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Original Article
Antisense Oligonucleotide-Mediated Terminal
Intron Retention of the SMN2 Transcript
Loren L. Flynn,
1,2,4
Chalermchai Mitrpant,
2,3,4
Ianthe L. Pitout,
1,2
Sue Fletcher,
1,2
and Steve D. Wilton
1,2
1
Centre for Comparative Genomics, Murdoch University, Perth, WA, Australia;
2
Perron Institute for Neurological and Translational Science, Perth, WA, Australia;
3
Department of Biochemistry, Mahidol University, Bangkok, Thailand
The severe childhood disease spinal muscular atrophy (SMA)
arises from the homozygous loss of the survival motor neuron 1
gene (SMN1). A homologous gene potentially encoding an
identical protein, SMN2 can partially compensate for the loss
of SMN1; however, the exclusion of a critical exon in the coding
region during mRNA maturation results in insufficient levels
of functional protein. The rate of transcription is known to in-
fluence the alternative splicing of gene transcripts, with a fast
transcription rate correlating to an increase in alternative
splicing. Conversely, a slower transcription rate is more likely
to result in the inclusion of all exons in the transcript. Target-
ing SMN2 with antisense oligonucleotides to influence the pro-
cessing of terminal exon 8 could be a way to slow transcription
and induce the inclusion of exon 7. Interestingly, following
oligomer treatment of SMA patient fibroblasts, we observed
the inclusion of exon 7, as well as intron 7, in the transcript.
Because the normal termination codon is located in exon 7,
this exon/intron 7-SMN2 transcript should encode the normal
protein and only carry a longer 30UTR. Further studies showed
the extra 30UTR length contained a number of regulatory
motifs that modify transcript and protein regulation, leading
to translational repression of SMN. Although unlikely to pro-
vide therapeutic benefit for SMA patients, this novel technique
for gene regulation could provide another avenue for the
repression of undesirable gene expression in a variety of other
diseases.
INTRODUCTION
With a frequency of 1 in 10,000 live births,
1
the neurodegenerative
disease spinal muscular atrophy (SMA) is the leading genetic cause
of infant death.
2
SMA arises from inadequate levels of the survival
motor neuron (SMN) protein that ultimately results in the death of
motor neurons. While the survival motor neuron 1 (SMN1) gene is
missing in most SMA patients, copies of the homologous gene,
SMN2, potentially compensate for SMN production
3
; however, a
C > T base change in SMN2 exon 7 results in exclusion of the exon
from 90% of neuronal SMN2 transcripts.
3,4
To date, the main RNA
therapeutic focus for SMA has been the use of antisense oligonucleo-
tides (AOs) to enhance SMN2 exon 7 inclusion and increase
SMN levels (for review, see Porensky and Burghes
5
). In particular, a
20O-methoxyethyl (MOE) AO covering the ISS-N1 splicing domain
(Anti-ISS-N1) has shown promise in clinical trials
6–8
and has recently
received approval by the U.S. Food and Drug Administration.
9
How-
ever, the therapy is by no means definitive, with unknown conse-
quences of long-term AO exposure and further improvements in
AO efficacy needed before this therapy can be considered a qualified
success. While other studies have focused on targeting AOs to in-
tronic splice silencing motifs to enhance exon 7 inclusion,
8,10–12
AO-mediated splice modification has broader potential.
The strategy described here was focused on targeting AOs to the last
exon in an attempt to slow transcription rates and concurrent pre-
mRNA processing to temporarily stall the spliceosome machinery.
Others have shown that a slow RNA polymerase II elongation rate
during transcription can increase the “window of opportunity”for
upstream splicing events, with alternative exons more likely to be
included in the mature transcript.
13,14
To determine whether slower
transcription elongation could be induced by an AO, we targeted
AOs to SMN2 exon 8 in an attempt to increase the inclusion of
SMN2 exon 7 in the transcript.
Unexpectedly, AOs targeting SMN2 exon 8 induced the retention of
exon 7 and intron 7 in the mature transcript. Interestingly, an AO
covering the exon 8 acceptor site has been reported by others to induce
exon 7 and intron 7 retention, yet this work was not pursued further.
15
Because the normal termination codon is located within exon 7, this
induced transcript should therefore encode the normal full-length
protein; however, the size of the 30UTR is increased. It is well docu-
mented that the length of the 30UTR can affect transcript stability
and protein translation, with longer 30UTRs having more opportunity
for the binding of microRNAs and regulatory elements (for review, see
Barrett et al.
16
). However, the consequences of intron retention within
the mature transcript, and more specifically within the 30UTRs, are a
more recently explored and less well understood area.
