Cellular and Molecular Life Sciences
https://doi.org/10.1007/s00018-020-03596-7
Cellular and Molecular Life Sciences
ORIGINAL ARTICLE
Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid
deadenylation and translation inhibition of mutant transcripts
Adam Ciesiolka1 · Anna Stroynowska‑Czerwinska1,2 · Paweł Joachimiak1 · Agata Ciolak1 ·
Emilia Kozlowska1 · Michal Michalak1 · Magdalena Dabrowska3 · Marta Olejniczak3 ·
Katarzyna D. Raczynska4,5 · Dominika Zielinska1 · Magdalena Wozna‑Wysocka1 · Wlodzimierz J. Krzyzosiak1
Agnieszka Fiszer1
·
Received: 30 December 2019 / Revised: 1 July 2020 / Accepted: 9 July 2020
© The Author(s) 2020
Abstract
Polyglutamine (polyQ) diseases are incurable neurological disorders caused by CAG repeat expansion in the open reading
frames (ORFs) of specific genes. This type of mutation in the HTT gene is responsible for Huntington’s disease (HD). CAG
repeat-targeting artificial miRNAs (art-miRNAs) were shown as attractive therapeutic approach for polyQ disorders as they
caused allele-selective decrease in the level of mutant proteins. Here, using polyQ disease models, we aimed to demonstrate
how miRNA-based gene expression regulation is dependent on target sequence features. We show that the silencing efficiency
and selectivity of art-miRNAs is influenced by the localization of the CAG repeat tract within transcript and the specific
sequence context. Furthermore, we aimed to reveal the events leading to downregulation of mutant polyQ proteins and found
very rapid activation of translational repression and HTT transcript deadenylation. Slicer-activity of AGO2 was dispensable
in this process, as determined in AGO2 knockout cells generated with CRISPR-Cas9 technology. We also showed highly
allele-selective downregulation of huntingtin in human HD neural progenitors (NPs). Taken together, art-miRNA activity
may serve as a model of the cooperative activity and targeting of ORF regions by endogenous miRNAs.
Keywords miRNA · CAG repeats · Polyglutamine diseases · Huntington’s disease · Translational inhibition
This work is dedicated in the memory of Wlodzimierz
Krzyzosiak, deceased in December 2017, who extensively
developed research on repetitive sequences and his wife,
Krystyna Krzyzosiak, deceased in May 2019, who greatly
supported our scientific work.
Adam Ciesiolka and Anna Stroynowska-Czerwinska Joint
Authors.
Wlodzimierz J. Krzyzosiak: Deceased.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00018-020-03596-7) contains
supplementary material, which is available to authorized users.
* Agnieszka Fiszer
agnieszka.fiszer@ibch.poznan.pl
1
Department of Molecular Biomedicine, Institute
of Bioorganic Chemistry, Polish Academy of Sciences,
Noskowskiego 12/14, Poznan, Poland
2
Laboratory of Structural Biology, International Institute
of Molecular and Cell Biology, Ks. Trojdena 4, Warszawa,
Poland
Abbreviations
16CAG cell line
98CAG cell line
art-miRNAs
ASO
ATN1
ATXN3
ATXN7
ddPCR
DRPLA
Stably expressing exon 1 of HTT with
16 CAG repeats
Stably expressing exon 1 of HTT with
98 CAG repeats
CAG repeat-targeting artificial miRNAs
Antisense oligonucleotide
Atrophin-1
Ataxin-3
Ataxin-7
Digital droplet PCR
Dentatorubral–pallidoluysian atrophy
3
Department of Genome Engineering, Institute of Bioorganic
Chemistry, Polish Academy of Sciences, Noskowskiego
12/14, Poznan, Poland
4
Department of Gene Expression, Institute of Molecular
Biology and Biotechnology, Adam Mickiewicz University
in Poznan, Wieniawskiego 1, Poznan, Poland
5
Center for Advanced Technology, Adam Mickiewicz
University, Wieniawskiego 1, Poznan, Poland
13
Vol.:(0123456789)
A. Ciesiolka et al.
Fluc
HTT
HD
ICC
iPSCs
miRNA
NP
ORF
polyQ
RISC
SCA3
sgRNA
siRNA
smFISH
Rluc
NlucP
RT-qPCR
UTR
Firefly luciferase
Huntingtin
Huntington’s disease
Immunocytochemistry
Induced pluripotent stem cells
MicroRNA
Neural progenitor
Open reading frame
Polyglutamine
RNA-induced silencing complex
Spinocerebellar ataxia type 3
Small guide RNA
Short interfering RNA
Single-molecule fluorescent in situ
hybridization
Renilla Luciferase
Nano luciferase with PEST domain
Quantitative reverse transcription PCR
Untranslated region
Introduction
Non-coding RNAs (ncRNAs) are a large, diverse group
of transcripts that do not contain information about protein sequence but mainly play a crucial role in the posttranscriptional regulation of gene expression. Examples of
ncRNAs are short interfering RNAs (siRNAs) and microRNAs (miRNAs), which constitute a large family of short
(~21 nt) RNAs [1–4]. SiRNAs activate RNA-induced silencing complex (RISC) to carry out the AGO2-mediated cleavage of a transcript within a perfectly matched siRNA-mRNA
duplex, followed by mRNA degradation [5]. In contrast, the
miRNA strand guides the miRNA-induced silencing complex (miRISC) to interact with only partially complementary sequences within the transcripts in animal cells, causing
translational inhibition and mRNA transcript decay following deadenylation [6–10].
Functional miRNA-binding sites are usually localized
within the 3′ untranslated region (UTR) but might also be
present within the open reading frame (ORF) [11–15] and
5′UTR [16–18]. These latter sites are considered as less
functional than those in the 3′UTR as miRISCs cannot avoid
collision with the scanning small ribosomal subunit and rapidly translocating ribosomes [19]. Importantly, the efficiency
of the miRNA-mediated regulation of gene expression may
depend on the number of miRNA-binding sites within the
regulated target [20] and the distance between these sites
[21]. The more target sites at an optimal distance on mRNA
there are, the higher the observed inhibitory effect is, caused
by cooperative interaction between miRISCs bound to
neighboring sites [22].
13
SiRNAs and miRNAs, as negative regulators of gene
expression, are often used in the development of therapeutic approaches. One of the examples are strategies
for incurable and progressive neurodegenerative polyglutamine (polyQ) diseases which include Huntington’s
disease (HD), spinal bulbar muscular atrophy (SBMA),
dentatorubral–pallidoluysian atrophy (DRPLA) or spinocerebellar ataxia (SCA) types 1, 2, 3, 6, 7 and 17
(Fig. 1a). These disorders are caused by the expansion of
CAG repeat sequences within the ORFs of specific genes,
so that the normal alleles contain 10–20 CAG repeats,
whereas mutant alleles usually 40–70 CAG repeats. Due to
a location of mutation within ORF, mutated gene encode
protein with an expanded polyQ tract [23, 24].
One promising therapeutic approach is the elimination of
mutant gene expression by directly targeting the mutation
site in the transcript, i.e., the expanded CAG repeat tract
[25, 26]. In a series of studies from David Corey’s and our
groups, the effects of particular oligonucleotides, hereafter
called CAG repeat-targeting artificial miRNAs (art-miRNAs), were tested in various polyQ disease models [27–40]
(Table S1). The common feature of these oligonucleotides is
the presence of specific mismatches in the interaction with
the targeted CAG repeat tract, making these oligonucleotides similar to miRNAs. Allele-selective downregulation of
mutant polyQ proteins by art-miRNAs most probably results
from preferential activation of the silencing mechanism
when multiple miRISCs are present on the expanded repeat
tract. The targeted transcript level was less affected than the
protein level as art-miRNAs did not induce the substantial
mRNA cleavage typical of siRNAs [27, 28, 30]. Moreover, a
study of the mechanism of action of art-miRNAs suggested
cooperative silencing by miRISCs located on the expanded
repeat tract, as revealed by dose-response experiments, and
the involvement of AGO2 and GW182, as shown by RNA
immunoprecipitation and siRNA-based knockdown experiments [30]. Intriguingly, the activities of art-miRNAs in various polyQ disease models differed significantly, and minor
differences in oligonucleotide sequence largely affected the
observed activity (Table S1). Therefore, we decided to investigate the details of the activated silencing process in the
context of further development of this approach and as an
example of miRNA-based targeting of ORF regions.
In this study, we aimed to elucidate the key factors affecting silencing efficiency of art-miRNAs and determine the
mechanism of their action. For this purpose we used cells
with endogenous mutant gene expression, including human
neural progenitors (NPs), as well as we constructed several
dedicated cellular models. We compiled the results of testing our most effective art-miRNA, A2 [33], to highlight the
Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and…
variance in its activity in different polyQ disease models. We
show that allele-selectivity of art-miRNAs is determined by
the localization of CAG repeat tract in ORF and strengthened by specific sequence of huntingtin (HTT) transcript.
