 min). One tenth of the immunoprecipitate was used for immunoblotting, the remainder
for Coomassie staining after SDS-PAGE. Gel bands from SDS-PAGE were excized and submitted for
protein identification. Bands were in-gel digested with trypsin and the resulting peptides analyzed by LCMS/MS on a 6520 Series Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) mass spectrometer
(Agilent Technologies, Santa Clara, CA) or, alternatively, on a LTQ Orbitrap XL ETD Spectrometer
(ThermoFisher Scientific, Waltham, MA). MS/MS spectra were analyzed with the Mascot search
algorithm (Matrix Science, London, UK) for protein identification against the IPI Human Database, using
the following parameters: trypsin as digesting enzyme with one missed cleavage allowed,
carbamidomethylation of cysteine as fixed modification, and methionine oxidation as variable
modification. Peptide tolerance was set at 10 ppm. Proteins were positively identified in at least 2 peptides
with significant score (p<0.05).
Biochemical analysis
Interaction assays. 0.2 g of one of the MBNL140-41-42-43 proteins were incubated with 0.1 g of Src or
Lyn in the absence or presence, respectively, of the synthetic Pro-rich peptide (KGGRSRLPLPPLPPPG),
prepared according to Weng (Weng et al., 1994), or of the recombinant Glutathione S-transferase (GST)fusion form of the SH3 domain of Lyn (GST-Lyn SH3 domain), expressed and purified according to
Takemoto (Takemoto et al.,1996), for 10 min at 30°C and then subjected to immunoprecipitation assays.
10
Immunoprecipitation. Src and Lyn kinases were immunoprecipitated either as purified proteins in
interaction assays, or from lysates of transfected cultured cells. Immunoprecipitation was performed in the
absence or presence of the GST-Lyn SH3 domain, or, when required, with the Pro-rich domain for 2 h at
4°C with the appropriate antibodies in competition assays. The resulting immunocomplexes were
recovered by incubation for 1 h with protein A/G-Sepharose previously saturated with BSA and washed
three times with 50 mM Tris/HCl, pH 7.5, 1 mM orthovanadate, phosphatase and protease inhibitor
cocktails. Samples were then subjected to WB analysis with the appropriate antibodies.
Cell lysis and nuclei isolation. For total lysates: 293T cells or myotubes samples were rapidly lysed in
62mM Tris/HCL buffer, pH6.8, 5% glycerol and 0.5% β-mercaptoethanol containing 0.5% SDS. For
nuclear isolation of 293T cells, 4 x 106 cells for each assay were disrupted on ice by sonication (3 cycles
of 5 s at intervals of 15 s) in 1 ml of isotonic buffer 0.25 M sucrose-TKM (50 mM Tris/HCl, pH 7.5, KCl
25 mM, MgCl2 5 mM), phosphatase inhibitor cocktail 1 and 2 (Roche Diagnostics, Mannheim, Germany),
1 mM sodium orthovanadate, and protease inhibitor cocktail (Sigma, St Louis, MO) and nuclei isolation
was performed as described (Aaronson et al. 1974). Briefly, homogenates were loaded on 200 µl of 2.3 M
sucrose-TKM and centrifuged at 600g for 10 min in a swinging-bucket rotor. After discarding the top
layer, the remaining supernatants were mixed with the cushions to give a final concentration of
approximately 1.6 M sucrose. The homogenous suspensions were then loaded on 300 µl of 2.3 M sucroseTKM and centrifuged for 60 min at 75,000g. The pellets represent the nuclear fractions.
Nuclear and cytosolic fractions from DM1 and control muscle biopsies were obtained by NE-PER
Nuclear and Cytoplasmic Extraction Reagent kit (ThermoFisher Scientific, Waltham, MA), in presence of
protease inhibitors cocktail (3.5% PIC, 2% PIC1 and PIC2; Sigma).
Immunoblot analysis Total lysates or the nuclear and cytosolic protein extracts were electrophoresed in
SDS-PAGE on 7.5-17.5%T 4%C lab-made gradient gels or on 4-12% NuPAGE precast gels (Invitrogen).
Proteins were blotted onto nitrocellulose or 0.45 µm PVDF membranes (Invitrogen) and probed the
appropriate antibodies. Anti-Lyn (sc-15) and anti-Src (sc-19) antibodies (Santa Cruz Biotechnology,
Heidelberg, Germany), anti-phosphotyrosine (p-Tyr) antibody (clone PY-20) (BioSource International,
Invitrogen, Paisley, UK), anti-nuclear Lamin B (Cell Signalling Technology, Danvers, MA), anti-β-actin,
anti-β-tubulin and anti-lactate dehydrogenase (LDH) (ab52488) (Abcam, Cambridge, UK). After
incubation with secondary HRP-conjugated antibodies (Calbiochem-Merck , Darmstadt, Germany),
positive bands were visualised by chemiluminescence (GE HealthCare). Integrated optical density of each
band was calculated with commercial software and normalized with reference to lamin or actin amounts.
11
Supplemental References
Fields,G.B., Noble,R.L.(1990).Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino
acids. Int. J. Pept. Protein Res. 35, 161-214.
Takemoto,Y., Sato,M., Furuta,M., Hashimoto,Y.(1996) Distinct binding patterns of HS1 to the Src SH2
and SH3 domains reflect possible mechanisms of recruitment and activation of downstream molecules. Int.
Immunol. 8, 1699-1705.
Weng,Z., Thomas,S.M., Rickles,R.J. et al.(1994).Identification of Src, Fyn, and Lyn SH3-binding
proteins: implications for a function of SH3 domains.Mol.Cell Biol.14, 4509-4521.
Aaronson,R.P.,Blobel,G.(1974). On the attachment of the nuclear pore complex. J. Cell Biol. 62, 746-754.