A study by Braunschweig and colleagues
17
reported that three-quar-
ters of mammalian multi-exon genes exhibit intron retention within
Received 20 December 2017; accepted 25 January 2018;
https://doi.org/10.1016/j.omtn.2018.01.011.
4
These authors contributed equally to this work.
Correspondence: Steve D. Wilton, Centre for Comparative Genomics, Health
Research Centre, Building 390, Murdoch University, 90 South Street, Perth, WA
6150, Australia.
E-mail: swilton@ccg.murdoch.edu.au
Molecular Therapy: Nucleic Acids Vol. 11 June 2018 ª2018 The Authors. 91
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
the mature transcript as a result of alternative splicing events. While
6%–16% of 30UTRs are suggested to contain introns,
18
it is unclear at
this stage what percentage of these have the propensity to retain an
intron within the mature message. While transcripts containing in-
trons within the 30UTR were once believed to be non-functional
due to nonsense-mediated decay,
19,20
there is now evidence to show
that intron retention within the mature message is an important
mechanism for transcript and protein regulation (for review, see
Bicknell et al.
18
and Ge and Porse
20
). Tissue-specific transcript regu-
lation by intron retention is particularly common in neuronal cells
during differentiation and maturity,
21
and recent studies have re-
vealed a role for intron retention in hematopoietic cellular differenti-
ation.
22
Furthermore, intron retention within the 30UTR has been
shown to play a role in transcript autoregulation to maintain protein
homeostasis, a mechanism that is particularly common in proteins
involved in forming the spliceosome and in regulating pre-mRNA
processing.
23,24
A number of factors have been reported to regulate splicing events re-
sulting in intron retention, with a correlation observed between
intron retention and the presence of certain regulatory cis elements.
17
Of particular interest, intron retention has been suggested to be the
result of stalling of the RNA polymerase II elongation due to poor
splicing factor recruitment and weakened splicing in non-essential
transcripts.
17,25
Other factors influencing this mechanism include
the position of the intron within the transcript, reduced intron length,
an increase in G/C content within the intron, and weak splice site
strength.
17
While factors that determine intron retention have been studied in
canonical splicing events, it is unknown what role they play in medi-
ating AO-induced intron retention and transcript expression. Conse-
quently, this study focused on gaining a further understanding of the
mechanisms influencing AO-induced intron retention and, further-
more, investigating how it can impact transcript and protein expres-
sion as a potential strategy in treating genetic disease.
RESULTS
Targeting AOs to Exon 8 Results in Exon 7 and Intron 7 Retention
in SMN2 Transcripts
SMA type I fibroblasts (Coriell GM03813) were transfected with
20O-methyl AOs targeting SMN2 exon 8 (for binding coordinates
and AO sequences, see Table 3) at 300, 150, and 75 nM and incubated
(37C) for 48 hr. RT-PCR analysis (Figure 1A) of SMN2 showed an
increase in abundance of an approximately 850-bp product, which
was confirmed by sequencing (Figure 1B) to be the SMN2 transcript
retaining exon 7, as well as intron 7 (848 bp). This product is referred
to as exon/intron 7-SMN2 and is labeled ex/in7 in the figures. Because
the stop codon is located within exon 7, the addition of an extra
444-bp intronic sequence should encode the same protein as SMN1,
but increases the length of the 30UTR (Figure 1C). These results
were reproducible in two unrelated SMA patient primary cell strains
(data not shown), including an SMA type II patient (prepared in-
house) and an SMA type I patient with only one copy of SMN2 (Cor-
iell GM00232). Two additional bands were observed at approximately
100 bp above and 100 bp below the exon/intron 7-SMN2 transcript.
The larger band was deemed to be a PCR artifact because it was un-
able to be re-amplified and disappeared following increasing primer
annealing temperature. The lower band was confirmed by sequencing
to be the naturally occurring D5-SMN2 transcript containing intron 7
(data not shown).
The initial screening of AO sequences 1–18 is shown in Figure S1.
Following preliminary screening, additional AOs were designed by
microwalking around promising AO target sites, shifting up or down-
stream of the original sites (Table 3). Analysis of SMN2 transcripts
following transfection with refined AO sequences showed an
improvement in AO-induced exon/intron 7 retention (Figure 1A).
A clear dose response was observed in all AO-treated cells, with
AOs 10, 18, 24, and 25 consistently inducing the highest levels of in-
clusion across experiments (n = 6). These promising AOs were there-
fore selected for further evaluation, including protein analysis.
Splice Site Analysis Shows a Weak Exon 7 Donor Splice Site
To further investigate the exon/intron 7-SMN2 transcript induced by
AOs targeting exon 8, we analyzed splice site scores (Table 1) using
the online Human Splicing Finder 3.0 website.