Moreover, we demonstrate that A2 induced rapid mRNA
deadenylation and translation inhibition and AGO2 was not
required in activated silencing mechanism.
Expression of SOX1, SOX2, PAX6, and NES markers was
confirmed by ICC (Supplementary Figure S4A) and by RTqPCR (Supplementary Figure S4B). All cell lines were cultured at an appropriate cell confluence at 37 °C in 5% CO2.
Cell banks were stored in liquid nitrogen.
RNA oligonucleotides
Materials and methods
Cell lines
HEK 293T (American Type Culture Collection) and host
Flp-In T-REx-293 cell lines (Thermo Fisher Scientific)
were cultivated in Dulbecco’s Modified Eagle’s Medium
(Sigma-Aldrich), containing 10% fetal bovine serum (Biowest), penicillin-streptomycin solution (Sigma-Aldrich),
2 mM L -glutamine (Sigma-Aldrich). Additionally, for
16CAG and 98CAG Flp-In T-REx-293 cell culture 100 µg/
ml hygromycin B (Thermo Fisher Scientific) and 5 µg/ml
blasticidin S (Thermo Fisher Scientific) was supplemented.
Patient-derived fibroblasts (Coriell Institute, SCA3
GM06153: 17/70 CAG repeats in ATXN3; HD GM04281:
17/68 CAG repeats in HTT, DRPLA GM13716: 16/68
CAG repeats in ATN1, SCA7 GM03561: 8/62 CAG repeats
in ATXN7; and control line GM05565) were grown in
Eagle’s Minimal Essential Medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich),
antibiotic–antimycotic solution (Sigma-Aldrich), 2 mM
GlutaMAX (Gibco) and MEM non-essential amino acids
(Sigma-Aldrich).
Human neural progenitors (NPs) were derived from HD
induced pluripotent stem cells (iPSC) ND42222 (19/109
CAG repeats in HTT) obtained from NINDS Human Genetics Resource Center (Coriell Institute). For neural induction STEMdiff SMADi Neural Induction Kit (STEMCELL
Technologies) was used according to monolayer protocol,
following manufacturer’s instructions. Briefly, iPSC were
grown in Essential 8 (Gibco) medium on Geltrex (Gibco)
coated 6-well plate until 70–80% confluence was reached.
Then, iPSCs were dissociated to single cells by incubation
with 0.5 mM EDTA in PBS for 10 min. Cells were counted
using TC20 Automated Cell Counter (Bio-Rad) and resuspended at 1 × 106 cells/ml density for seeding in STEMdiff
Neural Induction Medium with SMADi and 10 nM Y-27632
(all from STEMCELL Technologies). For further cultivation
cells were detached using Accutase (STEMCELL Technologies) and after third passage they were grown in STEMdiff
Neural Progenitors Medium (STEMCELL Technologies).
All siRNA oligonucleotides (Table S2) were synthesized
by Metabion or Future Synthesis, dissolved in water to
100 µM concentration and stored at − 80 °C. To obtain
20 µM duplexes sense and antisense strands were diluted
in annealing buffer, heated for 1 min in 90 °C and kept for
gradual cooling at room temperature for 45 min.
Transfection
Lipofectamine 2000 (Invitrogen) was used to transfect HEK
293T, Flp-In T-REx-293 cells and fibroblasts with plasmids
and oligonucleotides, accordingly to the manufacturer’s protocol. 24 h prior to transfection cells were plated after estimation of cell number. To optimize and monitor transfection
efficiency control fluorescent BlockIT siRNA (Invitrogen)
or control plasmid encoding GFP (System Biosciences) was
used. Cells were harvested at specific time points indicated
in figure legends. Briefly, HEK 293T line (120,000 cells/well
seeded into 24-well plate) was co-transfected with 100 ng of
plasmid of pmirGLO construct and 50 nM oligonucleotide
using 1.5 µl Lipofectamine 2000 in 300 µl medium. Generated Flp-In T-REx-293 lines (160,000 cells/well seeded into
12-well plate) were transfected with 100 nM oligonucleotide
using 4 µl Lipofectamine 2000 in 1.2 ml of medium. Transfection of NPs was performed at fourth or fifth passage using
2 μl of siPORT Amine (Ambion) per well of 6-well plate in
1 ml of complete medium. After 3 h medium was replaced
with fresh one and after next 24 h the medium was changed
for the media lacking Y-27632. NPs were harvested using
Accutase fixed 48 h post-transfection
Protein isolation and western blot
Cells were collected at specific time points for particular
experiments (which are given in Figure legends), e.g. time
points selected for most efficient downregulation of specific
proteins in fibroblasts and NPs. Cell pellets were washed
once with PBS and lysed with PB buffer (60 mM Tris-base,
2% SDS, 10% sucrose, 2 mM PMSF). Next, the cell extract
was heated in 95 °C for 5 min and protein concentration was
estimated based on measurement at 280 nm using DeNovix
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A. Ciesiolka et al.
spectrophotometer. Equal amounts (~30 µg) of total protein were diluted in loading buffer and heated in 95 °C for
5 min and run on SDS-polyacrylamide gels: 5% stacking,
10% resolving gel in Tris/glycine/SDS buffer for ataxin-3
and luciferase detection; 3–8% NuPAGE Tris acetate gels
(Thermo Fisher Scientific) in XT Tricine buffer (Bio-Rad)
with cooling in ice-water bath for atrophin-1 and huntingtin
detection. Next, proteins were wet-transferred to nitrocellulose membrane (GE Healthcare) and specific primary (antiataxin-3, anti-huntingtin, anti-atrophin-1, anti-vinculin and
anti-Fluc) and horseradish peroxidase-conjugated secondary antibodies (anti-rabbit or anti-mouse) were used. All
antibodies used are given in Table S3. The immunodetection was performed using WesternBright Quantum HRP
Substrate (Advansta). The chemiluminescent signals were
scanned from membranes using GBOX documentation system (Syngene) and the bands were quantified using Gel-Pro
Analyzer.
RNA isolation, RT‑qPCR and ddRT‑PCR
After cell lysis in TRI Reagent (ThermoFisher), Direct-zol
RNA MiniPrep kit (ZymoResearch) or Total RNA Zol-Out
kit (A&A Biotechnology) was used for total RNA isolation.
For Flp-In T-REx-293 cell lines, a fraction of lysates prepared in Cytoplasmic Lysis Buffer [PBS, 0.1% NP40, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)] was
mixed with four volumes of TRI Reagent for further isolation. The concentration of isolated total RNA was assessed
by measurement at 260 nm using DeNovix spectrophotometer. Reverse transcription was performed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems)
and random hexamer primers (Promega), according to the
manufacturer’s protocols. RT-qPCR was performed using
SsoAdvanced Universal SYBR Green Supermix (Bio-Rad)
and CFX Connect Real-Time System (Bio-Rad), according
to the manufacturer’s protocols and established guidelines
for qPCR. Digital droplet PCRs (ddPCRs) were prepared
using DG8 cartridges and gaskets, QX200 Droplet Generation Oil and QX200 EvaGreen Digital PCR Supermix
(BioRad) and performed on QX200 Droplet Digital PCR
System (BioRad), according to the manufacturer’s protocols.
All primer sequences are listed in Table S4.
Hill coefficient calculation
The obtained results of an average relative protein level
have been fitted by GraphPad Prism to the Hill equation
curve (y = a + (b − a)/[1 + (K/x)N], where x is oligonucleotide
concentration, y is relative protein expression, a is minimal
value of y, b is maximal value of y, K is fitting parameters
and N determines the slope of the curve and the value is the
Hill coefficient, nH).
13
smFISH
Probes, buffers and protocol from Stellaris RNA FISH
technology (Biosearch Technologies) were used. Probes
3′-labelled with Quasar 670 dye were used for human
HTT (cat # SMF-20836-5) and ATXN3 (Custom Stellaris
RNA FISH probes designed using online Stellaris probe
designer, sequences are listed in Table S5), and with CAL
Fluor 590 dye for GAPDH (cat # SMF-2026-1). Cells were
fixed in 4% paraformaldehyde in PBS for 20 min at RT,
then prehybridized in Wash Buffer A containing 10% formamide for 5 min at RT. Hybridization was performed in
Hybridization Buffer with 10% formamide at 37 °C overnight. Washing was performed with Wash Buffer A for
30 min at 37 °C and next with Wash Buffer B for 5 min
at RT. SlowFade Diamond Antifade Mountant (Thermo
Fisher Scientific) was used for nuclear staining. Images
were captured with Leica DMI6000 inverted fluorescence
microscope equipped with DFC360 FX camera. Excitation/emission filters sets were Leica A for DAPI, Chroma
49005 and 49009 for CAL Fluor 590 and Quasar670,
respectively. To visually examine data, a maximum intensity z-projection of all of slices in each stack were created
using ImageJ. Signals were simplistically attributed as
nuclear based on DAPI staining. Quantification of individual RNA FISH spots was done using the StarSearch
software (https://www.seas.upenn.edu/~rajlab/StarSearch
/launch.html).
