Ule et al., Supplementary Fig Legends
Supplementary Figure S1: General scheme to define and validate the RNA map and
relate it to the mechanisms of Nova-dependent splicing regulation.
Prior work established YCAY clusters as legitimate target sequences for analysis, based
on RNA selection, biochemistry/X-Ray crystallography1-7, and new methods for
identification and genetic validation of Nova splicing targets2, 6, 8. In the current paper,
these targets were first analyzed by a computational procedure to define the Nova RNA
map of splicing regulation, which in turn predicted the action of Nova on new splicing
targets (top triangle). The RNA map was related to the mechanism of Nova action
through biochemical studies in a reconstituted splicing system in vitro (circle). Two
examples illustrate Nova inhibition of exon inclusion by binding NESS2 element and
blocking U1 snRNP assembly on the pre-mRNA, and Nova upregulation of exon
inclusion by binding NISE2/3 element to enhance spliceosome assembly. These results
were generalized by quantification of splicing intermediates in Nova dKO brain, which
showed that the RNA map predicts the in vivo site of Nova action in all 19 pre-mRNAs
where we detected an asymmetry in Nova’s action on removal of the two introns flanking
the alternative exons.
Supplementary Figure S2: Analysis of the positions of YCAY motif enrichment in
previously validated Nova-target pre-mRNAs.
a, An outline of four regions within the pre-mRNA that were analyzed to define the RNA
map. The peaks demonstrate the enrichment of YCAY motifs in pre-mRNAs with exons
down-regulated (blue) or upregulated (red) by Nova.
b, YCAY motif frequency was calculated in 60 nucleotide sequences at 20 nucleotide
intervals on the pre-mRNAs containing Nova-regulated exons. Mouse sequences of 34
pre-mRNAs with exons up-regulated by Nova (Nova +), 21 pre-mRNAs with exons
down-regulated by Nova (Nova -) and 3865 pre-mRNAs with control exons (control, not
regulated by Nova, as shown by a previous study9) were analyzed. In addition, for each
60 nucleotide window in Nova-regulated pre-mRNAs, 25 shuffled sequences were
generated to generate over 500 sequences for each analyzed position in pre-mRNAs with
1
Ule et al., Supplementary Fig Legends
exons up-regulated by Nova (Nova + shuffle) and pre-mRNAs with exons downregulated by Nova (Nova - shuffle). Each dot on the graph represents the average values
(within each group) of 60 nucleotide sequences that are located at the same distance from
the splice site. The error value of the control pre-mRNAs was calculated by generating
100 random sets of 20 pre-mRNAs, and calculating standard deviation between the
average values of each set. The diagram below the graph illustrates the part of the premRNA that is being analyzed in the graph. At each significant peak of YCAY enrichment
in Nova-regulated versus control pre-mRNAs, the fold enrichment of YCAY motifs and
p-value (two-tailed t-test, unequal variance) is shown.
Supplementary Figure S1: Analysis of the YCAY cluster characteristics.
The highest score YCAY cluster was found within previously validated Nova regulated
pre-mRNAs (|YCAY cluster score| > 0.5, the clusters sequences are shown in
Supplementary Table S2).
a, Distribution of the YCAY cluster widths (nucleotides from the beginning of the first
YCAY to the end of the last YCAY motif are counted).
b, For each YCAY within the cluster, the distance from the next closest YCAY motif was
measured, and plotted in the distribution of inter-YCAY distance within the clusters. (-1
stands for cluster YCAYCAY, 0 for YCAYYCAY, 1 for YCAYNYCAY, etc.). Majority
of YCAY motifs are 2 or less nucleotides apart, and 90% of are 6 or less nucleotides
apart.