26
SMN2 exon 7 was
predicted to have a very strong acceptor site with a score of 98.2
out of a possible 100, while the donor splice site was weaker, scoring
82.81 out of 100. The exon 8 acceptor splice site had a predicted score
of 91.9 out of 100. While these splice site scores are only a predicted
measurement of the likelihood of the site being recognized by the
splicing machinery, the comparatively weaker exon 7 donor splice
site could lead to reduced splicing at the exon/intron 7 junction
when the intron 7/exon 8 junction is further compromised following
AO treatment.
PMO Delivery by Electroporation Improves Exon/Intron 7
Inclusion, Inducing a Decrease in SMN Protein
Previously identified optimal 20O-methyl AO sequences 10, 18, 24,
and 25 were resynthesized as phosphorodiamidate morpholino olig-
omers (PMOs) by Genetools (Philomath, OR, USA), and are now
cited as PMOs 10, 18, 24, and 25. PMOs were administered to cells
using nucleofection for optimal delivery at 1 and 0.5 mM for SMN
transcript and protein analysis by RT-PCR and western blot,
respectively. Nucleofection of PMOs showed increased levels of
exon/intron 7 retention in the mature transcript compared with the
same sequences tested as 20O-methyl AOs, with a clear reduction in
the levels of FL-SMN and D7-SMN transcripts. In particular, PMO-
10 induced almost 100% exon/intron 7 inclusion as determined by
RT-PCR (Figure 2A).
Interestingly, western blot analysis of SMN protein levels revealed a
significant decrease in the amount of SMN detected in samples trans-
fected with exon-8-targeting PMOs (Figures 2B and 2C). PMO-10
and PMO-24 were the most effective compounds inducing a respec-
tive 50% (p = 0.022) and 33% (p = 0.027) decrease in SMN protein
when compared with the level observed in untreated fibroblasts
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92 Molecular Therapy: Nucleic Acids Vol. 11 June 2018
(n = 4). The Anti-ISS-N1 PMO sequence was transfected as a positive
control and was shown to increase SMN levels by up to 80%
compared with that in untreated SMA patient fibroblasts (p = 0.032).
PMO-Induced SMN Knockdown Is Reproducible in Unaffected
Fibroblasts
PMO-24 and PMO-25 were evaluated in non-SMA fibroblasts to
determine the effects of intron 7 retention on SMN protein levels in
cells with a higher baseline of SMN. PMOs were transfected by nucle-
ofection at 1 and 0.5 mM, and incubated for 3 days prior to western
blot analysis. RT-PCR analysis of the total SMN transcripts confirmed
that exon-8-targeting AOs induce almost 100% exon/intron 7 reten-
tion, and hence this must represent both the SMN1 and the SMN2
transcripts (Figure 3A). Consistent with the findings in SMA patient
fibroblasts, western blot analysis (Figures 3B and 3C) demonstrated
that PMO-24 and PMO-25 effectively decreased the levels of SMN
protein in non-SMA fibroblasts by 55% (p = 0.041) and 38%
(p = 0.072), respectively (n = 3). Anti-ISS-N1 was transfected as a
positive control and was shown to increase the levels of SMN protein
by up to 35% as seen in non-SMA cells transfected with the low AO
dose; however, this was not statistically significant. Furthermore,
an AO designed to induce exon 7 skipping was transfected into
non-SMA fibroblasts as a positive control for downregulating SMN
levels. Fibroblasts transfected with this PMO show a 76% decrease
Figure 1. SMN Transcript Analysis following 20OMethyl AO Transfection
Analysis of SMA fibroblasts following transfection with exon 8 targeting 20O-methyl AOs, showing (A) RT-PCR analysis of SMN2 transcripts from transfected fibroblasts (300,
150, 75 nM). Anti-ISS-N1 was used as a positive control for transfection efficiency, and a sham control AO was used as a transfection negative control. A 100-bp marker was
used for comparison, and an RT-PCR no-template negative control was loaded in the final lane, (B) sequencing chromatogram across SMN2 exon 7 to intron 7 and intron 7 to
exon 8, confirming the presence of intron 7 within the mature transcript, and (C) schematic showing SMN2 transcripts identified within fibroblasts transfected with exon-8-
targeting AOs, including full-length SMN (FL-SMN), the D7-SMN transcript missing exon 7, and the exon/intron 7 retained transcript (ex/in7-SMN) with the extended 30UTR.
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Molecular Therapy: Nucleic Acids Vol. 11 June 2018 93
in SMN levels compared with sham control and untreated fibroblasts
(p = 0.030).
Functional SMN protein forms aggregates with the gemin proteins
that fluoresce as bright sparkling foci, reminiscent of gems after anti-
body staining. Therefore, the presence of gems within the cell indi-
cates functional localization of SMN protein. The percentages of
fibroblast nuclei staining positive for gems were counted (Figure 4A).
