Supplementary Information
Supplementary Methods ......................................................................................... 2
References ............................................................................................................... 5
Table S1. Overview of sequencing samples .......................................................... 6
Table S2. Genome coverage of each sequencing library by regions .................. 6
Table S3. List of ADAR1-binding transcripts identified in the study ................... 6
Table S4. List of primers used in this study .......................................................... 6
Legends to Supplementary Figures ....................................................................... 7
1
Supplementary Methods
Reagents and antibodies
All chemicals were purchased from Sigma (St. Louis, MO), except where otherwise
indicated. Anti-cleaved caspase 3 (polyclonal), anti-α-tubulin (polyclonal) and
anti-DNM1 (monoclonal) antibodies were from Cell Signaling Technology (Danvers,
MA, USA). Monoclonal antibodies against MyoG, MyoD, MHC, ADAR1, ADAR2, and
GAPDH, as well as polyclonal antibodies against PKR and phosphorylated form of
PKR were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Anti-ADAR1 rabbit polyclonal antibody was purchased from Abnova (Taiwan).
ARHGAP26 and XIAP rabbit polyclonal antibodies were purchased from Abcam
(Cambridge, MA, USA). Anti-Anxa4 antibody was obtained from EnoGene Biotech
(New York, NY, USA). Secondary antibodies used in the western blot assays were
from Jackson Laboratories (West Grove, PA, USA), whereas those used in
immunofluorescence analysis were obtained from Invitrogen.
RNA extraction and quantitative reverse transcription (RT)-PCR
Total RNA from cells was isolated using the TRIzol reagent (Invitrogen) according to
manufacturer’s instructions. cDNA and microRNA cDNA were synthesized by MMLV
reverse transcriptase (Invitrogen) using random hexamers and microRNA RT-loop
primers, respectively. Sequences for the microRNA RT-loop primers and primers for
real-time PCR assays are listed in Supplementary Table S4. Quantitative
determination of the cDNA levels was done by real-time PCR using the Bio-Rad iQ5
Gradient Real Time SYBR-Green PCR system. Levels of cDNA were normalized to
the GAPDH values of the respective samples, and microRNA cDNA was normalized
to U6 control. All results represent the mean ± SD of at least three independent
experiments.
Cell lysate preparation
Cells were harvested and washed twice in PBS. Whole cell extracts were prepared
using WCE buffer [20 mM HEPES, pH 7.4, 0.2 M NaCl, 0.5% Triton X-100, 5%
glycerol, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 2 mM Na3VO4, 1 mM
NaF, 1 mM DTT, and cocktail protease inhibitor (Roche)]. After 30-min incubation on
ice, whole cell extracts were collected by centrifugation (12000 g, 20 min).
Bromodeoxyuridine (BrdU) ELISA cell proliferation assay
The proliferative capacity of cells was measured using a BrdU ELISA cell proliferation
assay (Millipore) according to the manufacturer’s instructions. Cultured cells were
incubated with BrdU labelling solution for 12 hrs and incubated with monoclonal
antibody against BrdU for 1 hr. The BrdU absorbance was measured directly using a
spectrophotometric microplate reader at a test wavelength of 450 nm and a reference
wavelength of 540 nm. This provided a measure of the degree of proliferation of cells,
which we termed the proliferation index. Each sample was cultured in quadruplicate.
Western blot analysis and immunoprecipitation
2
Western blot analysis was performed after electrophoretic separation of polypeptides
by SDS-PAGE and transfer to Immobilon-P/PVDF membrane (Millipore). Blots were
probed with the indicated primary and appropriate secondary antibodies.
Immuno-bands were subsequently detected by the enhanced chemiluminescence
reaction (ECL) (PerkinElmer). All immunoprecipitations were performed with equal
amounts of cell extract protein incubated with the indicated antibodies (2.5 μg) at 4°C
for overnight with rotation. The immunocomplexes were captured with protein
G-sepharose (30 μl) (Millipore) for 2 hrs at 4°C with rotation. The protein
G-antigen-antibody complexes were washed six times with the WCE buffer, and
boiled in 2× urea sample buffer dye for subsequent PAGE and immunoblotting
analysis as described above.