Luciferase‑based plasmids containing CAG repeat
tracts
The plasmids were generated on the basis of the pmirGLO
Vector (Promega) encoding Firefly luciferase (Fluc) and
Renilla luciferase (Rluc). CAG repeat tract sequences were
inserted into Fluc gene, either downstream (“3′UTR” and
“3′ORF” plasmids; between SalI and XbaI restriction sites)
or upstream (“5′ORF” plasmids; between restriction sites for
EcoRI and NdeI inserted in two steps using QuikChange II
XL Site-Directed Mutagenesis Kit (Agilent Technologies)).
“3′ORF” plasmid was generated by mutation of “3′UTR”
plasmid in the Fluc gene STOP codon. ATXN3 and HTTspecific inserts with normal and mutant CAG repeat tracts
were obtained by PCR using cDNA from fibroblast cell
lines (SCA3 and HD) and primers with specific restriction
sites. Short synthetic inserts (containing 17 CAG repeats
and including specific restriction sites) were chemically
synthesized (Sigma-Aldrich) and annealed for cloning. For
longer synthetic inserts we used in vitro repeat expansion
method known as Synthesis of Long Iterative Polynucleotide
(SLIP) [41, 42] with the “17CAG” insert as initial template
for the repeat tract expansion. After obtaining first plasmid
Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and…
with expanded CAG tract, we further modified a protocol
and used two plasmids with various lengths of CAG tract in
SLIP. This approach allowed for generating longer expansion
at one step. Due to DNA polymerase slipping the lengths of
mutated constructs are slightly different. Ligation of inserts
with plasmids was performed using T4 Ligase (Promega)
according to the manufacturer’s procedure. Next, competent DH5α E. coli cells were transformed and plasmids were
isolated using Endotoxin-free MidiPrep kit (Qiagen). Due to
technical problems mutant synthetic inserts are not included
for the set of “5′ORF” constructs. Sequences of DNA oligonucleotides used for cloning are given in Table S6.
Flp‑In T‑REx‑293 cell lines for inducible and stable
expression of CAG repeat tracts
All components of the designed dual-luciferase system were
cloned into pcDNA5/FRT/TO vector (Invitrogen) for obtaining inducible expression in Flp-In T-REx-293 cell lines of
either normal or mutant HTT fragment. This vector was integrated into the genome via Flp recombinase-mediated DNA
recombination at the FRT site. Sequences of Fluc and Nano
luciferase with PEST domain (NlucP) were cloned from
pmirGLO and pNL1.2 (Promega) vectors, respectively. A
bidirectional inducible promoter system (BI-16) capable of
reproducible coexpression of two proteins was constructed
[43]. Additional SV40pA sequences were cloned from
pNL1.2 (Promega) at respective sites. The full exon 1 of
HTT containing either 16 or 98 CAG repeats was amplified
in PCR and these inserts were cloned upstream of NlucP
sequence to obtain expression of HTT-NlucP fusion gene.
Sequences of DNA oligonucleotides used for cloning are
given in Table S7. The pcDNA5/FRT/TO-based expression
constructs and pOG44 vector were co-transfected (at 1:9
ratio) into Flp-In T-REx-293 host cells using Lipofectamine
2000, according to manufacturer’s protocol. Selection of
hygromycin-resistant monoclones that contain stably integrated expression cassette was performed using 100 μg/
ml hygromycin B, according to manufacturer’s protocols.
Additional details on the DNA cloning procedure and the
generation of these stable cell lines are given in Supplementary Methods.
Luciferase assay
For the results presented in the Figs. 2 and S2 assays were
performed using Dual-Luciferase Reporter Assay System
(Promega), accordingly to the manufacturer’s protocol.
Briefly, cells were lysed 24 h after transfection in Passive
Lysis Buffer (Promega), followed by the luciferase activity
measurement using Centro LB 960 Luminometer (Berthold
Technologies). The Fluc measurement data was normalized
firstly to Rluc signal in the sample, next to Fluc/Rluc signal
ratio obtained in cells co-transfected with negative control
(NTC, non-targeting siRNA) and particular plasmid, and
finally to Fluc/Rluc signal ratio measured for cells treated
with plasmid lacking CAG repeat insert and particular
oligonucleotide.
For the results presented in the Figs. 4 and 5 cells were
lysed using Cytoplasmic Lysis Buffer [PBS, 0.1% NP40,
cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)]
according to the REAP method [44]. Next, the lysates was
used in the Nano-Glo Dual-Luciferase Reporter Assay System (Promega) and measured with Victor X4 Multilabel
Plate Reader (Perkin Elmer), according to the manufacturer’s instructions. Background values of NlucP and Fluc
signals at t = 0 h were subtracted. Nluc signals for A2- or
siHTT-treated cells were normalized to NlucP measurement obtained at specific time points for siRluc-treated cells.
Finally, NlucP signal was normalized to Fluc signal in a
respective sample.
Polysome profiling
The protocol was adapted from [45, 46]. Briefly, 5 × 106
Flp-In T-REx-293 16 CAG or 98CAG cells were seeded
into 55 cm2 plate in medium without antibiotics. After 24 h
cells were transfected using selected oligonucleotides at final
concentration of 100 nM. After additional 12 h HTT exogene
expression was induced using 1 µg/ml doxycycline (SigmaAldrich) and after 3 h 100 µg/ml cycloheximide (SigmaAldrich) was added to inhibit translation elongation and fix
ribosomes on transcripts. After 5 min of incubation at 37 °C
cells were washed with ice-cold PBS containing cycloheximide and harvested in 1.5 ml of this buffer by scraping.
Cells were collected by centrifugation at 300 rpm for 5 min
at 4 °C and lysed in 500 µl ice-cold lysis buffer (10 mM
HEPES pH 7,9; 1.5 mM MgCl2; 10 mM KCl; 0.5 mM DTT;
1% Triton X-100, 100 µg/ml cycloheximide) containing also
100 μ/ml of RNasin (Promega). After 10 min incubation
on ice lysates were centrifuged at 1500g for 5 min at 4 °C.
Supernatant was collected and OD was measured at 260 nm.
10–60% sucrose gradients were prepared using Gradient Station (BioComp) in buffer containing 100 mM KCl,
20 mM HEPES pH 7.6; 5 mM MgCl2, 100 µg/ml cycloheximide; 5 µ/ml RNasin and Protease Inhibitor Cocktail
(Roche). 10 OD was loaded onto cooled sucrose gradients
and centrifuged at 39,000 rpm for 2 h and 40 min at 4 °C
using ultracentrifuge and SW 41Ti rotor (Beckman Coulter)
About twenty 0.5 ml fractions were collected using Piston
Gradient Fractionator (BioComp). Next, 0.5 ml of TRI-reagent (Ambion) was added to each fraction and subsequently
RNA was isolated, including treatment with DNase I. Equal
volumes of total RNA were reverse transcribed and HTTNlucP and GAPDH expression levels were determined by
qRT-PCR.
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A. Ciesiolka et al.
Poly(A) tail length measurements
Statistical analysis
The analysis was performed based on a polyG/I extension method [47] using the Poly(A) Tail-Length Assay Kit
(Thermo Fisher Scientific). In these experiments 5 µg/ml
actinomycin-D (Sigma-Aldrich) was added to the medium
of 16CAG and 98CAG Flp-In T-REx-293 cells to stop
transcription. 200 ng of isolated RNA from selected time
points were taken for poly(A) tail length analysis that was
performed following manufacturer’s protocol. Specific primers used are listed in Table S4. For estimation of poly(A)
tail lengths, a product obtained using gene-specific reverse
primer was used as a reference. PCR products were analyzed
on 2100 Bioanalyzer using DNA 1000 Kit (Agilent).
Analyses were performed using GraphPad Prism software. Two-tailed p value < 0.05 was considered significant and is depicted on the graphs by: *0.05 > p > 0.01;
**0.01 > p > 0.001; ***p < 0.001. All experiments which
resulted in statistically-analyzed quantitative data were
repeated at least three times (the exact number of biological
replicates, n, is given in figure legends). Depending on the
experimental setup specific statistical tests were used and
are indicated in figure legends. The error bars in the graphs
represent standard deviations.
