At the heart of an innate immune response lies a tightly regulated gene expression program. This precise regulation is crucial because small changes can shift the balance from protective to destructive immunity. Here we identify a frequently used alternative splice site in the gene oligoadenylate synthetase 1g (Oas1g), a key component of the 2-5A antiviral system. Usage of this splice site leads to the generation of a transcript subject to decay, and removal of the site leads to increased expression of Oas1g and an improved antiviral response. However, removal of the splice site also leads to an increase in apoptotic cell death, suggesting this splicing event exists as a compromise between the pathogen protective benefits and collateral damage associated with OAS1g activity. Across the innate immune response, we show a multitude of alternative splicing events predicted to lead to decay exist and thus, have the potential to play a significant role in the regulation of gene expression in innate immunity

Baltimore, David

Frankiw, Luke

Joglekar, Alok

Li, Guideng

Mann, Mati

English

Caltech Authors - Main

Ctl Upf1
shRNA-1
Upf1
shRNA-2
0.00
0.01
0.02
0.03
0.04
0.05
N
or
m
al
iz
ed
 E
xp
re
ss
io
n
Oas1g Expression
8 hrs Poly(I:C)
**
***
C.	
#	
of
	In
tr
on
s
MaxEntScore
Ctl Upf1
shRNA-1
Upf1
shRNA-2
0.010
0.015
0.020
0.025
0.030
N
or
m
al
iz
ed
 E
xp
re
ss
io
n
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
Co
ntr
ol
Fix
ed
 
Cl
on
es
Co
ntr
ol
Fix
ed
 
Cl
on
es
0.00
0.02
0.04
0.06
0.08
0.10
N
or
m
al
iz
ed
 E
xp
re
ss
io
n
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., 2009 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 targeted 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 splice 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 independent 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 right. 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 BMDMs 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 assembly.  (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 
denotes 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 


Alternative Splicing Coupled with Transcript Degradation Modulates OAS1g Antiviral Activity

https://authors.library.caltech.edu/100036/2/Supplemental_Figures_Table.pdf

Alternative Splicing Coupled with Transcript Degradation Modulates OAS1g Antiviral Activity

Abstract

Similar works

Full text

Available Versions

Caltech Authors - Main

Caltech Authors - Main