Figure legends
Figure 1. Analysis of NF1 pre-mRNA splicing.
(A) Schematic overview of NF1 exon trap constructs derived as
part of this study, showing the approximate location of the tested
variants. Exonic sequences are shown as boxes, intronic sequences as
horizontal lines; intronic sequence representing a pseudo-exon is
indicated by the shaded box. § indicates a variant also subjected to
functional assessment (see Supplementary Table S2). Variant nomenclature
is according to reference transcript NM_000267.3, except ¶: reference
transcript NM_001042492.2.
(B) Approximate location of the NF1 variants investigated with
RT-PCR and Sanger sequencing in patient RNA in relation to the NF1
reference transcript (NM_000267.3).
(C) Exon trap and RT-PCR analysis of the
NM_000267.3(NF1):c.2710T>A variant. Agarose gel
electrophoresis of the NF1 exon 21 wild-type (WT) and
c.2710T>A exon trap RT-PCR products (left) and RT-PCR
products from the index patient and 3 control individuals (center). A
indicates the canonical exon 21 product; B indicates the product
obtained due to the cryptic splice site at c.2706 created by the
c.2710T>A substitution. Electropherogram of Sanger sequence
analysis of the RT-PCR products, confirming the abnormal r.2707_2850del
transcript.
(D) Exon trap and RT-PCR analysis of the
NM_000267.3(NF1):c.3871-3T>G and c.3974+5G>C
variants. Agarose gel electrophoresis of the NF1 exon 29 wild-type (WT)
and c.3871-3T>G, c.3974+5G>C and
c.3871-3T>G + c.3974+5G>C exon trap RT-PCR
products (indicated with the red box). Note the complete loss of the
wild-type product from the constructs containing both substitutions.
Figure 2. In vitro functional assessment of RAS GAP
activity of the NF GRD.
(A) Schematic overview of the pull-down assay for NF RAS GAP activity.
Variants were first introduced into the wild-type NF expression
construct by site-directed mutagenesis (SDM), co-transfected into
mammalian cells in culture, together with a RAS reporter expression
construct and, after 5 minutes stimulation with EGF, the cells were
lysed and GTP-bound RAS subjected to GST-RAF-RBD pull-down with
glutathione-agarose beads. Lysate and pull-down fractions were
subsequently analyzed by immunoblotting.
(B) Schematic overview of NF (above), showing the different proposed
functional domains (CSRD, orange; TBD, green; SPRED1 interaction,
violet; SEC14-PH, cyan; HEAT, pink; SBD, blue) and the NF GRD expression
constructs used in this study (below). Amino acid changes are given
according to reference transcript NM_000267.3. Variants derived in the
NF V5-p.1180_1504 expression construct [Thomas et al., 2012] are
indicated in cyan; other variants were derived from the wild-type NF
p.V5-1180_1504-V5 expression construct.
(C) Relative RAS GAP activity of the NF p.V5-1180_1504 variants. NF
V5-p.1180_1504 variants were co-expressed with HA-H-RAS and RAS GAP
activity was estimated in a pull-down assay using recombinant
GST-RAF-RBD to specifically bind RAS-GTP. The signals for HA-H-RAS in
the pull-down fractions were determined in 3 independent experiments.
The mean estimated RAS GAP activity is shown relative to the wild-type
(V5-GRD); = 1.0). Error bars represent the standard error of the mean.
Variants showing significantly reduced RAS GAP activity are indicated in
red (see main text for details).
(D) Relative RAS GAP activity of the NF p.V5-1180_1504 variants.
Pull-down assay was performed as in (C); RAS GAP activity was estimated
relative to the wild-type control (V5-GRD-V5). Variants showing no
evidence for RAS GAP activity are indicated in red; variants with
reduced RAS GAP activity are shown in orange; active variants are
indicated in black (see main text for details). Quantification data of
at least 3 independent experiments is shown.
(E) Representative immunoblot showing the GST-RAF-RBD pull-down
fractions.
(F) Cell lysate fractions corresponding to the samples shown in (E).
Figure 3. In vitro functional assessment of NF RAS GAP
activity.
(A) Schematic overview of neurofibromin (NF) (top) and the expressed
truncated NF proteins used for the functional experiments (below). The
C-terminal region absent from the expressed NF p.1_2069-myc and
p.420ins10-myc proteins is indicated and the approximate positions of
the different variants are shown, including the 10 amino acid insertion
(420ins10) that differentiates between the two expressed wild-type
proteins. Variants derived in the NF p.1_2069ins10-myc construct are
indicated in cyan; variants derived in the NF p.1_2069-myc construct
are shown in black. To determine the RAS GAP activity of the variants,
the scheme shown in Figure 2A was used. Briefly, NF p.1_2069-myc and NF
p.1_2069ins10-myc variants were co-expressed with HA-H-RAS. RAS GAP
activity was estimated in a pull-down assay using recombinant
GST-RAF-RBD in at least 4 independent experiments, relative to the
wild-type NF p.1_2069-myc or NF p.1_2069ins10-myc (indicated in cyan)
proteins.
(B) Quantification of NF1 variant RAS GAP activity. Variants
showing evidence of disruption of RAS GAP activity (> 50%
reduction in activity; P < 0.05) are indicated in red;
variants with reduced RAS GAP activity are shown in orange (<
50% reduction; P < 0.05); active variants (no evidence
for reduced RAS GAP activity)(P > 0.05) are
indicated in black (see main text for details). IT/RC: NF1
p.Ile1799Thr/Arg1809Cys (double cis variant).
(C) Representative immunoblot showing the lysate (above) and GST-RAF-RBD
pull-down (below) fractions.
Figure 4. Functional assessment of the effects of NF1and SPRED1 variants on NF-SPRED1 interaction and NF expression.
(A) Schematic overview of the in vitro functional assessment of
the NF1-SPRED1 interaction. Variants were introduced into the wild-type
(WT) expression construct by site-directed mutagenesis (SDM) and the NF
p.1_2069-myc or p.1_2069ins10-myc and FLAG-SPRED1 expression
constructs co-transfected into mammalian cells. NF-SPRED1 complexes were
isolated by immunoprecipitation (IP) using anti-FLAG agarose beads.
Lysate and IP fractions were subsequently analyzed by immunoblotting.
Signals for the variants relative to the WT proteins (NF p.1_2069-myc,
NF p.1_2069ins10-myc or FLAG-SPRED1) were determined in at least 3
independent experiments.
(B) Quantification of the IP signals for the NF1 (left) andSPRED1 (right) variants. Variants showing a reduced interaction
(> 50% reduction in signal in the coIP fraction; P< 0.05) are indicated in red; variants showing <
50% reduction (P < 0.05) are shown in orange; variants
showing a comparable interaction to wild-type NF-SPRED1 (P> 0.05) are indicated in black (see main text for details).
IT/RC: NF1 p.Ile1799Thr/Arg1809Cys (double cis variant).
(C) Quantification of the signals for the expressed NF1 (left)
and SPRED1 (right) variants. Variants showing > 50%
reduction (P < 0.05) are indicated in red; variants
showing < 50% reduction (P < 0.05) are shown
in orange; variants expressed at levels comparable to the wild-type
control (P > 0.05) are indicated in black (see main
text for details). IT/RC: NF1 p.Ile1799Thr/Arg1809Cys (double,cis variant).
(D) Representative immunoblot showing the anti-FLAG IP (above) and
lysate (below) fractions for NF1 variants.