c, Analysis of the YCAY cluster conservation. To analyze the conservation of YCAY
clusters in Nova-target pre-mRNAs, we compared it with conservation of YCAY clusters
located at corresponding positions within control pre-mRNAs (containing alternative
exons not regulated by Nova, as determined by a previous study9). 37 YCAY clusters
with |YCAY cluster score| > 0.5 were found within 200 nucleotides of the alternative
exon or flanking exons in Nova-regulated pre-mRNAs, and 152 were found in 1388
control pre-mRNAs in mouse genome. In control pre-mRNAs, 65% of the exonic YCAY
clusters and 33% of intronic clusters are conserved. Higher conservation of exonic
clusters would be expected due to coding constraint. 97% of the YCAY clusters in Nova-
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regulated pre-mRNAs are conserved, and there is little difference between the
conservation of intronic and exonic clusters, suggesting that the main conservation
constraint to these sequences is their role in binding Nova. Independent analysis of these
exons and their corresponding mouse and human cDNAs suggests that those exons that
contain conserved YCAY clusters are similarly regulated in human; therefore, we
estimate that ~97% of the exons found to be regulated by Nova in mouse are conserved
alternative exons, regulated by Nova also in human pre-mRNAs.
Supplementary Figure S4: Analysis of the predictive accuracy of net YCAY cluster score
at different stages of the algorithm.
The graphs in a-f show analysis of 48 Nova-regulated exons that were identified by
previous studies2, 6, 8, in addition to 1388 control exons. 1388 control exons were a subset
of the 4738 exons that showed little or no splicing change by splicing microarray analysis
of Nova2–/– brain9, where we were able to collect orthologous sequences from all the
regions on the RNA map from zebrafish, Xenopus, chicken, opossum, dog, mouse and
human genomes. I9 (splicing change), as determined using splicing microarray or RTPCR2, 6, 8, is shown on the x axis, and the net YCAY cluster score, calculated at different
stages of the algorithm, on the y axis. A positive net YCAY cluster score predicts Novadependent exon inclusion, and negative score predicts Nova-dependent exon skipping. To
analyze the accuracy of the net YCAY cluster score at each stage, we have analyzed two
values. First, as an estimate for accuracy (regular text in the figure), we determine the
cutoff level where roughly a 25% of the Nova-regulated exons are predicted, and
calculate the percentage of exons predicted above this cutoff that are regulated by Nova
(relative to control exons). Second, as a measure for efficiency (italic text in the figure),
we determine a cutoff level that distinguishes between exons that are up- or downregulated by Nova (that means, a positive cutoff above which no down-regulated exon is
predicted, and negative cutoff below which no up-regulated exon is predicted), and
calculate the percentage of Nova-regulated exons that are predicted above this cutoff
(relative to all Nova-regulated exons).
3
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a, YCAY score was calculated using only the mouse sequence, by subtracting the
maximum value of a cluster in silencer regions from maximum value of a cluster in
enhancer regions. Accuracy is 17% (12/72), and efficiency is 35% (17/48).
b, YCAY score was calculated using only the mouse sequence, by subtracting the
maximum value of a cluster in silencer regions from maximum value of either a single
cluster in enhancer regions, or 2/3 of a sum of maximum clusters in NISE2 and NISE3
regions. Accuracy is 19% (14/74), and efficiency is 42% (20/48). This step exploits a
common feature of Nova targets to contain YCAY clusters both in NISE2 and NISE3
region, and thus increases the number of predicted up-regulated exons, with no effect on
control exons, which rarely contain two YCAY clusters in flanking intron (see
Supplementary Fig. S4b)
c, YCAY score was calculated using the 2x minimum value of YCAY clusters in
orthologous mouse, human and dog sequences. Accuracy is 79% (11/14), and efficiency
is 54% (26/48). This step shows the important of orthologous sequences for the accuracy
of the algorithm, since it increases the ability to distinguish between Nova-regulated and
control exons. It also increases efficiency, since it has a better ability to distinguish
between exons that are up- or down-regulated by Nova.
d, YCAY score was calculated using the average value of YCAY clusters in orthologous
mouse, human and dog sequences + average value of YCAY clusters in orthologous
opossum, chicken, Xenopus and Zebrafish sequence. Accuracy is 69% (11/16), and
efficiency is 63% (30/48). Thus, this average value has a slightly lower accuracy than the
minimum value (in c), but slightly higher efficiency, since minimum value doesn’t
predict exons with clusters in all but one species.
e, YCAY score was calculated using the 2x minimum value of YCAY clusters in
orthologous mouse, human and dog sequences + the average value of YCAY clusters in
orthologous mouse, human and dog sequences + average value of YCAY clusters in
orthologous opossum, chicken, Xenopus and Zebrafish sequence. Accuracy is 76%
(13/17), and efficiency is 63% (30/48). Thus, combining the minimum and average
values gains optimum accuracy and efficiency.
f, The score was calculated as in e, but only using the cluster values greater than 0.6
(values <0.6 were changed to 0). Accuracy is 76% (13/17), and efficiency is 66% (31/48).