Results
Generation of AGO2 knockout and AGO2(D597A)
mutant stable cell lines and transient AGO2
overexpression
C R I S P R- C a s 9 - m e d i a t e d AG O 2 k n o cko u t a n d
AGO2(D597A) mutant cell lines were established using
previously generated Flp-In T-REx-293 98CAG cells. For
AGO2 knockout Cas9_sg1 and Cas9_sg2 plasmids, encoding sgRNA1 and sgRNA2 which are specific for target
sequences within exon 2 of AGO2 gene, were used. Cas9_
sg3 encoding sgRNA3, binding to sequence within exon 14,
was used for AGO2(D597A) mutant cell line. To generate
these plasmids, sense and antisense DNA strands of sgRNAs were annealed and ligated into pSpCas9(BB)-2A-GFP
(PX458) (Addgene) plasmid, digested with the FastDigest
BpiI (Thermo Fisher Scientific). Chemically competent E.
coli GT116 cells (InvivoGen) were transformed with the
plasmids, plated onto ampicillin selection plates (100 μg/
ml ampicillin) and incubated overnight at 37 °C. The plasmids were isolated using the Gene JET Plasmid Miniprep
kit (Thermo Fisher Scientific) and analyzed by Sanger
sequencing. For nucleofection Flp-In T-REx-293 98CAG
cells were electroporated with the Neon Transfection System (Invitrogen). Briefly, 1 × 105 cells were harvested, resuspended in Buffer R and electroporated with 1 μg of plasmid
DNA (500 ng of each Cas9_sg1 and Cas9_sg2 plasmids) in
10 μl tips using the following parameters: 1100 V, 20 ms,
two pulses. For AGO2(D597A) cell line generation, cells
were electroporated with 1 μg of plasmid DNA and 1 μl
of 100 μM single-stranded donor oligonucleotide (ssODN)
(IDT) harboring GAC to GCC codon change. Selection of
clones is described in Supplementary Materials and Methods. The oligonucleotide sequences are included in Table S8.
For AGO2 overexpression, AGO2 coding sequence was
amplified using PCR from pIRESneo FLAG/HA Ago2 plasmid (Addgene, #10822) [48] and cloned into pcDNA3.1(+)
(Invitrogen) between HindIII/BamHI sites.
13
A2‑mediated silencing of different polyQ
disease‑related genes is varied
A large set of art-miRNAs have been tested in fibroblasts derived from patients with several polyQ diseases
(Table S1). We have now complemented our results obtained
with the A2 oligonucleotide [33, 38, 39] and present a direct
comparison of its activity when used at the same concentration in cell lines bearing similar repeat tract lengths in
mutant alleles, i.e., 62–70 CAG repeats (Fig. 1a). Overall,
we observed that the efficiency and allele-selectivity of A2
art-miRNA differed in various models of polyQ diseases.
The highest degree of allele-selectivity was achieved for
the downregulation of HTT and ATXN7, where mutant
huntingtin and ataxin-7 proteins were lowered to ~ 20% of
control level, without reduction in normal protein levels.
For ataxin-7 we also observed significant increase in normal protein level after A2 treatment [39]. Normal ATN1 and
ATXN3 alleles were more susceptible to downregulation, but
levels of normal atrophin-1 and ataxin-3 was decreased by
no more than 50% of the control level with a relatively high
concentration of A2 (50 nM) (Fig. 1a). In all of the examined disease models, A2 caused an allele-selective decrease
in mutant protein levels at a wider range of concentrations
used, as shown in the DRPLA model example (Fig. 1b).
Mutant atrophin-1 was downregulated with 20 nM A2 to
less than 20% of the control level without a reduction in normal protein level. Additionally, we analyzed the activity of
A2 at a very wide range of concentrations in HD and SCA3
models to assess potential cooperative activity, as was previously reported for other art-miRNAs [30, 31]. We obtained
a Hill coefficient (nH) value considerably > 1 (~ 1.7) what
suggests cooperative activity of the silencing machinery for
the downregulation of mutant huntingtin (Figs. 1c, S1A). On
the other hand, among the models investigated, the ataxin-3
protein was decreased in the least allele-selective manner,
and the obtained nHs suggest cooperative silencing of both
Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and…
Fig. 1 A2 activity in patient-derived fibroblasts and the characteristics of targeted transcripts. a Upper panel: table with information
about investigated models of polyQ diseases. In the last column
CAG repeat tract lengths (normal/mutant allele), present in fibroblasts cells used, are given. Middle panel: sequence of art-miRNA
A2 and predicted base-pairing of two strands within a duplex. Lower
panel: results compiled from the western blot analysis showing HTT,
ATN1, ATXN3 or ATXN7 protein levels in HD, DRPLA, SCA3 or
SCA7-patient-derived fibroblasts, respectively, after transfection
with 50 nM A2. Vinculin, GAPDH and plectin were used as reference proteins. NTC—cells treated with non-targeting siRNA. The
results of HTT and ATXN7 downregulation are from published studies [33, 39]. The following statistical tests were used: one-sample t
test with a hypothetical value = 1 for allele expression level; unpaired
t test with Welch’s correction for comparisons of normal and mutant
allele expression. n = 3 b Western blot analysis of atrophin-1 levels in
DRPLA-patient-derived fibroblasts lysed 48 h after transfection with
5, 20 or 50 nM A2. NTC—cells treated with non-targeting siRNA.
Data were analyzed using one-way ANOVA (Bonferroni multiple
comparisons test). n = 3. c Western blot analysis of huntingtin levels
in HD fibroblasts lysed 72 h after transfection with the indicated concentration of A2. The results are presented as dose–response curves
that were used to calculate the indicated Hill coefficient. NTC—cells
treated with non-targeting siRNA. See Figure S1A for more data.
n = 3 d Non-allele-specific quantification of HTT, ATN1, ATXN3
and ATXN7 transcripts with ddPCR. The results were obtained from
two sets of cDNA from independent cultures of each of five fibroblast cell lines. See Figure S1C for more data. e, f Representative
smFISH images for HTT (E) and ATXN3 (f) mRNAs in HD patientand SCA3 patient-derived fibroblasts, respectively. DAPI was used
for nuclear staining. Middle panels: GAPDH transcripts detected in
the same cells. Right panels: non-allele-specific quantification of HTT
and ATXN3 signals in healthy and patient-derived fibroblasts. Signals
were counted from at least 50 cells. NTC used in experiments presented in this figure was BlockIT siRNA
the normal and mutant alleles by A2 (Fig. S1B). Overall,
these and previous observations clearly show the targeted
transcript-dependent activity of art-miRNAs.
The cellular level of polyQ disease‑related
transcripts is relatively low
To characterize several polyQ disease-related transcripts
in more detail, we first performed their quantification in
patient-derived fibroblast cells using ddPCR. The respective transcripts were present at very low levels relative to
13
A. Ciesiolka et al.
Fig. 2 Impact of repeat tract length, location and sequence surrounding the targeted region on efficiency of downregulation by A2. a
Scheme of HTT (NM_002111.8), ATN1 (NM_001007026.2), ATXN3
(NM_004993.6) and ATXN7 (NM_000333.3) transcripts with CAG
repeat tract locations, UTRs and ORFs marked. b Scheme of dualluciferase-based constructs containing the Fluc gene fused with the
indicated CAG repeat tracts in various locations: the 3′UTR and 3′
and 5′ sites of the ORF. The exact lengths of the tracts in constructs
containing CAG repeats without (‘Pure’) or with gene-specific sur-
rounding sequences (from ATXN3 or HTT) are given in a table. c
Luciferase assay performed 24 h after cotransfection of HEK 293T
cells with 50 nM A2 and 100 ng of the indicated plasmids. NTC—
light gray: cells treated with pmirGLO plasmid and non-targeting
siRNA, dark gray: “5′ORF”-modified pmirGLO plasmid treated
with non-targeting siRNA (signal normalization details are given in
Materials and Methods). Data were analyzed using one-way ANOVA
(with Bonferroni multiple comparisons test). n = 3
GAPDH, and similar expression levels of HTT and ATXN7,
slightly lower ATXN3 levels and considerably higher ATN1
levels were observed (Fig. 1d). Five separately analyzed
fibroblast cell lines showed some variation in transcript levels but without outstanding tendency for fibroblasts with the
mutation in specific gene, e.g., the HTT mRNA level in HD
fibroblasts (Fig. S1C).
In addition, we performed single-molecule fluorescent
in situ hybridization (smFISH) for the precise quantification and visualization of selected transcripts in fibroblasts.