PMOs 10, 18, 24, and 25 were all transfected into non-SMA cells by
nucleofection at 1 mM and incubated for 3 days prior to fixation
and immunofluorescent staining. Interestingly, the sham control
PMO induced an increase in nuclei containing gems from 16.3% in
untreated fibroblasts to 20.6% following control AO transfection.
However, PMOs 10, 18, 24, and 25 all decreased the number of nuclei
containing gems, with as low as 7.3% of fibroblasts containing gems
following transfection with PMO-10. In comparison, 25.9% of fibro-
blasts transfected with the Anti-ISS-N1 PMO sequence express gems,
while only 3.3% of those transfected with the exon skipping control
PMO express gems. Representative images of fibroblasts transfected
with each PMO are shown in Figure 4B.
PMOs targeting exon 8 induce more efficient retention of exon and
intron 7 in both the SMN1 and the SMN2 transcripts compared
with the 20O-methyl AOs of the same sequence, and analysis of the
SMN protein by western blot and immunofluorescence shows a
further decrease in SMN expression following transfection. While
the exon/intron 7-SMN transcript occurs naturally at low levels in un-
transfected cells, it appears that the extended 30UTR introduces a
number of new regulatory mechanisms into the transcript that nega-
tively impact on protein expression.
Intron Retention Introduces Negative Regulatory Elements to
the 30UTR
In silico analysis of the extended 30UTR was carried out using the
online tools UTRscan,
27
miRBase,
28
Polyadq,
29
and DNA Functional
Site Miner (DNA FS Miner).
30
Table 2 lists the potential regulatory
elements identified within intron 7 from each of these databases,
Figure 2. SMN Transcript and Protein Analysis in
SMA Fibroblasts following PMO Nucleofection
SMN transcript and protein levels in SMA fibroblasts
transfected with PMOs by nucleofection at 1 and 0.5 mM,
showing (A) RT-PCR analysis of SMN2 products con-
firming exon/intron retention, (B) western blots showing
SMN protein levels compared with b-tubulin levels, and (C)
densitometric analysis showing changes in SMN protein
levels normalized against b-tubulin. SMN levels in trans-
fected fibroblasts are shown as an n-fold change
compared with those in samples from untreated cells.
Error bars represent the SEM.
Table 1. Exons 7 and 8 Splice Site Predictions Using Human Splicing Finder
3.0
Exon Splice Site Type Splice Site Motif Consensus Value (0–100)
7 acceptor ttttccttacagGG 98.2
7 donor GGAgtaagt 82.81
8 acceptor tctcatttgcagGA 91.9
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94 Molecular Therapy: Nucleic Acids Vol. 11 June 2018
and Figure 5A illustrates the location of these elements within
intron 7. In silico analysis of the extended 30UTR by the online
tool UTRscan drew attention to two possible regulatory motifs known
as the bearded (BRD) box and the K box. Each of these motifs has
been shown by others to disrupt translation of neuronal gene tran-
scripts during Drosophila development by recruiting microRNAs.
31,32
The BRD box consensus sequence is AGCUUUA and for K box is
UGUGAU. A search for microRNA recognition sites using miRBase
revealed three potential microRNA binding sites within intron 7,
with E values below 10, that suggests that these sites are active. The
microRNAs hsa-miR-3118, hsa-miR-3976, and hsa-miR-5580-3p all
have complementary bases to the SMN intron 7 sequence within
the seed region. It is therefore possible that these microRNAs or the
BRD and K box motifs could disrupt SMN translation.
The exon/intron 7-SMN transcript was further analyzed for polyade-
nylation [poly(A)] signals using two online tools, Polyadq
29
and DNA
FS Miner.
30
Each tool identified two potential poly(A) sites with cor-
responding CA cleavage sites within intron 7. A potential poly(A)-1
(ATTAAA) signal was identified at 132 bases into intron 7, and a po-
tential poly(A)-2 (AATAAA) signal was identified at 238 bases into
intron 7. To determine whether early polyadenylation could destabi-
Figure 3. SMN Transcript and Protein Analysis in
Unaffected Fibroblasts following PMO
Nucleofection
SMN transcript and protein levels in unaffected fibroblasts
transfected with PMOs by nucleofection at 1 and 0.5 mM,
showing (A) RT-PCR analysis of SMN products confirming
exon/intron retention, (B) western blots showing SMN
protein levels compared with b-tubulin levels, and (C)
densitometric analysis showing changes in SMN protein
levels normalized against b-tubulin. SMN levels in trans-
fected fibroblasts are shown as an n-fold change
compared with those in samples from untreated cells.