4
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This last step removes the background noise, with no negative effect on ability to predict
Nova target exons.
Supplementary Figure S5: YCAY cluster position in individual Nova-regulated premRNAs.
Conserved YCAY cluster score was determined for each 45 nucleotide sequence along
each individual Nova-regulated pre-mRNA. The pre-mRNAs with similar pattern of
YCAY cluster positions were grouped together and plotted onto the same graph.
a, 9 pre-mRNAs containing YCAY clusters both in NISE2 and NISE3.
b, 7 pre-mRNA containing YCAY clusters in NISE2, but not NISE3.
c, 2 pre-mRNAs containing YCAY clusters in NISE3, but not NISE2.
d, 2 pre-mRNAs containing YCAY clusters both in NISS1 and NESS.
e, 3 pre-mRNAs containing YCAY clusters in NISS2 (and NESS)
f, 4 pre-mRNAs containing YCAY clusters in NESS.
Pre-mRNAs containing two YCAY clusters (shown in a and d) were 58 fold more
common in Nova-regulated pre-mRNAs (35%, 9/31) than in control pre-mRNAs with
YCAY clusters (0.6%, 1/158), suggesting that cooperative action via different binding
sites might contribute to Nova-dependent splicing regulation. However, two binding sites
do not seem to be a prerequisite, as evident by cases shown in b, c, e, f (see also
Supplementary Figure S8).
Supplementary Figure S6: Functions of YCAY clusters in the context of consecutive
alternative exons, alternative splice sites or terminal exons.
YCAY clusters predicted 5 cases of Nova-dependent regulation of consecutive alternative
exons (a-c), and 4 cases of Nova-dependent regulation of alternative splice sites (d, Fig.
2d-e).
a, In two pre-mRNAs, a YCAY cluster is located between two alternatively skipped
exons. In both cases, the cluster acts as a NISE2 element to promote inclusion of the
upstream exon (E14 in CP110 and E9 in Bcas1). Notably, E14 of CP110 is by
bioinformatic criteria considered constitutive, since no cDNA or EST in the database
skips E14 and in wild-type brain E14 is rarely skipped. Only analysis of Nova knockout
5
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brain shows that inclusion of this exon requires a splicing factor, suggesting that analysis
of mutants in splicing factors could reveal new regulated alternative exons that do not
appear alternative by bioinformatic criteria. In both CP110 and Bcas1, the YCAY cluster
also acts as NISS1 to promote an isoform that skips the downstream exon (it promotes
isoform E14-E15 in CP110, skipping E14a, and isoform E9-E11 in Bcas1, skipping E10).
However, there is a crucial difference between the two RNAs. In case of CP110, splicing
of E14 to E14a is very inefficient (see Supplementary Fig. 14g), therefore the main action
of the cluster is to silence E14a inclusion (as NISS1) by promoting splicing of E14 to
E15. On the other hand, Nova can promote splicing of E9 to E10 in Bcas1 (see
Supplementary Fig. 14a), therefore the main action of the cluster is to enhance E10
inclusion.
b, The cases of Sh2bpsm1 and Map4 demonstrate the specificity of Nova’s action at
YCAY clusters, because only the exon in the vicinity of NISS2 element is regulated, but
not the alternative splice sites of the flanking exons.
c, The case of Dab1 exons 7b and 7c shows that Nova can silence two consecutive
alternative exons when both contain a NISS2 element.
d, The ability of RNA map to predict Nova’s action on alternative splice sites (NISE2
enhances and NESS1 silences a splice site) suggests local action of Nova via YCAY
clusters.
e, Two examples of Nova-dependent regulation of terminal exons predicted by the RNA
map. All bands were confirmed by sequencing.
Supplementary Figure S7: Position of NISS2, NISE1 and NISE3 elements relative to
putative branch sites.