Microscopic analyses showed approximately 30 HTT transcripts and approximately 20 ATXN3 transcripts per cell
(Figs. 1e, f, S1D). Interestingly, the ratio of transcripts in
the cytoplasm to those in the nucleus was approximately
4:1 and 2:1 for HTT and ATXN3, respectively. This suggests
that larger cytoplasmic fraction of HTT transcripts, in comparison to ATXN3 transcripts, is available for the activation
of RISC-mediated processes in fibroblast cells. No substantial differences in specific mRNA copy number or localization were observed in the healthy cell line in comparison to
mutant cell line (Figs. 1e, f, S1D). Therefore, we conclude
that there are no significant differences in number or localization of normal vs. mutant variants of specific mRNA in
human fibroblasts, although we were able to use only nonallele-specific quantification of transcripts by ddPCR and
smFISH.
13
The presence of the targeted region in an ORF
and an HTT‑specific flanking sequences improve
the allele‑selectivity of art‑miRNAs
The results in human fibroblasts demonstrated that activity
of art-miRNAs is dependent on specific features of targeted
transcripts (Fig. 1a). PolyQ disease-related transcripts differ
considerably in the lengths of their ORFs and UTRs as well
as the localization of CAG repeat tracts. In details, CAG
repeat tract is located in HTT and ATXN7 at the 5′ end of the
ORF, while in ATXN3 at the 3′ end of the ORF, and in ATN1
in the middle of the ORF (Fig. 2a). Therefore, we decided to
elucidate how CAG repeat tract localization and sequences
Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and…
flanking CAG repeats influence silencing efficiency and
allele preference by art-miRNAs. For this purpose, we generated pmirGLO-based plasmids encoding the Fluc gene,
fused to a normal (~ 17 CAG repeats) or mutant (~ 65 CAG
repeats) tract, and Rluc as internal reference (Fig. 2b). The
repeat tract was placed at two sides of the Fluc ORF: at the
HTT-like 5′ side (“5′ORF”) or at the ATXN3-like 3′ side
(“3′ORF”), as well as in the 3′UTR, which is a typical region
for miRNA-binding sites. Inserts contained either pure CAG
repeats (P CAG) or ~ 50 nt-long HTT or ATXN3 mRNA
sequences flanking both sites of the CAG repeat tract, giving a total of 17 constructs (Fig. 2b).
First, we confirmed the expression of fusion proteins in
HEK 293T cells (Fig. S2A). Next, we co-transfected the
designed plasmids with selected oligonucleotides (artmiRNAs, siRNA targeting Fluc-siFluc or non-targeting
siRNA-NTC) and performed a dual-luciferase assay. Typical siRNA, siFluc, caused efficient reduction of expression
of all the constructs, to ~ 10% of the control level, regardless the location of the target site (Fig. S2C). In contrast,
for A2 we observed varied activity for particular constructs
(Fig. 2c). The most prominent downregulation of Fluc by
A2 was obtained for the targeted sequence location in the
“3′UTR” constructs (Fig. 2c, bars with blue background).
The luciferase signal was decreased to ~ 35% of the control
level regardless of repeat tract length and the sequence flanking CAG repeats. Downregulation of constructs expression
with the target sequence localized in the ORF of Fluc was
less efficient (Fig. 2c, bars with a red and green background),
however for constructs containing HTT flanking sequence
(both “3′ORF” and “5′ORF”) we observed significant alleleselectivity of A2 activity. In these cases, expression of the
mutant construct was decreased to ~ 50% of the control level,
whereas normal construct expression remained unchanged or
decreased to only ~ 90% of the control level (Fig. 2c). Similar results were obtained for the other art-miRNAs analyzed:
A4, G2 and G4 (Fig. S2b, c). Together, these observations
demonstrate that the allele-selectivity of art-miRNAs was
achieved only for constructs with HTT-specific sequences.
In agreement with the results obtained in patient-derived
fibroblasts (Fig. 1a), we conclude that better art-miRNAs
allele-selectivity of HTT downregulation in comparison to
ATXN3, is a combination of two effects: (1) increased downregulation of the mutant HTT allele as compared to mutant
ATXN3 and (2) decreased silencing of the normal HTT allele
in comparison with normal ATXN3.
We also considered additional features of HTT and
ATXN3 mRNAs that could affect the discrepancy in artmiRNA allele-selectivity in HD and SCA3 models (Supplementary Text). For example, the presence of rare codons,
upstream to miRNA-binding site, was shown to improve
the efficiency of miRNA silencing for targets present in
ORFs, possibly due to the decreased rate of translation [49].
Therefore, we analyzed the codon usage values in HTT and
ATXN3 transcript sequences upstream of the CAG repeat
tracts (Fig. S3), but no significant differences were found for
these mRNAs in this aspect (Supplementary Text).
A2 and siHTT caused a decrease in HTT mRNA
in the cytoplasm of HD NPs
To investigate A2 activity in a more disease-relevant cell
type, we included an additional model of human NP cells,
derived from iPSCs. As A2 acted with high allele-selectivity
for HTT silencing, we generated HD NPs. First, we characterized this cell line for the expression of neural stem cells
markers (Fig. S4a, b) and optimized oligonucleotide delivery (Fig. S4c). Next, we investigated the efficiency of HTT
expression silencing and changes in transcript abundance in
HD NPs after transfection with HTT-specific siRNA (siHTT)
or A2 art-miRNA. Similarly to results in HD fibroblasts
(Fig. 1a), in HD NPs A2 allele-selectively downregulated
mutant proteins to ~ 30% of the control level, without reduction in normal protein level, whereas siHTT decreased both
alleles of the huntingtin protein to ~ 25% of the control level
(Fig. 3a). Next, we performed a microscopic observation of
endogenous HTT transcripts targeted with A2. Using nonallele-selective smFISH, we observed a cytoplasm-specific
decrease in the huntingtin transcript number by ~ 45% after
treatment with A2 and a more substantial reduction by ~ 70%
after treatment with siHTT (Fig. 3b, c). This observation
stays in agreement with the classical model of RISC activity in cytoplasm. Cellular localization of targeted transcript
could affect the efficiency of its targeting by miRNA or siRNAs. In recent study, a larger fraction of HTT transcripts
was detected in the nuclei of healthy human neuronal cells
compared to non-neuronal cells [50]. In agreement with this
observation, we also observed an increased ratio of nuclear
to cytoplasmic HTT mRNAs in NPs, relative to fibroblasts
(Figs. 1e, 3c). It is worth to notice that, after treatment with
A2, we did not observe the retention of transcripts in the
nucleus or cytoplasmic aggregation of mRNAs, that could
result in decreased huntingtin synthesis and suggesting additional mechanisms of art-miRNA activity. Taken together,
the results obtained in HD NPs show the therapeutic potential of A2, as its allele-selective activity was achieved not
only in patient-derived fibroblasts but also in the neuronal
cell line.
Kinetic analysis of transcript and protein
downregulation shows the early events
of translation inhibition induced by A2
To verify crucial factors affecting art-miRNAs activity and
mechanistic details, we decided to include additional models
with the exogenous expression of the targeted transcripts.
13
A. Ciesiolka et al.
Fig. 3 A2 activity in human HD
neural precursors. a Western
blot analysis of huntingtin
allele levels in HD NPs lysed
48 h after transfection with
100 nM non-targeting siRNA
(NTC, BlockIT siRNA),
siHTT (siRNA for HTT) or
the art-miRNA A2. n = 4. b
Representative images showing
the smFISH-based detection of
HTT and GAPDH mRNAs in
non-treated (NT) HD NP cells
and HD NP cells treated with
100 nM fluorescent siRNA
(BlockIT), non-targeting siRNA
(NTC, siRluc), siHTT or A2.
Cells were fixed 48 h after
transfection with the indicated
oligonucleotides. c Quantification of smFISH images for the
experiments described in b. Signals were counted from at least
200 cells for each treatment.
Data were analyzed using oneway ANOVA (with Bonferroni
multiple comparisons test)
For this purpose, we designed a dual-luciferase system for
the inducible expression of HTT reporters. We generated
Flp-In T-REx-293 cell lines stably expressing exon 1 of HTT
(with 16 or 98 CAG repeats, hereafter called “16CAG” and
“98CAG” cell lines, respectively) fused with the NlucP
reporter, named HTT-NlucP (Fig. 4a). Fluc expression was
used as a normalization control, and both reporters were
placed under a bidirectional doxycycline-inducible promoter. First, we confirmed the similar expression of the
reporters and non-significant Bl-16 promoter leakage in the
absence of doxycycline (Fig. S5A, B). Next, we transfected
the 16CAG and 98CAG cell lines with the art-miRNA A2,
siRNA specific for HTT, siHTT, or non-targeting siRNA,
followed by induction of reporter expression and subsequent
analysis of the transcript and protein levels at particular time
points (Fig. 4b).