Sequence alignments and microRNA target prediction
The potential microRNA target sites within the 3’UTR of ADAR1 were predicted using
web-based bioinformatics database TargetScan (http://www.targetscan.org).
Synthetic microRNA mimics
For ectopic expression of miRNA mimics and antagomers, C2C12 myoblasts or HeLa
cells were transiently transfected with the indicated synthetic RNA oligonucleotides
and corresponding control (Thermo Fisher Scientific) using Lipofectamine 2000
(Invitrogen). Overexpression of miR-206 was also performed by a plasmid-based
system (pcDNA6.2) harboring coding sequence for either miR-206 or LacZ and
subsequent transfection using Lipofectamine 2000 (Invitrogen).
Promoter and 3’UTR reporter constructs, mutagenesis, and luciferase reporter
assay
To generate 3’UTR reporter, a 2,268-bp PCR fragment corresponding to mouse
ADAR1 3’UTR was subcloned into the luciferase vector pMIR-REPORT (Ambion).
Site-directed mutagenesis was employed to generate a mutant version that harbor
altered miR-1/206 target site (predicted seed complement GGAATGT mutated to
GGTTAGT; Mt). For 3’UTR reporter assay, C2C12 cells were seeded in 12-well plate
before transfection. Co-transfections were done with the reporter constructs and the
indicated synthetic miRNA using Lipofectamine 2000 (Invitrogen). After 48 hr
incubation, cells were lysed in Reporter Lysis 5× Buffer (Promega) and centrifugated
to remove cell debris. Luciferase activity was measured by a Dual-Luciferase
Reporter Assay System (Promega). The co-expressed β-gal was used as loading
control.
Over-expression and ectopic expression constructs
Plasmid construction was based on the pFLAG-CMV (Sigma), pCMV-SPORT6 (for
C-terminal FLAG tagging), and pIRES2-EGFP system (Clontech), which is also driven
by the CMV promoter. For over-expression during initial differentiation, cDNAs
encoding full-length mouse ADAR1 p150 and p110 were expressed from
pFLAG-CMV. Mutations were created by PCR-based mutagenesis in the dsRBDs
3
(K618/619/622/726/727/730A) or the catalytic domain (E613A for p110 or E861A for
p150) of ADAR1, and corresponding constructs were generated as described
previously (Lai et al., 1995; Valente and Nishikura, 2007). PCR mutagenesis was
done using HiFi Hotstart DNA Polymerase (KAPA Biosystems) with mutagenic
oligonucleotides creating specific codon changes (mutations in bold): dsRBD-EAA2,
5’-GTGAGCGCCCCCAGCGAGGCGGTAGCAGCGCAGATGG-3’;
dsRBD-EAA3,
5’-CGTGTGTGCACACAGCGAGGCACAGGGCGCGCA-3’;
and
CT-EA,
5’-GTCAATGACTGCCATGCCGCAATCATCTCCCGGAGGGGCTTC-3’.
For ectopic expression of ADAR1 during late differentiation, we modified the
pFLAG-CMV and pIRES2-EGFP vector by replacing the CMV promoter with
muscle-specific MCK upstream regulatory sequence (Takeshita et al., 2007). The
mouse MCK promoter (nucleotides -1256 to 0 relative to transcript start site) was
PCR-amplified from genomic DNA isolated from C2C12 cells. The CMV promoters in
pFLAG-CMV and pIRES-EGFP were released by restriction digestion subsequently
replaced with the MCK promoter amplicon, products of which were respectively
termed pFLAG-MCK and pIRES2-MCK.
