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Cryptic RNA-binding by PRC2 components EZH2 and SUZ12
Supplementary Figures
Supplementary Figure 1. EZH2 and SUZ12 are RNA-binding proteins. A. Purified
recombinant components of PRC2 purified from HEK293T cells. ps: HRV3C
(PreScission) protease cleavage site. B. Western blot of purified PRC2 core proteins shows
minimal cross contamination with endogenous PRC2 components, except for SBP-SUZ12
that was co-purified with RbAp48. C. Representative competition experiments with
increasing concentration of non-radiolabeled 1 rep Wt and Mut. Related to Figure 1G.
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Cryptic RNA-binding by PRC2 components EZH2 and SUZ12
Supplementary Figure 1. Computational prediction of the minimum free energy (MFE)
secondary structures of each of the 8.5 stem loop structures of Xist (left). The figures on
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Cryptic RNA-binding by PRC2 components EZH2 and SUZ12
the right side correspond to the structures of mutant versions of the RNA fragments after
the introduction of point mutations that disrupt the secondary structures. See
supplementary table 2 for detailed descriptions of the mutations. Predictions were
perfomed using RNAfold with default parameters (Gruber et al., 2008). The color scale
indicates base-pairing probabilities. In the case of unpaired regions the scale refers to the
probability of being unpaired.
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Supplementary table 1. Sequences of all RNAs used in this study. Underlined sequences correspond to mutated nucleotides.
Name
8.5 rep
Wt
8.5 rep
Mut
Length Sequence
UCUUCCACUCUCUUUUCUAUAUUUUGCCCAUCGGGGCUGCGGAUACCUGGUUUUAUUAUUUUUUCUUUGCCCAACGGGGCCGUGGA
UACCUGCCUUUUAAUUCUUUUUUAUUCGCCCAUCGGGGCCGCGGAUACCUGCUUUUUAUUUUUUUUUCCUUAGCCCAUCGGGGUAU
CGGAUACCUGCUGAUUCCCUUCCCCUCUGAACCCCCAACACUCUGGCCCAUCGGGGUGACGGAUAUCUGCUUUUUAAAAAUUUUCU
424
UUUUUUGGCCCAUCGGGGCUUCGGAUACCUGCUUUUUUUUUUUUUAUUUUUCCUUGCCCAUCGGGGCCUCGGAUACCUGCUUUAAU
UUUUGUUUUUCUGGCCCAUCGGGGCCGCGGAUACCUGCUUUGAUUUUUUUUUUUCAUCGCCCAUCGGUGCUUUUUAUGGA
424
UCUUCCACUCUCUUUUCUAUAUUUUAGCAAUCGGGGCUGCAGAUACAUAGUUUUAUUAUUUUUUCUUUAGCAAACGGGGCCGUAGA
UACAUACCUUUUAAUUCUUUUUUAUUCAGCAAUCGGGGCCGCAGAUACAUACUUUUUAUUUUUUUUUCCUUAAGCAAUCGGGGUAU
CAGAUACAUACUGAUUCCCUUCCCCUCUGAACCCCCAACACUCUGAGCAAUCGGGGUGACAGAUAUAUACUUUUUAAAAAUUUUCU
UUUUUUGAACAAUCGGGGCUUCAGAUACAUACUUUUUUUUUUUUUAUUUUUCCUUAGCAAUCGGGGCCUCAGAUACAUACUUUAAU
UUUUGUUUUUCUGAACAAUCGGGGCCGCAGAUACAUACUUUGAUUUUUUUUUUUCAUAACACAUCGAUGCUUUUUAUGGA
184
UCUUCCACUCUCUUUUCUAUAUUUUGCCCAUCGGGGCUGCGGAUACCUGGUUUUAUUAUUUUUUCUUUGCCCAACGGGGCCGUGGA
UACCUGCCUUUUAAUUCUUUUUUAUUCGCCCAUCGGGGCCGCGGAUACCUGCUUUUUAUUUUUUUUUCCUUAGCCCAUCGGGGUAU
CGGAUACCUGCU
4 rep
Mut
184
UCUUCCACUCUCUUUUCUAUAUUUUAGCAAUCGGGGCUGCAGAUACAUAGUUUUAUUAUUUUUUCUUUAGCAAACGGGGCCGUAGA