The kinetics of HTT reporter transcript and protein downregulation by A2 were clearly different in the
16CAG and 98CAG cell lines (Fig. 4c). For the mutant
HTT reporter transcript, we observed a maximum of ~ 50%
13
downregulation starting 3 h after induction, whereas for the
normal HTT reporter transcript, we detected only a slight
decrease (Fig. 4c, upper panel). In contrast to the transcript
levels, repression of the HTT-NlucP protein by A2 was more
prominent, and the mutant protein level was decreased up to
~ 30%, while the normal protein was decreased up to ~ 60%
of the control level at selected time points (Fig. 4c, lower
panel). Interestingly, at early time points (up to 2 h postinduction), A2 significantly lowered only the level of mutant
protein, suggesting that translational repression preceded
mRNA decay in the allele-selective inhibition of the mutant
HTT allele. As a reference for the typical RNAi mechanism,
we performed the same analysis with siHTT. We observed
rapid transcript and protein downregulation with no apparent
difference in activity towards the normal and mutant alleles
(Fig. 4d), suggesting the AGO2-mediated cleavage of both
transcripts and, as a result, a decrease in the protein levels.
Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and…
A2 causes a shift of mutant transcripts from heavy
polysome fractions
To assess translation inhibition caused by art-miRNA in
more detail, we performed polysome profiling analysis of
HTT reporter transcripts. The 98CAG and 16CAG cell lines
were treated with A2 or control siRNA, and lysates were
prepared 3 h after the induction of luciferase expression
(Fig. 4b, e, representative UV absorbance profiles, including a profile following disruption with EDTA, are shown
in Fig. S6a). In 98CAG cells A2 caused a statistically significant shift in HTT reporter transcript distribution in the
analyzed fractions, as referred to non-targeting siRNA treatment. We observed an approximately two-fold change in
mutant HTT-NlucP transcript abundance in selected fractions, i.e., increased cosedimentation with 40S, 60S, 80S and
first light polysome fraction and decreased cosedimentation
with heavier polysomes, as compared to the treatment with
control siRNA (Fig. 4e, upper panels). This results suggest
that A2 inhibited mutant HTT-NlucP translation at initiation and/or early elongation step. In these experiments, some
HTT-NlucP transcripts remained associated with heavier
polysomes after A2 treatment, probably because not all HTT
reporter transcripts were bound by this art-miRNA. Our conclusions are supported by analogous control experiments
performed in the 16CAG cell line which results did not show
any significant difference in the cosedimentation of HTTNlucP transcripts across the collected fractions between
A2- and control siRNA-treated cells (Fig. 4e, lower panels). Moreover, as expected, no significant changes in Fluc
expression relative to GAPDH expression were observed
in experiments using both, 98CAG and 16CAG cell lines
(Fig. S6b). Together, we conclude that observed translation
inhibition occurred very rapidly and efficiently for mutant
transcript as a result of A2 activity.
A2 induces rapid shortening of the targeted mRNA
poly(A) tail
We aimed to explain in more detail the observation that
mutant HTT reporter protein level was decreased after A2
treatment already at early time points (1–2 h) after induction, without a change in the level of its transcript (Fig. 4c).
This can be explained as the effect of direct translation inhibition or transcript deadenylation, which in turn results in
reduced translation due to disrupted transcript circulation.
To verify the latter mechanism, we performed transcription
pulse-chase experiment and examined the length of poly(A)
tails in HTT reporter transcripts using poly G/I extension
followed by resolution of the PCR products in a microfluidic chip (Fig. S7a). In details, after transfection of the
98CAG and 16CAG cell lines with A2 or control siRNA, we
induced expression of the HTT reporter for 1 h, stopped the
transcription and then analyzed poly(A) tail length profiles
at three time points (Fig. 4b, f).
In the 98CAG cell line, A2 caused significant deadenylation of HTT-NlucP transcript already at the 60 min time
point, when a substantial pool of HTT reporter transcript
deadenylation intermediates with a short (~ 30 A) poly(A)
tail appeared (Fig. 4f, upper panels). The formation of transcripts with shortened poly(A)-tails further accelerated at
the 80 min and 240 min time points (Fig. 4f, upper panels).
In contrast, we did not observe these deadenylation intermediates at even the 240 min time point after treatment of
the 98CAG cell line with control siRNA. Analogous experiments in the 16CAG cell line showed no significant difference in the poly(A) tail length profiles of A2- and control
siRNA-treated cells at the 60 and 80 min time points and
only a slight difference at the latest 240 min time point
(Fig. 4f, lower panels). In both cell lines and following treatment with A2 and control siRNA, we observed significant
changes in the length of the poly(A) tail at 240 min compared to that at earlier time points (Fig. S7b), indicating
events typical of transcript decay after transcription arrest.
Taken together, A2 caused rapid deadenylation only of
mutant HTT-NlucP transcript, suggesting crucial role of
this process in the art-miRNA-mediated repression. Deadenylation is expected to lead to mutant transcript degradation
observed at later time points (3–24 h) (Fig.4c).
AGO2 is dispensable for the A2‑mediated silencing
of HTT expression
Our next step was to elucidate the role of AGO2 in artmiRNA mechanism. We wanted to determine if AGO2
presence or the activity of its catalytic subunit is required
for art-miRNAs-mediated silencing. To verify this, we modified the 98CAG cell line using CRISPR-Cas9 technology.
We created homozygous cell lines in which AGO2 gene
was knocked out (AGO2del) or a cleavage-deficient AGO2/
D597A protein was expressed (AGO2mut) (Fig. S8a, b). The
D597A mutation in AGO2 is known to abolish RNA cleavage without affecting efficiency of siRNA binding or translational repression [51, 52]. Two sgRNAs were designed to
knock out AGO2 by deletion of a gene fragment in exon 2
leading to premature STOP codons (Fig. S8a, b), whereas an
approach using one sgRNA and a donor template containing a specific nucleotide mutation was used to introduce a
catalytic mutation in AGO2 (Fig. S8c, d). We selected the
final clones based on the results of DNA sequencing (Fig.
S8b, d) and AGO2 immunoblotting (Fig. 5b).
To analyze the requirement of AGO2 in A2 and siHTT
activities, we analyzed both the transcript and protein levels
of the mutant HTT reporter in 98CAG-AGO2mut and -AGO2del cell lines. In addition, we performed rescue experiments and transfected cell lines with plasmids encoding WT
13
A. Ciesiolka et al.
13
Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and…
◂Fig. 4 Mechanistic details of A2 activity in stable cell lines with
inducible expression of the HTT fragment. a Constructs used to generate Flp-In T-REx-293 cell lines with two-directional, inducible
expression of the HTT fragment (exon 1 with 16 or 98 CAG repeats)
fused with NlucP. Fluc expression was used as a reference. b Timeline of the experiments presented in this figure. Specific treatment
and cell lysis time points are indicated. c, d Results of RT-qPCR
(upper panels) and dual-luciferase assay (lower panels) to determine the HTT-NlucP transcript level and HTT-NlucP protein signal,
respectively, after transfection of the 16CAG and 98CAG cell lines
with 100 nM A2 (c) or siHTT (d) at the indicated time points. The
results were normalized to the mRNA level/protein signal of Fluc
in the same sample and are shown as the relative expression level/
relative protein signal of HTT-NlucP in cells transfected with 100 nM
control siRNA (NTC, siRLuc). n = 3. e Results of RT-qPCR to assess
HTT-NlucP expression levels in the indicated fractions containing
ribosomal subunits (40S and 60S), the 80S monosome and polysomes
(P-P9) after transfection of the 98CAG (upper panels) or 16 CAG
(lower panels) cell lines with 100 nM A2 or control siRNA (NTC,
siRLuc) at 3 h after induction. Data from each fraction were normalized to GAPDH expression and are presented as the % of HTT-NlucP
expression in which 100% is the sum of the obtained values for all
fractions. Graphs in the right panels show data with values calculated
as the % difference in values obtained for separated fractions for control siRNA vs. A2. n = 3. The data for c–e were analyzed using twoway ANOVA. f Analysis of the poly(A) tail length of the HTT-NlucP
transcript in the 98CAG (upper panels) or 16CAG (lower panels) cell
lines at the indicated time points (60, 80, 120 min) after transfection
with 100 nM A2 or control siRNA (NTC, siRLuc). Estimated poly(A)
tail lengths are indicated. The experiment was repeated (n = 2), and
similar results were obtained. A0—peak obtained with reporter-specific primers to amplify a region upstream of the polyadenylation site.
*An internal standard peak (1500 bp upper marker)
AGO2 (Fig. 5b). Based on previous experiments (Fig. 4c),
we selected early time point of 3 h after the induction of
HTT-NlucP expression (Fig. 5a) which we found suitable for
analysis of the details of A2 activity. As in previous experiments, in this time point in the 98CAG cell line (Fig. 4c,
d), both A2 and siHTT repressed HTT-NlucP expression
up to 50% of control level (Fig. 5c). As expected for the
AGO2mut and AGO2del cell lines transfected with typical
siRNA, siHTT, repression of HTT-NlucP was completely
abolished at both the transcript and protein levels and could
be restored after WT AGO2 overexpression (Fig. 5c, blue
bars). In contrast, after A2 transfection into AGO2mut and
AGO2del cell lines, efficient lowering of both, transcript and
protein levels of HTT-NlucP was achieved (Fig. 5c, orange
bars). Additionally, we observed no substantial effects of
WT AGO2 overexpression on A2 and siHTT activities.