Indirect immunofluorescence and confocal microscopy
C2C12 cells were fixed in 4% paraformaldehyde and permeabilized in 0.5% Triton
X-100. Cells were then blocked in blocking buffer (1% BSA in PBS) and probed with
the indicated primary antibodies and species-specific secondary antibodies (Alexa
488- or 594-conjugated anti-mouse or anti-rabbit IgG). Cell nuclei were
counter-stained with DAPI (Sigma); F-actin was stained with phalloidin. Images were
captured on a Zeiss LSM 510 Meta confocal laser-scanning microscope (Carl-Zeiss,
Feldbach, Switzerland), using a 63×/NA 1.4 oil immersion objective lens. The fusion
index was calculated as the ratio of nuclei number in myocytes versus the total
number of nuclei. Each data point was generated from at least 1,500 randomly
chosen MHC-positive cells or myotubes.
Nuclear run-on assay
The nuclear run-on assay was carried as described previously (Hsieh et al., 2011).
Cells were suspended in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM
MgCl2,
Digitonin). After 10-min incubation, nuclei were collected and then
washed with lysis buffer devoid of Digitonin. To perform run-on reactions, aliquots of
nuclei were mixed with 100 µl of 2× reaction buffer (20 mM Tris-HCl, pH 8.0, 5 mM
MgCl2, 200 mM KCl, 4 mM dithiothreitol, 1 mM each of ATP, CTP, and GTP, 200 mM
sucrose and 20% glycerol) and biotin-16-UTP (Roche) in a final volume of 200 µl at
29°C for 30 min. A total of 60 U of RNase-free DNaseI (Fermentas; Burlington,
Ontario, Canada) and 6 µl 250 mM CaCl2 were added, and the reaction mixture was
incubated for an additional 10 min at 37°C. The nuclear run-on RNA and total RNA
were then digested with DNase I (Ambion) to further remove contaminating genomic
DNA. Biotinylated RNA was purified by Dynabeads M-280 (Invitrogen), a magnetic
bead covalently linked to streptavidin. Beads were washed once with 500 µl 2×
4
SSC-15% formamide for 10 min and twice with 1 ml 2× SSC for 5 min each. Random
hexamer-primed cDNA was synthesized using 10 µl biotinylated RNA and 500 ng total
RNA, and subsequently subjected to reverse transcription-quantitative PCR to assay
for RNA transcription rate. To ensure the efficiency of the reverse transcription, the
intensities of PCR products were normalized to those of U6 snRNA.
Nuclear and cytoplasmic fractionation
Cells were washed once with PBS and lysed with the nuclear fractionation buffer
containing 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 0.2% NP-40, 3 mM MgCl2, protease
inhibitor (Roche), 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate and 1
mM sodium fluoride for 10 min at 4°C. Cells were centrifuged at 800 g for 5 min at 4°C.
The supernatant was removed and used as a cytoplasmic fraction. The pellet was
washed once with nuclear fractionation buffer and centrifuged again at 800 g for 10
min at 4°C. The pellet was used as a nuclear fraction. Cytoplasmic and nuclear
fractions were controlled using GAPDH and Lamin B as respective markers.
References
Hsieh, C.L., Lin, C.L., Liu, H., Chang, Y.J., Shih, C.J., Zhong, C.Z., Lee, S.C., and Tan,
B.C. (2011). WDHD1 modulates the post-transcriptional step of the centromeric
silencing pathway. Nucleic Acids Res 39, 4048-4062.
Lai, F., Drakas, R., and Nishikura, K. (1995). Mutagenic analysis of double-stranded
RNA adenosine deaminase, a candidate enzyme for RNA editing of glutamate-gated
ion channel transcripts. J Biol Chem 270, 17098-17105.
Takeshita, F., Takase, K., Tozuka, M., Saha, S., Okuda, K., Ishii, N., and Sasaki, S.
(2007). Muscle creatine kinase/SV40 hybrid promoter for muscle-targeted long-term
transgene expression. Int J Mol Med 19, 309-315.
Valente, L., and Nishikura, K. (2007). RNA binding-independent dimerization of
adenosine deaminases acting on RNA and dominant negative effects of nonfunctional
subunits on dimer functions. J Biol Chem 282, 16054-16061.
5
Table S1. Overview of sequencing samples
(Please see separate MS Excel file.)
Table S2. Genome coverage of each sequencing library by regions
(Please see separate MS Excel file.)