Error bars represent the SEM.
lize the extended SMN transcript following
PMO treatment, we designed specific poly(A)
primers to target the predicted cleavage sites
and downstream sequences to amplify polyade-
nylated products (Figure 5B). Following nucleo-
fection of PMOs into unaffected fibroblasts,
RNA was collected at multiple time points
including 12, 24, 48, and 72 hr. Samples were
DNase treated and RNA was amplified using
the exon/intron 7 forward primer with the spe-
cific poly(A)-R1 and R2 primers (Figure 5C).
No differences were observed within each treat-
ment group over the 72-hr duration of the time
course.
Specific poly(A)-R1 primer binding to the first
ATTAAA poly(A) site amplified a faint product
in some samples, suggesting this site could
initiate polyadenylation. RT-PCR using the specific poly(A)-R2
primer directed to the second AATAAA site resulted in two products,
a stronger amplicon amplified by the second cleavage site, as well as a
fainter non-specific amplification of the first cleavage site. The stron-
ger amplicon was sequenced and confirmed to have a poly(A) tail
extending past the primer annealing site (Figure 5D). This result
suggests that early polyadenylation is occurring at this second
AATAAA site within intron 7, and as a result could destabilize the
exon/intron 7-SMN transcript and therefore result in decreased pro-
tein levels.
To compare the stability and cleavage of the exon/intron 7-SMN and
FL-SMN transcripts, we designed primers to target downstream of the
poly(A) sites, and we tested them with a forward primer targeting the
exon/intron 7 boundary (Figure 5C). Interestingly, both primer sets
produce a strong amplicon extending beyond the poly(A) signal
and do not show diminished expression of the transcript following
AO treatment. Taken together, these results show that while early
polyadenylation appears to occur at the second poly(A) signal, the
transcript level remains stable, suggesting that transcription may be
occur at a faster rate than polyadenylation and cleavage, or that the
early polyadenylation is not destabilizing the transcript.
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Molecular Therapy: Nucleic Acids Vol. 11 June 2018 95
DISCUSSION
The original intent of this study was to influence the rate of SMN2
transcription by targeting AOs to the terminal exon in an attempt
to increase exon 7 inclusion. However, another splice-switching
mechanism for manipulating expression was revealed. Selected AOs
targeting SMN2 exon 8 promoted exon 7 and intron 7 inclusion in
the mature SMN message, revealing a novel AO application: inducing
terminal intron retention. In silico analysis of the SMN2 exon 7 splice
sites suggests that this action may be the result of a strong acceptor
splice site (scoring 98 out of 100) and a weaker donor splice site
Figure 4. Immunofluorescence Staining and Analysis of SMN Protein following PMO Nucleofection
Immunofluorescence analysis of SMN shown as gems in PMO nucleofected unaffected fibroblasts (1 mM), compared with untreated fibroblasts. (A) Graph displaying the
percentages of cell nuclei containing gems, as indicated above each bar, and (B) representative images showing anti-SMN- (green) and Hoechst (blue)-stained SMA fi-
broblasts following PMO nucleofection. Typical gems stained within the cell nucleus are indicated with white arrows. Images were taken at 20objectiv e. Scale bar, 25 mm.
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96 Molecular Therapy: Nucleic Acids Vol. 11 June 2018
(scoring 83 out of 100). The weaker donor site might be subject to
poor recognition by the splicing machinery, abrogating splicing be-
tween exon 7 and the following exon, especially when the splicing
of exon 8 is compromised by AO binding.
The correlation in splice-switching efficacy between 20O-methyl and
PMO compounds of the same sequence is well established for induced
exon skipping in dystrophin transcripts as a therapy for Duchenne
muscular dystrophy,
33
as well as for the efficacy of the Anti-ISS-N1
sequence.
34
Furthermore, the PMO chemistry has been shown to be
safe, with no significant adverse events reported in a 5-year study of
Duchenne muscular dystrophy patients receiving weekly treatment
with Exondys51, a PMO drug granted accelerated approval by the
U.S. Food and Drug Administration (FDA) in 2016 for amenable dys-
trophin mutations.
35,36
Consistent with the results of exon 8 targeting 20O-methyl AOs,
PMOs of the same sequence were effective at inducing exon/
intron 7 retention. Interestingly, while PMOs targeting exon 8
increased the levels of the exon/intron 7-SMN transcript, these
PMOs also induced a 50% decrease in SMN protein as assessed by
western blot. This result was reproducible between SMA patient fi-
broblasts and unaffected fibroblasts following AO transfection. Simi-
larly, immunofluorescence staining of the transfected fibroblasts
showed fewer nuclei containing functional SMN in the form of
“gems”when compared with sham-control PMO transfected cells.
It is likely that the observed decrease in SMN protein could be due
to the longer than normal 30UTR within the exon/intron 7-SMN
transcript, and therefore many regulatory factors could contribute
to protein downregulation.
