Ctl
Upf1
shRNA-1
Upf1
shRNA-2
0.00
0.01
0.02
0.03
0.04
0.05
Normalized Expression
Oas1g Expression
8 hrs Poly(I:C)
**
***
C
.
# of Introns
MaxEntScore
Ctl
Upf1
shRNA-1
Upf1
shRNA-2
0.010
0.015
0.020
0.025
0.030
Normalized Expression
Upf1 Expression
No Stimulation
***
***
A.
B
.
D
.
B
.
Figure S1. Related to Figure 1.
E.
Figure S2. Related to Figure 2.
Clone 1
Allele A
Allele B
Clone 2
Allele A
Allele B
Clone 3
Allele A
Allele B
Clone 4
Allele A
Allele B
Clone 5
Allele A
Allele B
Clone 6
Allele A
Allele B
Clone 7
Allele A
Allele B
Control
Alternative Splice Site
A.
B.
D
.
C.
Oas1a
0
hr
1
hr
4
hr
8
hr
12
hr
49
4
33
4
41
9
114
16
134
10
Control
Fixed
Clones
Control
Fixed
Clones
0.00
0.02
0.04
0.06
0.08
0.10
Normalized Expression
Oas1a qPCR
n.s.
Unstimulated
8 hrs. Poly(I:C)
n.s.
Genomic coordinate (chr5), “
-
” strand
Figure S3.
Related
to Figure 2.
Figure S4.
Related to Figure 2.
Clone 1
Allele A
Allele B
Clone 2
Allele A
Allele B
Clone 3
Allele A
Allele B
Clone 4
Allele A
Allele B
Clone 5
Allele A
Allele B
Clone 6
Allele A
Allele B
Clone 7
Allele A
Allele B
Control
Alternative Splice Site
Supplemental Table 1: Oligonucleotides
OLIGONUCLEOTIDE
SOURCE
IDENTIFIER
EMCV
Forward qPCR Primer (5'
-
CCTCTTAATTCGACGCTTGAA
-
3')
Pérez
et. al., 200
9
N/A
EMCV
Reverse qPCR Primer (5'
-
GGCAAGCATAGTGATCGAAG
-
3')
Pérez
et. al., 2009
N/A
Rpl32 Forward qPCR Primer (5'
-
GCCTCTGGTGAAGCCCAAG
-
3')
Ruiz
-
Villalba et. al., 2017
N/A
Rpl32 Reverse qPCR Primer (5'
–
TTGTTGCTCCCATAACCGATGT
-
3')
Ruiz
-
Villalba et. al., 2017
N/A
Oas1g
TaqMan
Thermo Fisher Scientific
Mm01730198_m1
Rpl32
TaqMan
Thermo Fisher Scientific
Mm02528467_g1
Oas1a
TaqMan
Thermo Fisher Scientific
Mm00836412
_m1
Supplemental Figure Legends
1
Figure S1. The Alternative 5’ Splice Site Mediating the AS Event is of Similar Strength to the
2
Consensus 5’ Splice Site
.
(A)
RT
-
qPCR analysis of
Upf1
mRNA levels in
unstimulated
3
macrophages expressing a scrambled shRNA (light blue) or one of two
Upf1
targeted shRNAs
4
(darker blue).
(B)
RT
-
qPCR analysis of
Oas1g
mRNA levels in
poly(I:C) stimulated (8 hrs.)
5
macrophages expressing a scrambled shRNA (light blue) or one of two
Upf1
tar
geted shRNAs
6
(darker blue).
(
C
)
Histogram representing the 5’ splice site strength (MaxEntScore) of introns of
7
expressed in BMDMs. The bin with which the consensus splice site falls is shown by the light blue
8
line. The bin with which the alternative spl
ice site falls is shown by the dark blue line.
(
D
)
Pie chart
9
representing
alternative junction usage for all expressed junctions upon 4 hrs. of poly(I:C)
10
stimulation
.
The slice including the alternatively spliced third junction of
Oas1g
is
labelled (Alt.
11
Junction Usage 0.41
-
0.60)
.
(
E
)
Same as (D) but for 12
hrs.
of poly(I:C) stimulation
.
The slice
12
including the alternatively spliced third junction of
Oas1g
is
labelled (Alt. Junction Usage 0.21
-
13
0.40).
Data is representative of two indepen
dent experiments
(A, B
)
from three
biological replicates
14
(error bars indicate SEM). * denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001 using
15
a Student’s t test. Results are presented relative to those of
Rpl32
.
16
17
Figure S2.
Oas1g
Macrophage Cell Line Genotyping.
Sanger sequencing gDNA from a control
18
sample (very top) and the
Oas1g
SS KO clones. Sequencing is centered around the
Oas1g
alternative
19
splice site. Sequencing is oriented such that the negative strand runs left to righ
t.
20
21
Figure 3.
Oas1a
has a Similar Frequently Utilized AS
-
NMD Event. (A)
Schematic depiction
22
showing the homology between
Oas1a
and
Oas1g
at the alternatively spliced third junction.
(B)
23
(left)
Sashimi plots centered at the third junction of
Oas1g
from BM
DMs stimulated with poly(I:C)
24
for 0, 1, 4, 8, and 12 hrs.
Sequenced RNA was derived from the total nuclear fraction.
The y
-
axis
25
represents Reads Per Kilobase of transcript, per Million mapped reads (RPKM). Genomic
26
coordinates represent the mm9 genome as
sembly.
(right)
Posterior distributions
of the
Ψ
value for
27
each individual time point. The mean
Ψ
is depicted by the red line. Mean and 95% confidence
28
intervals are labelled to the right of the posterior distribution.
(C)
RT
-
PCR upon stimulation with
29
poly(I:C) confirming alternative splice site usage in control populations and forced productive
30
splicing in fixed clones.
(D)
RT
-
qPCR analysis of
Oas1g
mRNA levels in
unstimulated and
31
stimulated (8 hrs poly(I:C)) macrophages.
Control samples are represented in light blue, SS KO
32
clones are represented in dark blue.
Data is representative of two independent experiments
(D
-
F)
33
and is shown as mean (error bars indicate SEM). * denotes p < 0.05, ** denotes p < 0.01, and ***
34
denote
s p < 0.001 using a Student’s t test. Results are presented relative to those of
Rpl32
(D)
35
36
Figure S4
.
Oas1a
Macrophage Cell Line Genotyping.
Sanger sequencing gDNA from a control
37
sample (very top) and the
Oas1a
SS KO clones. Sequencing is centered around the
Oas1a
alternative
38
splice site. Sequencing is oriented such that the negative strand runs left to right.
39
40
Supplemental Table 1: Oligonucleotides
41
42