The sequences upstream of the 3’ splice site containing NISE1, NISS2 or NISE3
elements in different Nova-target pre-mRNAs were aligned between mouse, human and
opossum pre-mRNAs. YCAY motifs were colored in purple, and conserved YCAY
clusters were labeled by purple rectangles.
a, NISS2 elements are located within 5 nucleotides, or downstream of the conserved
branch site (labeled by blue rectangle).
6
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Two examples of NISS2 elements are the YCAY clusters present upstream of the
alternative exons 7b and 7c (termed 555* in a previous study10) of Dab1 RNA. In human,
mouse and opossum, these two exons are ~50 bp long, separated by an intron of ~90 bp.
The peptide sequences encoded by the two exons are highly related, suggesting a
duplication event during evolution10. This observation agrees with finding that chick and
lizard mRNAs contain only the second of the two exons10. Interestingly, these species
also lack one of the NISS2 clusters, suggesting that YCAY clusters have co-evolved with
the exons 7b and 7c. Exons 7b and 7c are included in mRNA of proliferating neuronal
precursors, but are absent from mRNA of differentiated neurons, with silencing starting
in embryonal day 12 brain10. Chick retinal cultures transfected with a neuronal Dab1
isoform (lacking exon 7b and 7c, and containing another neuron-specific exon) were able
to form elongated processes and activate Src family kinases, whereas transfection of the
isoform containing exons 7b and 7c had no effect11. This suggests that exons 7b and 7c
might encode an inhibitory region. We observe that sequence of YCAY clusters (present
upstream of exon 7b and 7c) is more conserved than sequence of exons 7b and 7c,
agreeing with the possibility that these exons inhibit Dab function, therefore the main
evolutionary pressure acts to preserve ability of Nova to silence their inclusion in brain.
b, NISE1 elements are located 13 or more nucleotides upstream of the conserved branch
site (labeled by red rectangle). Note that in case of Ank3, the YCAY cluster is located at
a position where it can either act as NISS1 to silence proximal branch site, or as NISE1 to
enhance the distal branch site.
c, NISE3 elements are generally located upstream of the conserved branch site (labeled
by black rectangle). In the three cases shown, there is another conserved branch site
located 5-8 nucleotides downstream of the YCAY cluster; the significance of this second
branch site is unknown.
Supplementary Figure S8: Three examples of single YCAY cluster within short introns
acting as NISE2 or NISE3.
Even though two YCAY clusters in NISE2 and NISE3 are a common feature of Novadependent exon inclusion, two clusters do not seem to be a prerequisite. Three examples
are shown here of the whole intronic sequence with a single YCAY cluster. Two introns
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are very short, containing a single YCAY cluster roughly in the middle of the intron (Falz
and Plcl3). The third example (Lrp1b) has a clear NISE2 element, but no YCAY cluster
close to the distal part of the intron (no NISE3).
Supplementary Figure S9: Correlation of YCAY cluster location with exon size, or with
the location of a putative exonic splicing enhancer.
a, 56% of exons regulated by Nova via NISE2 elements are shorter than 50 nucleotides,
whereas only 14% of the rest of the Nova-regulated exon is as short. The distribution of
Nova-regulated exon sizes that are flanked by NISE2 elements is significantly different
from the rest of the Nova regulated exons (p=0.003, two-tailed t-test, unequal variance).
b, The most common pentamer in the 9 NESS elements with highest YCAY cluster score
is CACCA. The sequences of the 9 NESS elements are shown, with the CACCA motif
marked in red, and the flanking YCAY motifs in blue.
Supplementary Figure S10: Bioinformatic analysis of Nova-regulated exons.
a,
Analysis
of
splice
site
scores,
as
determined
by
MaxEnt
(http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq_acc.html)
score12
shows
no
significant difference in splice site scores of Nova-regulated exons as compared to
constitutive exons in the same RNAs.
b, Analysis of the number of enhancer and silencer elements/100 nucleotides of exonic
sequence,
as
determined
by
RESCUE-ESE
and
FAS-ESS
(http://hollywood.mit.edu/Dexon.php)13 shows a 22% decrease in the average density of
enhancer elements in Nova-regulated exons as compared to constitutive exons in the
same RNAs. However, neither the difference in the number of predicted enhancer nor
silencer elements is significant.
c, Analysis of the conserved alternative exon score as predicted by ACEScan
(http://hollywood.mit.edu/Dexon.php)13 shows a significant difference in the score of
Nova-regulated exons as compared to constitutive exons in the same RNAs. The ability
of ACEScan to predict that Nova-regulated exons are conserved alternative exons might
be due to long regions of conserved intronic sequence flanking Nova-regulated exons,
which is a characteristic of conserved alternative exons12.