Endogenous intron retention has been shown by others to act as
a form of gene repression, often through the introduction of a
premature termination codon, rendering the transcript susceptible
to nonsense-mediated decay.
17,37
In the study presented here,
nonsense-mediated decay is unlikely to be the cause of SMN downre-
gulation due to the retained intron occurring after the normal termi-
nation codon. However, as a result of the extended 30UTR, influences
of downstream sequences could lead to this transcript not being effi-
ciently translated.
The length of the 30UTR can be a critical factor in regulating tran-
script stability and protein expression. A longer 30UTR can increase
the opportunity for sequence-specific recognition motifs to recruit
regulatory factors, including microRNAs.
16
Furthermore, the possi-
bility of altered mRNA secondary structures can influence the avail-
ability of such sequences to these factors.
38
A number of online
databases can identify potential microRNA and regulatory factor
binding sites in NCBI documented transcripts. However, there is a
lack of appropriate resources whereby an altered 30UTR sequence
can be analyzed, limiting the search possibilities for this study. It is
probable that there are many factors influencing the translational
knockdown observed for the exon/intron 7-SMN transcript.
The microRNA prediction database miRBase allows the user to input
an mRNA sequence for analysis, and analysis of the SMN intron 7
sequence revealed three potential microRNA binding sites. Of these,
miR-3976 is a validated microRNA and is reportedly overexpressed in
pancreatic cancer.
39
However, how miR-3976 impacts translation,
and whether it is expressed in the dermal fibroblast used in this study,
is yet to be determined. Further in silico analysis of the sequence using
the online UTRscan database
27
drew attention to a number of motifs,
including the BRD and K box motifs, as well as two alternative poly-
adenylation signals. The BRD box and K box motifs have been shown
to recruit microRNAs that act as translational inhibitors of certain
proteins during Drosophila development.
31,32,40
The functionality of
these motifs in human sequences is unknown and, therefore, any in-
fluence on SMN translation is only speculative.
Others have tested the use of AOs to prevent microRNAs from
binding to a transcript, by either binding of the microRNA as an
antagomir, or to act as a decoy, binding directly to the transcript of
interest.
41
To further investigate the mechanism of translational
knockdown presented here, future studies could use AOs targeting
the microRNAs themselves, their binding sites, as well as the BRD
and K box motifs. Examining SMN expression in such studies could
indicate whether these regulatory elements play a role in inhibiting
SMN translation in the exon/intron 7-SMN transcript. However, it
is unlikely that this will be of clinical benefit to SMA patients.
Further in silico analysis of the SMN2 intron 7 sequence identified two
potential polyadenylation signals with corresponding CA cleavage
sites that could prematurely cleave the mRNA, potentially resulting
in transcript and protein destabilization. Premature polyadenylation
and mRNA cleavage have been shown by others to cause less efficient
Table 2. In Silico Analysis of Potential Regulatory Elements within SMN
Intron 7
The prediction value or score indicates the strength of the regulatory site within the
SMN sequence. For poly(A) signals, scores >0.5 are true predictions for Polyadq and
scores >0.6 are true predictions for DNA FS Miner. For miRBase, E values <10 may
have binding potential.
NA, not applicable.
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Molecular Therapy: Nucleic Acids Vol. 11 June 2018 97
processing of transcripts when compared with those cleaved at the
distal 30or canonical poly(A) site.
42
In this study, primers were
designed to anneal to the sites identified within intron 7, and should
amplify a product only if polyadenylation has occurred. The primer
designed to anneal to the ATTAAA site failed to generate a product,
while the primer annealing to the AATAAA site amplified a clean,
consistent product whose levels increased with an increase in intron
7 retention.
It appears that the early AATAAA site within intron 7 initiates poly-
adenylation prior to use of the canonical poly(A) site within exon 8,
and as such negatively affects SMN translation. However, primers
amplifying downstream of the early poly(A) site produced a strong
and consistent product that is not diminished following AO treat-
ment at any time point, indicating that mRNA cleavage does not
Figure 5. Analysis of Predicted SMN Intron
Regulatory Elements
Analysis of predicted SMN intron 7 regulatory elements,
showing (A) a schematic of the in silico analysis of regu-
latory elements within intron 7, (B) a schematic of the
location of potential poly(A) signal and cleavage sites and
the primer binding sites, (C) RT-PCR across SMN and
Cyclin-D (housekeeping gene) transcripts in fibroblasts
nucleofected with 1 mM (1) PMO-24, (2) PMO-25, (3)
control PMO, and (4) an untreated sample, using indicated
primers, and (D) sequencing chromatogram of the RT-
PCR product amplified using the exon 6/7 forward and
poly(A)R2 reverse primers.
occur downstream of either of the poly(A) sig-
nals. Taken together, these results show that
while early polyadenylation appears to occur at
the second poly(A) signal, the transcript level re-
mains stable, suggesting that transcription in
this case may occur at a faster rate than polyade-
nylation and cleavage.