Together, our results show that A2-mediated downregulation of HTT reporter expression is mostly independent of
AGO2-mediated slicer activity but is rather a consequence
of transcript deadenylation and translation inhibition. Moreover, considering canonical miRNA-related mechanisms,
our observations suggest that other AGO proteins (AGO1,
AGO3, AGO4) and their respective miRISCs are sufficient
in mutant transcript repression caused by A2, in the absence
of AGO2.
Discussion
Normal and mutant alleles of polyQ diseases‑related
genes show varied susceptibility to regulation
by art‑miRNAs
Art-miRNAs were designed to target mutation site, i.e.
expanded CAG repeat tract, in several transcripts implicated
in polyQ diseases [25, 26]. The main rationale behind such
design was to generate the universal treatment for these rare
disorders. However, during research we and others (Supplementary Table 1) observed varied susceptibility of targeted
transcripts to regulation by art-miRNA (Fig. 1a).
The common feature of polyQ diseases-related mRNAs
is their rather low cellular level, including the brain regions
mostly affected in polyQ diseases [53]. We confirmed the
relatively low expression of four selected genes in patientderived fibroblasts (HTT, ATN1, ATXN3 and ATXN7), however, we also found substantial differences between the quantities of these mRNAs (Fig. 1d). In previously published
studies, turnover rate and cellular abundance of transcripts
were found to influence the efficiency of downregulation
mediated by RNAi [54, 55]. Therefore, these factors might
contribute to the observed differences in A2 activity for various transcripts, i.e., higher efficiency of ATN1 silencing may
result, at least partially, from higher expression level of this
gene.
In addition to the cellular factors, we looked at polyQ
disease-related transcripts and noticed that they differ in
their arrangement of specific regions as well as the location
of the repeat tract within the ORF (Fig. 2a). To address specific questions concerning the impact of CAG repeat tract
localization on differences in allele-selective silencing by
art-miRNAs, we developed cellular models with exogenous
expression of the designed constructs (Fig. 2b). Silencing of
exogenes expression may differ from that of endogenes, as
higher expression levels of exogenes are obtained, and all
additional sequences that could affect silencing efficiency
are only present in endogenes. Nevertheless, we observed
clear tendencies in the potency of art-miRNAs depending on the location of their targeted site and its flanking
sequences (Fig. 2c). Clearly, CAG repeat tract length also
determines art-miRNA activity, as large differences were
observed for normal and mutant alleles silencing. Moreover, we revealed that it is crucial that the targeted sequence
is present in the ORF, as the presence of the targeted tract
in the 3′UTR caused efficient silencing of both normal and
mutant alleles (Fig. 2c). Possibly this preference is caused
by the lack of ongoing translation in 3′UTRs, in contrast to
ORF regions where ribosomes interfere with the miRISC
complexes [56]. Additionally, unique features of each transcript and the context of the targeted sequence can affect
13
A. Ciesiolka et al.
could contribute to diversity in efficiency of polyQ diseasesrelated genes silencing by A2 [58, 59].
Art‑miRNAs activate events of mRNA deadenylation
and translation inhibition
Fig. 5 Verification of the involvement of AGO2 in A2 activity. a
Timeline for the experiments presented in this figure. Specific treatment and cell lysis time points are indicated. b Western blot analysis
of AGO2 protein levels in 98CAG Flp-In T-REx-293 stable cell lines
(98CAG standard cell line, AGO2del deletion of endogenous AGO2,
AGO2mut abolished catalytic activity of slicer domain). +AGO2 cell
lines after transfection with AGO2 WT plasmid to rescue protein.
c Results of RT-qPCR and luciferase assays to detect HTT-NlucP
mRNA and protein levels, respectively, after transfection of the
98CAG cell lines (including AGO2mut and AGO2del) with 100 nM
A2 or siHTT as well as the indicated plasmids. Data were normalized
to Fluc expression levels in the same sample and HTT-NlucP expression levels after transfection with 100 nM control siRNA (NTC,
siRLuc). The data were analyzed using two-way ANOVA (with Bonferroni multiple comparisons test among a set of samples for each of
the cell lines). n = 3
efficiency and allele-selectivity of art-miRNAs. One such
factor may be the structure formed by the CAG repeat tract,
the stability of which was shown in vitro to be dependent on
the flanking sequence (reviewed in [57]). Moreover, additional factors, like varying distance of the targeted site from
STOP codon or from 3′ and 5′-ends of transcript (Fig. 2a),
13
MiRNA-mediated regulation is known to occur in many
ways depending on the activating miRNA and targeted
mRNA, which can affect each other through multiple
miRISC components [60, 61]. As the repertoire of miRISC
proteins activate various cellular processes, the detailed
analysis of a particular gene silencing mechanism is complex [3, 62]. Briefly, this silencing mechanism involves
AGO-mediated recruitment of the GW182/TNRC6 protein
family [51, 63], followed by subsequent binding of poly(A)binding protein (PABPC), mRNA deadenylase complexes
PAN2-PAN3 and CCR4-NOT, catalyzing deadenylation of
the mRNA target, eventually leading to target decapping
and transcript degradation ([64–67], reviewed in [2]). We
show that art-miRNAs targeting expanded CAG repeats
in ORF regions cause translation inhibition (Fig. 4e, top
panel) and activate rapid mRNA deadenylation (Fig. 4f,
top panel), similarly to classical miRNA pathway. Moreover, mRNA deadenylation and translational repression in
typical miRISC-mediated gene silencing were also shown
to be interconnected when AGO-miRNAs bound 3′UTRs
(reviewed in [3, 62]). Nevertheless, the vast majority of
endogenous miRNAs target 3′UTR sequences and cause
mRNA decay (estimated at 66–90%) that is directly responsible for the protein downregulation [62, 68, 69]. In this
study, we initially observed stronger lowering of protein
level, than mRNA level (Fig. 4c), suggesting that for a
pool of transcripts targeted by art-miRNAs in ORF region,
translation was inhibited without activation of mRNA decay.
However, when we performed detailed poly(A) tail length
analysis after A2 art-miRNA treatment, deadenylation was
observed already at the earliest time point analyzed after
transgene induction (60 min) (Fig. 4f, top panel). On the
other hand, our data obtained from polysome profiling suggested that A2-activated translational repression occurs
also very rapidly, 3 h after transgene induction (Fig. 4e, top
panel). Therefore, we cannot exclude that deadenylation preceded or was concurrent with translation inhibition. Indeed,
in some cases, translational inhibition was shown to precede poly(A) tail shortening and mRNA decay. Such cases
were: a study performed in HeLa cells [8] and a study using
a Drosophila S2 cell-based controllable expression system
[70] where miRNA-targeted sequences were localized in the
3′UTR. Here, to understand the investigated mechanism, it
was also crucial to determine if ribosome complexes are
formed on targeted transcripts as the result of art-miRNA
activity. Our data obtained from polysome profiling suggest
that A2-activated translational repression can occur at the
Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and…
elongation step and/or at the initiation step (Fig. 4e). This
conclusion is supported by the comparison of the shift of
HTT transcript, observed after A2 treatment, with the profiles of mRNA distributions in monosome and polysome
fractions characteristic for global inhibition of translation
at the initiation or elongation step [71, 72].
Transcript-dependent factors, affecting the activation
of deadenylation and translation inhibition processes, may
also contribute to differences in the effectiveness of artmiRNA in different models of polyQ diseases. According
to translation-dependent closed-loop model [73, 74], it can
be assumed that, although CAG repeats are in a very large
distance from the poly(A) tail (especially for HTT transcript
~ 13 kb), art-miRNA-bound miRISCs are in close proximity
to the poly(A) tails of polyQ disease-related mRNAs in cells
(Fig. 6). In this case, miRISC-mediated translational repression can also occur through recruitment of the RNA helicase DDX6, which acts as both a translational inhibitor and
decapping activator [75–77]. Moreover, it was also shown
that DDX6 can act by displacing the eukaryotic translation
initiation factors eIF4A-I and eIF4A-II from the targeted
transcripts, thereby preventing translation initiation [10, 78,
79]. These processes are dependent on features of 3′UTR
region of targeted transcript, as various cis- and trans-acting elements in specific 3′UTRs were found to influence
miRNA-mediated gene expression regulation [80].