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Supplementary Figure S11: Analysis of 10 alternative exons with net YCAY score near
zero.
An independent analysis of 10 alternative exons with net YCAY score near zero revealed
that none were regulated by Nova (comparing Nova2–/–/Nova1–/– vs. wildtype brain).
Supplementary Figure S12: Recombinant Nova represses the inclusion of exon 4 in vitro.
a, Schematic of the substrate used (E-I-EYCAY-I-E): exons 1 and 3 are derived from
human -globin; the lines and block in gray represent introns 3 and 4 and exon 4 of
Nova1, respectively. Each vertical red line represents a YCAY motif.
b, Purity of recombinant Nova1 employed in the present study: tagged Nova1 protein was
purified by two affinity steps (Nickel column and anti-T7 monoclonal antibody resin). An
aliquot of purified protein was separated by electrophoresis on a SDS polyacrylamide gel
(5-20%) and visualized by Coomassie blue staining.
c, In vitro splicing assay: Radiolabeled E-I-EYCAY-I-E (lanes 2-5) and E-I-EYAAY-I-E
(lanes 6-8) RNA substrates were incubated in the absence (lane 2) and presence of ATP
(lanes 3 to 8) in the presence of HeLa nuclear extract in splicing conditions. Recombinant
Nova1 was included in lanes 4 and 7 (0.06 µM) and 5 and 8 (0.12 µM). After 4 hours of
incubation, RNA was extracted and separated by electrophoresis on a denaturing
polyacrylamide gel. The gel was dried and RNA visualized by Phosphoimager.
d, Reproducibility of Nova action on EYCAY-I-E RNA in vitro. This experiment is a
separate reproduction (from a total of three similar experiments) of Figure 3a. EYCAY-I-E
(lanes 1 to 5; vertical red lines in cartoon represent YCAY motifs) or EYAAY-I-E (lanes 6
to 10) RNA was incubated in nuclear extract in absence of ATP (lanes 1 and 6) or in
splicing conditions (lanes 2 to 5 and 7 to 10). Recombinant Nova1 was included in
concentrations ranging from 0.3 to 0.12 µM (lanes 3 to 5 and 8 to 10). After incubation,
RNA was extracted and analyzed on denaturing polyacrylamide gel, which was then
dried and exposed by Phosphoimager.
e, UV-cross-linking assay. Radiolabeled RNA substrate I-EYCAY-I (1 nM) was incubated
in HeLa nuclear extract without or with Nova (0.12 µM), treated with UV254 followed by
RNAse digestion, separation by SDS-PAGE and autoradiography.
9
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f, Quantitation of co-transfection experiments in 293 cells. Results refer to four
independent experiments; error bars represent standard deviations. While overexpression
of hnRNPK had only a marginal effect on E4 inclusion (11% decrease in exon inclusion,
p <1.4 X 10-5), expression of Nova1, even in the presence of an excess of hnRNPK, led to
E4 exon skipping (80% decrease in exon inclusion, p<1.7 X 10-6).
g, Quantitation of the psoralen-cross-linking assay. Incubation with 0.12 µM Nova results
in a 2.5 folds decreased formation of the U1 snRNA cross-linking product with the
labeled substrate. The mutation of YCAY to YAAY decreases the effect of Nova (1.2
folds reduction).
h, Effects of Nova1 on the formation of the spliceosome. In the presence of Nova1 the
formation of complexes B and C is inhibited (lanes 5 to 8).