Table S3. List of ADAR1-binding transcripts identified in the study
(Please see separate MS Excel file.)
Table S4. List of primers used in this study
(Please see separate MS Excel file.)
6
Legends to Supplementary Figures
Figure S1.
C2C12 cells were transfected with plasmid construct encoding C-terminally
FLAG-tagged wild-type p150. The empty vector was used as a control (ctrl). Cells
were analyzed for expression levels of the indicated proteins. Expression of the
FLAG-p110 form from this construct, denoted by black arrowheads, may be an
indication of alternative translation via internal start codon.
Figure S2.
Comparison of the proliferation status of the ADAR1 knockdown (left) and
over-expression (right) cultures in the C2C12 differentiation experiments (Figure 1, c
& e). Proliferation status was determined as described in the Supplementary Methods
based on the relative proportion of replicating cells.
Figure S3.
Relative expression levels of miR-1 and miR-206 in differentiating C2C12 (0, 24, 48,
72, and 96 hrs post-DM culture), as determined by real-time RT–PCR.
Figure S4. Genotyping results for the Adar1 transgenic mice (related to Figure
3d).
(a) Genotyping analysis of genomic DNA isolated from the amnion of 13.5-day old
embryos. Samples were analyzed by PCR detecting ADAR1 wild-type (Wt) and
Transgenic (Tg) alleles. DNA size marker (M), genotyping positive control (ctrl;
transgene construct as the template), and no-template controls (NTC) are as
indicated. Lengths of the product amplicons are shown on the bottom.
(b) WISH on wild-type (Wt) and transgenic (Tg; with MCK-Adar1 p110) embryos of
identical somite numbers at embryonic stage E13.5. Probe corresponding to Adar1
was used to monitor marker expression and somite structure, as indicated. Lateral
view of whole embryo is shown on the left of each panel, and magnification of boxed
thoracic regions on the right. The expression domain of Adar1 in interlimb somoites is
denoted by brackets,
(c) Quantitative assessment of MyoD WISH signals shown in Figure 3d. Relative
extent of MyoD expression in interlimb somites, in terms of domain length, was
determined for Tg versus Wt mice. Numbers on the x-axis denote arbitrary somite
regions, in the anterior-to-posterior organization.
Figure S5. RIP-Seq-based identification of ADAR1-binding targets (related to
Figure 4).
(a) Overrepresentated GeneGo Maps in the edited gene sets, as revealed by pathway
analysis using MetaCore.
(b) Read density plots for additional targets showing representation in the four
sequencing libraries. Corresponding gene structure is depicted on top of each plot. X
7
axis denotes relative position of the gene whereas y axis corresponds to number of
distinct reads.
(c) Preponderance of differentially bound gene transcripts in distinct biological
processes, as revealed by analysis using MetaCore. The top GeneGo process
networks, based on statistical significance of enrichment, are shown for DM-0 h
targets.
(d) Additional candidate targets of ADAR1 were confirmed by RIP-qRT-PCR.
Analyses for RIP enrichment and stage-dependent enrichment of ADAR1 transcript
binding were done as in Figure 4, d & e, respectively.
(e) Differentiation-associated mRNA expression profiling of selected ADAR1-targeted
candidates. C2C12 cells were cultured in GM or in DM for the indicated time lengths.
RNA was isolated from these cells and subjected to real-time RT-PCR with primers
specific to the indicated genes. The bar graphs show normalized values with GM cells
represented as 1.
(f) Effect of ADAR1 down-regulation on the mRNA levels of selected candidate targets,
as shown. C2C12 cells were transiently transfected with siRNAs targeting Adar1 or
control siRNA (ctrl; targeting GFP), and harvested at DM-48 h for qRT-PCR analysis.
Figure S6. Sanger sequencing analysis of target gene transcripts.
Sanger sequencing analysis was performed for cDNA isolated from C2C12 cells in
DM-0 hr vs. DM-72 hr culture. Candidate editing sites in DNM1 and DNM2 genes are
highlighted by grey shades and genomic coordinates (mm9).
8