Interestingly, the product amplified by the spe-
cific poly(A)-R2 primer was also observed in un-
treated samples, suggesting polyadenylation
may be a natural mechanism for controlling
SMN levels. Furthermore, it has been reported
that the canonical poly(A) signal and cleavage
site in SMN exon 8 is inefficient at recruiting
cleavage factors, and consequently SMN polya-
denylation is subjected to additional regulation
by U1A, a component of the U1 snRNP.
43
The
study showed that overexpression of U1A can
inhibit SMN polyadenylation and cleavage,
decreasing the levels of SMN protein.
43
Given
the inefficiency of the canonical poly(A) site, it
is probable that the intron 7 poly(A) signal could
be more favorable for initiating polyadenylation
and cleavage. However, the presence of U1A
may still inhibit cleavage at this site, and therefore explain the lack
of mRNA cleavage observed in the RT-PCR experiments within the
current study.
The presence of polyadenylation signals in terminal introns could
contribute to the process of protein regulation by intron retention.
To examine this theory further, future studies should assess the
occurrence of polyadenylation in alternatively spliced 30UTR-intron
retention transcripts. It would be interesting to investigate whether
this effect of “premature polyadenylation”occurs in other gene
transcripts.
AOs targeting the terminal exon to induce intron retention could be
useful in those diseases where protein repression is essential for treat-
ment, including many types of cancer. We speculate that in the study
Molecular Therapy: Nucleic Acids
98 Molecular Therapy: Nucleic Acids Vol. 11 June 2018
presented here, the extended SMN transcript is regulated by a number
of motifs within intron 7 that are involved in translational repression
due to the retention of this sequence in the 30UTR. However,
this mechanism may only apply to a select number of genes.
Alternatively, if the stop codon were to be in the final exon, intron
retention could disrupt the reading frame or introduce a premature
termination codon. To identify genes where intron retention could
be applied, it will be necessary to look at a number of factors within
the gene, most importantly the splice site scores for the flanking
exons. If the donor site is strong, then intron retention may not be
possible.
Aside from weakened splice sites of retained introns, additional cis
and trans factors suggested to influence natural intron retention
include the position of the intron in the transcript, an increase in
G/C content, and reduced intron length.
17
Interestingly, while the
G/C content of SMN intron 7 is only 33.8%, being positioned adjacent
to an alternatively spliced exon increases the probability of the intron
being retained. Furthermore, at 444 nt long, intron 7 is relatively short
compared with the median human intron length of 1,334 nt in the
coding region and 1,303 nt within the 30UTR.
44
It will be interesting
in future studies to compare the relevance of these factors across tran-
scripts to identify markers that could predict the likelihood of effec-
tive AO-induced intron retention.
In this study we present a novel application for splice-switching
PMOs in initiating terminal intron retention. It is unfortunate that
this model is unlikely to provide therapeutic benefit to SMA
patients, yet further work could see intron retention being applied
to a number of other diseases. This study is reflective of the ever-ex-
panding complexity of gene regulation and undoubtedly sheds new
light on splicing and AO mechanisms that may offer new avenues
of therapy.
MATERIALS AND METHODS
AO Design and Synthesis
AOs were designed to target the exon 8 acceptor splice site and exon
splice enhancers (ESEs) as predicted by the online SpliceAid predic-
tion tool,
45
available at http://www.introni.it/splicing.html.AO
nomenclature was based on that described by Mann et al.
46
All
20O-methyl PS-AOs were synthesized in-house on an Expedite
8909 nucleic acid synthesizer with a phosphorothioate backbone.
Following identification of optimal 20O-methyl AO sequences, these
AOs were prepared as PMOs, purchased through Genetools (Philo-
math, OR, USA). Table 3 lists the details of all AOs used in this study.
20O-Methyl AO Transfection
SMA type I patient fibroblasts (GM03183; Coriell Cell Repositories,
Camden, NJ, USA) and normal human dermal fibroblasts prepared
in-house (Murdoch University Human Research Ethics Committee
Approval #2013/156) were proliferated and seeded in 10% fetal
bovine serum (FBS) DMEM and incubated at 37C for 24 hr prior
to transfection. All 20O-methyl PS-AOs were transfected using
Lipofectin (Life Technologies, Melbourne, Australia) at a 2:1 ratio
of lipofectin to total AO, according to manufacturer’s protocols,
and incubated for 48 hr.