i, Analysis of spliceosomal complexes formation on EYCAY-I-E RNA by electrophoresis
on an agarose/polyacrylamide native gel. In the presence of Nova, while complexes B
and C were inhibited, a complex migrating at roughly the same rate as complex A was
still observed. This Nova-resistant complex still formed, although with reduced
abundance, when the first 15 nucleotides of U2 snRNA were digested with RNAse H
(lane 3-4). No discrete complex was observed when nucleotides 28-42 (lanes 5 and 6) or
both 1-15 and 28-42 (lanes 7-8) were digested. Analogously, in extracts that had been
depleted of U2 snRNP, no such complex was observed (lanes 13-14). In contrast, in
extracts that had been depleted of U1 snRNP (lanes 15 and 16) or where nucleotides 1-15
of U1 snRNA were digested (lanes 9-10) a complex migrating with a similar rate as
complex A is still observed, even in the presence of Nova. Summarizing, we observe a
Nova-resistant complex migrating at about the same rate as complex A. U2 snRNP (lanes
13 and 14) and nucleotides 28-42 of U2 snRNA (lanes 5 to 8), which span the residues
required for the recognition of the branch site, are required for the formation of this
complex. The first 15 nucleotides of U2 snRNA (lanes 3 and 4), which are required for
the interaction of U2 snRNP with U6, are not required (lanes 3 and 4). These findings,
together with the observation that this complex still forms in the absence of U1 (lanes 15
and 16) are consistent with the previous descriptions of complexes of similar mobility
forming on splicing substrates where the 5' splice site was mutated or absent (Query et
al., 1997)14. These data are consistent with the localized action of Nova in regulating the
10
Ule et al., Supplementary Fig Legends
recognition of the 5' splice site by U1, without interference with the recognition of the
branch site by U2.
j, The level of digestion of U1 and U2 snRNAs was verified on an ethidium bromide
agarose gel and by Northern Blot of the same gel probed with a mix of labeled
oligonucleotides complementary to U1, U2, U4, U5 and U6 snRNA.
Supplementary Figure S13: Analysis of Nova enhancement of exon inclusion in in vitro
splicing reactions.
a, The in vitro splicing of the substrate E-I-E-IYCAY-E was repeated multiple times using
different preparations of HeLa nuclear extract and recombinant Nova1. Three repeats of
one such experiment are shown, where the substrate (1nM) was incubated in the absence
or in the presence of 60 nM recombinant Nova1. The upper panel is a longer exposure of
the upper part of the gel, showing the two-intron lariats.
b, the ratio of the long (L) and short (S) products of splicing was measured by
Phosphoimaging from the gel shown in panel (a).
c, The identity of the bands in (a) was confirmed by a de-branching assay: RNA was
eluted from the gel, incubated in the presence of buffer D (see methods) or S100
cytoplasmic extract, 8mM EDTA at 30C for 45 minutes, and analyzed by denaturing
PAGE. The de-branched products ("S100" lanes) correspond to the predicted size of the
two introns and the alternative exon.
d, RT-PCR analysis of the in vitro splicing reaction using EE-IYCAY-E as substrate (as
shown in Fig 4b). RNA from the reaction shown in figure 5B was reverse transcribed and
amplified using primers complementary to the first and last exons. In presence of 0.12
µM Nova1, the spliced product normalized by the unspliced precursor increases by 2.1
fold.
e, Psoralen cross-linking on E-IYCAY-E. Body-labeled E-IYCAY-E (1nM) was incubated in
splicing conditions in the absence (lanes 1to 6) or in the presence (lanes 7 to 12) of
ATP/CP. After 25 minutes incubation at 30C, samples were treated with UV336, RNA
was extracted with Trizol, and analyzed on a denaturing gel. When nuclear extract was
omitted (lanes 1 and 7), close to 100% of the RNA migrates more slowly (indicated by
the star sign) than the non-cross-linked RNA (compare 1 to 2, where psoralen was
11
Ule et al., Supplementary Fig Legends
omitted, and 7 to 8), suggesting that most of the substrate folds in a secondary structure
stabilized by psoralen (lanes 1 and 7 are shown from a shorter exposure for clarity,
because the signal in these lanes is much stronger than in the lanes where nuclear extract
was included, due to nuclease activities present in the extract). Specific bands appear in
the presence of nuclear extract (lane 3, indicated by arrows). These bands are dependent
on the annealing of snRNA U1 and U2 to the RNA, as the DNA oligonucleotide-RNase
H-mediated digestion of the first 15 residues of U1, or of the nucleotides 28 to 42 of U2
abolishes them (compare lane 3 to 5 and 6; bands are indicated by arrows). None of these
bands are affected by the presence of Nova (lanes 4 and 10). The presence of Nova
drastically reduces the nuclear extract-independent bands (indicated by a star) both in the
absence and in the presence of ATP. Compare bands 4 to 3 and 10 to 9). These results
suggest that Nova affects the splicing of this substrate not by directly modulating the
annealing of snRNP U1 and U2, but by affecting the tri-dimensional folding of the
substrate RNA. Examples of secondary structures of RNA substrates affecting the
outcome of splicing have been reported in the literature (reviewed by Buratti and Baralle,
2004)15.