The art‑miRNA mechanism as a model
for the AGO‑dependent cooperative activities
of miRNAs within ORF regions
It is known that miRNAs regulate genes expression mostly
by recognition of sites within 3′UTR, however numerous
miRNA-binding sites were revealed by global approaches
also in the ORFs of human mRNAs [81, 82]. The functionalities of approximately twenty sites of this type in
specific transcripts have been experimentally confirmed
so far (reviewed in [83]), but precise mechanisms have not
been extensively investigated. Recently, a specific type of
miRNA recognition elements exclusive to ORF regions
was described, and a mechanism of gene expression regulation by temporary ribosome stalling was proposed for
DAPK3 kinase [84]. Based on the results of our study, we
can extrapolate mechanistic details of ORF regions-targeting by miRNAs, especially when multiple binding sites are
present. Indeed, for miRNA-based regulation within ORFs,
multiple binding sites have been found frequently [83]. One
such example is the regulation of the expression of a family
of genes containing C2H2 zinc-finger domains by a group
of miRNAs [85, 86]. Additionally, a general role of repeat
tracts localized in ORFs in post-transcriptional regulation
was suggested based on predicted interactions [86]. Overall,
these data and results regarding art-miRNA activity suggest
Fig. 6 Model of art-miRNA activity targeting transcripts containing expanded CAG repeat tracts within the ORF region. Art-miRNA
loaded into AGO binds to mutant CAG repeat tract with a mismatch
formed in the central region of this interaction. Multiple binding or
shuttling of the AGO protein results in the formation of the miRISC,
which affects translation by the inhibition of its initiation or early
elongation. Shortening of the poly(A) tail is activated for a pool of
targeted transcripts that leads to the subsequent degradation of mRNA
that for the efficient regulation of a gene’s expression by
targeting its ORF, multiple binding sites are required, resulting in cooperative action. Some mechanistic details of the
cooperative activity of miRNAs were revealed, e.g., a FRETbased method was used to show that AGO2 dissociation is in
kinetic competition with lateral diffusion, resulting in shuttling between adjacent target sites [87].
In general, human cells express four AGO paralogs
(AGO1-4) that act to regulate miRNA-based gene expression [88]. AGO2 is the most abundant, as it accounts for
~ 70% of the total AGO pool in HEK 293T and fibroblast
cells [89, 90], in which we performed most of mechanistic experiments. Previously, discrimination between the
normal and mutant alleles of HTT mRNA by art-miRNAs
was reported to be highly sensitive to the cellular pool of
AGO2 and GW182 family proteins [30]. Our analysis in total
AGO2 knockout and AGO2 D597A endonuclease-deficient
cell lines, showed that the absence of AGO2 does not affect
the observed silencing activity of exemplary A2 art-miRNA
(Fig. 5c). These results suggest that other slicer-deficient
AGOs (AGO1, 3 and/or 4) can act in the cooperative repression of the mutant allele. Nevertheless, we do not rule out
that AGO2 is a key, most abundant miRISC core protein, but
we show it may be replaced by other AGOs. These results
are consistent with previous observations that the majority
of human miRNAs associate with all four AGOs and do not
have a preference for a particular AGO paralog [20, 91, 92].
We assume, that after art-miRNA-AGO complex binding
to mRNA, the subsequent co-recruitment of the GW182/
TNRC6 with other effector miRISC proteins results in alleleselective inhibition of the mutant allele. Our data suggest
that art-miRNAs can be additionally recruited by slicerdeficient AGO proteins (AGO1, 3 and/or 4) to the expanded
13
A. Ciesiolka et al.
CAG repeats (Fig. 6) what might turn out to be advantageous
for the experimental therapy based on art-miRNA reagents.
This is due to the previous RNA-seq and mass spectrometry
analysis that clearly indicate that in brain tissue the relative
abundance of AGO1, AGO3 and/or AGO4 (when related
to the total AGO pool) are higher than in many other cells
and tissues analyzed [89, 93]. This particularly applies to
AGO1 protein as quantitative proteomic approach revealed
in HEK 293T cells the following proportions of AGO proteins: ~ 17% AGO1, ~ 75% AGO2, ~ 6% AGO3 and ~ 2%
AGO4, whereas similar mass spectrometry analysis conducted on mouse brain lysates showed proportions: ~ 35%
AGO1, ~ 55% AGO2, ~ 9% AGO3 and < 1% AGO4 [89].
Art‑miRNA activity in the context of other
therapeutic strategies for polyQ diseases
We are now witnessing large advances in antisense oligonucleotide (ASO)- and RNAi-based strategies in clinical trials [94, 95]. The most advanced clinical trial of a causative
therapy for HD involves the intrathecal delivery of RNaseH-activating ASOs targeting HTT (ClinicalTrials.gov Identifier: NCT03842969). The results obtained thus far are very
promising, as a decrease in huntingtin in the spinocerebellar fluid was reported [96]. Nevertheless, many challenges
remain to be faced in the developed therapy, among which
the allele-selectivity of silencing is one of the major points
[97]. Preservation of the level of the normal allele might be
required, as its long-term downregulation, which would be
the result of long-term treatment, could have many adverse
effects [98, 99]. Additionally, in the case of HD, some
reports suggest that targeting exon 1 of HTT, which contains
the CAG tract, may be crucial to eliminate key toxic entities
causing HD pathogenesis [100, 101]. For these reasons, we
find an approach using art-miRNAs to be desirable. This
CAG repeat-targeting strategy offers an option for the preferential silencing of several mutant alleles responsible for
polyQ diseases and would be applicable to a larger group
of patients than an allele-selective SNP-targeting-based
approach. Moreover, due to somatic instability, mosaicism
of highly expanded CAG repeats in the brain is likely a common effect largely responsible for brain-specific pathology,
as shown in HD [102–104]. In this case, transcripts containing increasingly expanded CAG repeat tracts are expected to
be more efficiently targeted by art-miRNAs. This assumption
remains to be proven experimentally but would clearly allow
preferential targeting of the RNAs which translation leads
to pathogenesis.
The potential universality of targeting the mutation site
in RNA was also shown for the activity of ASOs acting as
translation blockers or splicing modulators that were tested
for several polyQ diseases [105–107]. According to recent
findings, an art-miRNA-based therapeutic strategy might
13
not be applicable for SCA1 [108], and the development of a
universal molecule for several polyQ diseases might require
further research. Although more demanding than initially
assumed, the applicability of this strategy in at least a few
disorders remains feasible. Art-miRNAs possess additional
advantages as they can be chemically modified [31, 38] or
expressed from vectors [33, 109], and can include application of novel approaches for delivery to the brain [110,
111]. Interestingly, CRISPR-Cas9- and ZFP-based CAG
repeat-targeting strategies were recently successfully tested
in HD models [112–116]. These approaches offer an alternative solution for mutant HTT inhibition after the binding of
specifically designed molecules to mutant DNA, but some
challenges remain before their clinical testing.
In summary, our model of art-miRNAs activity show
potential versatility in the miRNA-based regulation of
gene expression. Although this model (Fig. 6) is based on
results obtained for artificial miRNA, it contributes to a better understanding of the mechanisms of action of natural
miRNAs which interact with sequences located in ORFs
with adjacent multiple binding sites. These mechanisms
have been harnessed to activate the therapeutically beneficial silencing of mutant genes with CAG repeat expansions,
showing the great flexibility of RNAi-based mechanisms
in cells.
Acknowledgements This work was supported by Grants from National
Science Centre [2014/15/B/NZ1/01880 to WJK/AF, 2015/17/D/
NZ5/03443 to AF, 2015/19/B/NZ2/02453 to WJK/ACie, 2015/17/N/
NZ2/01916 to EK]; and Polish Ministry of Science and Higher Education [DI 2011 0278 41 to ASC and 01/KNOW2/2014, the KNOW
program]. Microscopic images were obtained in the Laboratory of
Subcellular Structures Analysis, IBCH PAS. The authors would like
to thank former and current members of the Department for discussions
and contributions to the preliminary analyses of this study.
Author contributions Conceptualization: WJK, AF, ACie and ASC.
Transfections and western blot on fibroblasts: AF, ASC and DZ.
DdPCR: AF and MWW. Design of luciferase-based plasmids, relevant
experiments and bioinformatics analyses of transcripts features: ASC.
Design of luciferase-based plasmids used for Flp-In T-REx cell line
generation, luciferase assay experiments: ACie. Flp-In cell lines, RTqPCR and poly(A) tail length assay experiments: ACie, PJ. Optimization of polysome profiling method: ACio, KDR and DZ. Polysome
profiling experiments and relevant RT-qPCR: ACio. Generation of NPs
and relevant smFISH: EK. SmFISH on fibroblasts: MM. AGO2 mutant
and knockout cell lines: MD, MO and ACie. Manuscript writing: AF,
ASC and ACie, with the input and revision from all authors.
Compliance with ethical standards
Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long
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