Nucleofection of PMOs
PMO delivery by nucleofection was performed using a Nucleofection
X unit with the Nucleofection P2 kit, using the CA-137 program
(Lonza, Melbourne, Australia). PMOs were transfected at 1 and
0.5 mM, as determined by the final transfection volume, supplemented
with 5% FBS DMEM and incubated for 72 hr.
RNA Extraction and PCR
RNA was extracted using the MagMAX-96 Total RNA Isolation Kit,
including a DNase treatment (Life Technologies), according to the
manufacturer’s instructions. RT-PCRs were performed using the
One-Step SuperScript III RT-PCR kit with Platinum Taq Polymerase
(Life Technologies) according to manufacturer’s instructions. All
primer sequences used in this study are detailed in Supplemental
Materials and Methods. Products were amplified with the tempera-
ture profile, 55C for 30 min, 94C for 2 min, followed by 25–30 cycles
of 94C for 30 s, 56C for 30 s, and 68C for 1 min. Amplicon se-
quences were identified by Sanger sequencing at the Australian
Genome Research Facility (AGRF, Perth, Australia).
Western Blot Analysis
Cell lysates were prepared in 125 mM Tris/HCl (pH 6.8), 15% SDS,
10% glycerol (v/v), 1.25 mM PMSF (Sigma-Aldrich), protease inhibi-
tor cocktail (3 mL/100 mL; P8340; Sigma-Aldrich), 0.004% bromophe-
nol blue, and 2.5 mM dithiothreitol. Pellets were sonicated six times
for 1-s pulses and samples denatured at 94C for 5 min.
Approximately 10 mg of total protein (as determined by BCA assay)
was loaded per sample on a NuPAGE Novex 4%–12% BIS/Tris gel
(Life Technologies). Proteins were transferred onto a Pall Fluorotrans
polyvinylidene fluoride (PVDF) membrane at 350 mA for 1 hr
in western transfer buffer. MANSMA7 (1:1,000; Developmental
Studies Hybridoma Bank) and b-tubulin (1:20,000; DSHB) mono-
clonal primary antibodies were incubated overnight at 4C prior
to detection using a Western Breeze Chemiluminescent Immunode-
tection System (Life Technologies), according to the manufacturer’s
instructions. Western blot images were captured on a Vilber
Lourmat Fusion FX system using Fusion software, and Bio-1D soft-
ware was used for densitometry analysis. All p values were calculated
using a paired two-tailed t test, and SE bars were used to represent
the SEM.
Immunofluorescence
Cells on coverslips were fixed using ice-cold acetone-methanol (1:1),
then blocked in 10% filtered goat serum in PBS containing 0.2%
Triton-X (PBT). SMN was detected with MANSMA1 (1:100;
DSHB) antibody, incubated overnight at 4C in PBT. Cells
were stained with Hoechst (Sigma-Aldrich) for nuclei detection
(1 mg/mL diluted 1:125), and the MANSMA1 primary antibody
was detected using Alexa Fluor 488 (1:400; Thermo Fisher Scientific).
Photos were overlaid, and the number of gems per nuclei was counted
www.moleculartherapy.org
Molecular Therapy: Nucleic Acids Vol. 11 June 2018 99
and recorded as a percentage of total nuclei, with at least 300 cells
counted per slide.
In Silico Analysis
A number of online databases were used to analyze the extended
30UTR sequence to identify potential regulatory elements. Splice
site scores were analyzed by Human Splice Finder version 3.0 avail-
able at http://www.umd.be/HSF3/.
26
Regulatory element binding
was predicted using UTR Scan available at http://itbtools.ba.itb.
cnr.it/.
27
Polyadenylation signals were analyzed using Polyadq avail-
able at http://rulai.cshl.edu/tools/polyadq/polyadq_form.html
29
and
DNA FS Miner available at http://dnafsminer.bic.nus.edu.sg/.
30
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Materials and
Methods and one figure and can be found with this article online at
https://doi.org/10.1016/j.omtn.2018.01.011.
AUTHOR CONTRIBUTIONS
Conceived and designed experiments: L.L.F., C.M., S.F., and S.D.W.
Performed experiments: L.L.F., C.M., and I.L.P. Wrote and edited
manuscript: L.L.F., C.M., I.L.P., S.F., and S.D.W.
ACKNOWLEDGMENTS
The authors would like to acknowledge Project funding from
the Parry Foundation, Spinal Muscular Atrophy Association of
Australia, and the NHMRC (project grant 1086311). L.L.F. and
I.L.P. received scholarships from Team Spencer and Muscular Dys-
trophy WA.
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10 hSMN8A(+59+83) AUC UUC UAU AAC GCU UCA CAU UCC A 25
11 hSMN8A(+84+108) AUA UUU UGA AGA AAU GAG GCC AGU U 25
12 hSMN8A(+152+178) CAU AAC UUU UAA UCA AGA AGA GUU ACC 27
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