Supplementary Figure S14: Analysis of splicing intermediates of Nova-regulated RNAs.
Quantification of splicing intermediates, which contain one of the introns flanking the
alternative exon, and two spliced exons. RNA from Nova dKO relative to wild-type
littermate brain was used to quantify the effect of Nova on removal of the two introns
flanking each alternative exon. The radioactively labeled, RT-PCR amplified RNAs were
separated on denaturing polyacrylamide gels. The abundance of splicing intermediate can
be compared to the pre-mRNA. In addition to the primer pair that amplifies the splicing
intermediate, each RT-PCR contained two additional primers to monitor the efficiency of
all primers (except of the cases where the intron is shorter than 1.5kb, where the same
primer pair amplifies both the intermediate as well as the pre-mRNA: see panel a, Ddr1).
We also used real-time PCR to quantify Nova’s effect on the accumulation of splicing
intermediates, which gave similar data, and was also able to quantify effects onto
intermediates that were undetectable on the gel. Data from real-time PCR is shown as
12
Ule et al., Supplementary Fig Legends
PCR Baseline subtracted CF RFU (Y axis) versus PCR amplification cycle (X axis). The
graphs show quantification of bands representing splicing intermediates, normalized to
the pre-mRNA bands. Since the two splicing intermediates represent the two alternative
pathways leading to exon inclusion, we have normalized the added value of both
intermediates to 1. When Nova promotes exon inclusion, the added value of bands in WT
samples was normalized to 1, and when Nova silences exon inclusion, the added value in
dKO samples was normalized to 1. Color-coded objects present between each gel piece
represent the pre-mRNA and splicing intermediates. The small triangles indicate the
position of the primers used for RT-PCR. The circles represent Nova bound to YCAY
clusters. Each RT-PCR experiment was done in biologic triplicate and the identity of
bands was verified by sequencing.
a, In the Ddr1 pre-mRNA containing NISE1 element, Nova promotes splicing of the 5’
intron, increasing the amount of 5’ splicing intermediate. No significant effect of Nova is
seen on Smarcc2 pre-mRNA (possibly due to a smaller effect of Nova on the fold change
in final isoform ratio, see Table 1).
b, Analysis of 6 pre-mRNAs containing NISE2 elements.
c, Analysis of 10 pre-mRNAs containing both NISE2 and NISE3 elements or just NISE3
(Gabrg2). In b-c, Nova promotes primarily splicing of the 3’ intron, increasing the
amount of 3’ splicing intermediate.
d, Analysis of 2 pre-mRNAs containing NISS2 elements, where Nova primarily
decreases the amount of 5’ splicing intermediate.
e, Analysis of 2 pre-mRNAs containing NESS1 element, where Nova primarily decreases
the amount of 5’ splicing intermediate.
f, Analysis of 3 pre-mRNAs containing NESS2 element, where Nova primarily decreases
the amount of 3’ splicing intermediate.
g, Analysis of 2 pre-mRNAs containing two elements on both sides of the exon (NISS2
and NESS2 element in case of Gphn E7a, and NISS2 and NISS2 in case of Dab1 exons
7b and 7c) shows symmetric action of Nova on both the 5’ and 3’ splicing intermediates.
h, Analysis of 3 pre-mRNAs containing NISS1 element showing Nova–dependent
decrease in the amount of 3’ splicing intermediate. Assuming that Nova acts locally, the
NISS1 cluster should act to locally enhance recognition of the 5’ splice site of the exon
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Ule et al., Supplementary Fig Legends
upstream of the alternative exon. Such action is evident in case of CP110, where the
NISS1 YCAY cluster (which silences inclusion of exon 14a) also acts locally as NISE2
element to promote inclusion of exon 14 (Supplementary Fig. S6a). Analysis of splicing
intermediates shows that joining of CP110 exon 14 to 14a is not efficient, therefore the
NISS1/NISE2 element promotes joining of exon 14 to exon 15. Thus, it is possible that
the decrease in the 3’ splicing intermediate of exon 14a is an indirect result of the
enhancing action of Nova that promotes joining of exon 14 to 15, and thereby skipping of
exon 14a. However, the effect on the 5’ splicing intermediate varies, suggesting that the
mechanism of NISS1 action may depend on other elements in the pre-mRNA (for
example, Rap1 pre-mRNA contains a high score, conserved YCAY cluster in the middle
of the 5’ intron, which may act together with NISS1 to inhibit splicing of the 5’ intron;
data not shown).
i, Quantification of the Nova-dependent fold change in the amount of 5’ and 3’ splicing
intermediates for all of the pre-mRNAs shown in panels a-h
References for Supplementary figure legends:
1.
2.
3.
4.
5.
6.
7.
8.
Buckanovich, R. J. & Darnell, R. B. The neuronal RNA binding protein Nova-1
recognizes specific RNA targets in vitro and in vivo. Mol Cell Biol 17, 3194-201
(1997).
Jensen, K. B. et al. Nova-1 regulates neuron-specific alternative splicing and is
essential for neuronal viability. Neuron 25, 359-71 (2000).
Jensen, K. B., Musunuru, K., Lewis, H. A., Burley, S. K. & Darnell, R. B. The
tetranucleotide UCAY directs the specific recognition of RNA by the Nova Khomology 3 domain. Proc Natl Acad Sci U S A 97, 5740-5 (2000).
Lewis, H. A. et al. Sequence-specific RNA binding by a Nova KH domain:
implications for paraneoplastic disease and the fragile X syndrome. Cell 100, 32332 (2000).
Dredge, B. K. & Darnell, R. B. Nova regulates GABA(A) receptor gamma2
alternative splicing via a distal downstream UCAU-rich intronic splicing
enhancer. Mol Cell Biol 23, 4687-700 (2003).
Ule, J. et al. CLIP identifies Nova-regulated RNA networks in the brain. Science
302, 1212-5 (2003).
Dredge, B. K., Stefani, G., Engelhard, C. C. & Darnell, R. B. Nova autoregulation
reveals dual functions in neuronal splicing. EMBO J 24, 1608-20 (2005).
Ule, J. et al. Nova regulates brain-specific splicing to shape the synapse. Nat
Genet 37, 844-52 (2005).
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Ule et al., Supplementary Fig Legends
9.
10.
11.
12.
13.
14.
15.
Ule, J. et al. Nova regulates brain-specific splicing to shape the synapse. Nat
Genet 37, 844-52 (2005).
Bar, I., Tissir, F., Lambert de Rouvroit, C., De Backer, O. & Goffinet, A. M. The
gene encoding disabled-1 (DAB1), the intracellular adaptor of the Reelin
pathway, reveals unusual complexity in human and mouse. J Biol Chem 278,
5802-12 (2003).
Katyal, S. & Godbout, R. Alternative splicing modulates Disabled-1 (Dab1)
function in the developing chick retina. Embo J 23, 1878-88 (2004).
Yeo, G. W., Van Nostrand, E., Holste, D., Poggio, T. & Burge, C. B.
Identification and analysis of alternative splicing events conserved in human and
mouse. Proc Natl Acad Sci U S A 102, 2850-5 (2005).
Holste, D., Huo, G., Tung, V. & Burge, C. B. HOLLYWOOD: a comparative
relational database of alternative splicing. Nucleic Acids Res 34, D56-62 (2006).
Query, C. C., McCaw, P. S. & Sharp, P. A. A minimal spliceosomal complex A
recognizes the branch site and polypyrimidine tract. Mol Cell Biol 17, 2944-53
(1997).
Buratti, E. & Baralle, F. E. Influence of RNA secondary structure on the premRNA splicing process. Mol Cell Biol 24, 10505-14 (2004).
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