UACAUACCUUUUAAUUCUUUUUUAUUCAGCAAUCGGGGCCGCAGAUACAUACUUUUUAUUUUUUUUUCCUUAAGCAAUCGGGGUAU
CAGAUACAUACU
2 rep Wt
94
UCUUCCACUCUCUUUUCUAUAUUUUGCCCAUCGGGGCUGCGGAUACCUGGUUUUAUUAUUUUUUCUUUGCCCAACGGGGCCGUGGA
UACCUGCC
2 rep
Mut
94
UCUUCCACUCUCUUUUCUAUAUUUUAGCAAUCGGGGCUGCAGAUACAUAGUUUUAUUAUUUUUUCUUUAGCAAACGGGGCCGUAGA
UACAUACC
28
UUGCCCAUCGGGGCCACGGAUACCUGCU
UUAGCAAUCGGGGCCACAGAUACAUACU
4 rep Wt
1 rep Wt
1 rep
Mut
28
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Name
HOTAIR
full
length
(FL)
HOTAIR
-A
HOTAIR
-B
HOTAIR
-C
Length Sequence
GACUCGCCUGUGCUCUGGAGCUUGAUCCGAAAGCUUCCACAGUGAGGACUGCUCCGUGGGGGUAAGAGAGCACCAGGCACUGAGGC
CUGGGAGUUCCACAGACCAACACCCCUGCUCCUGGCGGCUCCCACCCGGGACUUAGACCCUCAGGUCCCUAAUAUCCCGGAGGUGC
UCUCAAUCAGAAAGGUCCUGCUCCGCUUCGCAGUGGAAUGGAACGGAUUUAGAAGCCUGCAGUAGGGGAGUGGGGAGUGGAGAGAG
GGAGCCCAGAGUUACAGACGGCGGCGAGAGGAAGGAGGGGCGUCUUUAUUUUUUUAAGGCCCCAAAGAGUCUGAUGUUUACAAGAC
CAGAAAUGCCACGGCCGCGUCCUGGCAGAGAAAAGGCUGAAAUGGAGGACCGGCGCCUUCCUUAUAAGUAUGCACAUUGGCGAGAG
AAGUGCUGCAACCUAAACCAGCAAUUACACCCAAGCUCGUUGGGGCCUAAGCCAGUACCGACCUGGUAGAAAAAGCAACCACGAAG
CUAGAGAGAGAGCCAGAGGAGGGAAGAGAGCGCCAGACGAAGGUGAAAGCGAACCACGCAGAGAAAUGCAGGCAAGGGAGCAAGGC
GGCAGUUCCCGGAACAAACGUGGCAGAGGGCAAGACGGGCACUCACAGACAGAGGUUUAUGUAUUUUUAUUUUUUAAAAUCUGAUU
UGGUGUUCCAUGAGGAAAAGGGAAAAUCUAGGGAACGGGAGUACAGAGAGAAUAAUCCGGGUCCUAGCUCGCCACAUGAACGCCCA
GAGAACGCUGGAAAAACCUGAGCGGGUGCCGGGGCAGCACCCGGCUCGGGUCAGCCACUGCCCCACACCGGGCCCACCAAGCCCCG
CCCCUCGCGGCCACCGGGGCUUCCUUGCUCUUCUUAUCAUCUCCAUCUUUAUGAUGAGGCUUGUUAACAAGACCAGAGAGCUGGCC
AAGCACCUCUAUCUCAGCCGCGCCCGCUCAGCCGAGCAGCGGUCGGUGGGGGGACUGGGAGGCGCUAAUUAAUUGAUUCCUUUGGA
2145 CUGUAAAAUAUGGCGGCGUCUACACGGAACCCAUGGACUCAUAAACAAUAUAUCUGUUGGGCGUGAGUGCACUGUCUCUCAAAUAA
UUUUUCCAUAGGCAAAUGUCAGAGGGUUCUGGAUUUUUAGUUGCUAAGGAAAGAUCCAAAUGGGACCAAUUUUAGGAGGCCCAAAC
AGAGUCCGUUCAGUGUCAGAAAAUGCUUCCCCAAAGGGGUUGGGAGUGUGUUUUGUUGGAAAAAAGCUUGGGUUAUAGGAAAGCCU
UUCCCUGCUACUUGUGUAGACCCAGCCCAAUUUAAGAAUUACAAGGAAGCGAAGGGGUUGUGUAGGCCGGAAGCCUCUCUGUCCCG
GCUGGAUGCAGGGGACUUGAGCUGCUCCGGAAUUUGAGAGGAACAUAGAAGCAAAGGUCCAGCCUUUGCUUCGUGCUGAUUCCUAG
ACUUAAGAUUCAAAAACAAAUUUUUAAAAGUGAAACCAGCCCUAGCCUUUGGAAGCUCUUGAAGGUUCAGCACCCACCCAGGAAUC
CACCUGCCUGUUACACGCCUCUCCAAGACACAGUGGCACCGCUUUUCUAACUGGCAGCACAGAGCAACUCUAUAAUAUGCUUAUAU
UAGGUCUAGAAGAAUGCAUCUUGAGACACAUGGGUAACCUAAUUAUAUAAUGCUUGUUCCAUACAGGAGUGAUUAUGCAGUGGGAC
CCUGCUGCAAACGGGACUUUGCACUCUAAAUAUAGACCCCAGCUUGGGACAAAAGUUGCAGUAGAAAAAUAGACAUAGGAGAACAC
UUAAAUAAGUGAUGCAUGUAGACACAGAAGGGGUAUUUAAAAGACAGAAAUAAUAGAAGUACAGAAGAACAGAAAAAAAAUCAGCA
GAUGGAGAUUACCAUUCCCAAUGCCUGAACUUCCUCCUGCUAUUAAGAUUGCUAGAGAAUUGUGUCUUAAACAGUUCAUGAACCCA
GAAGAAUGCAAUUUCAAUGUAUUUAGUACACACACAGUAUGUAUAUAAACACAACUCACAGAAUAUAUUUUCCAUACAUUGGGUAG
GUAUGCACUUUGUGUAUAUAUAAUAAUGUAUUUUCCAUGCAGUUUUAAAAUGUAGAUAUAUUAAUAUCUGGAUGCAUUUUC
GACUCGCCUGUGCUCUGGAGCUUGAUCCGAAAGCUUCCACAGUGAGGACUGCUCCGUGGGGGUAAGAGAGCACCAGGCACUGAGGC
CUGGGAGUUCCACAGACCAACACCCCUGCUCCUGGCGGCUCCCACCCGGGACUUAGACCCUCAGGUCCCUAAUAUCCCGGAGGUGC
308
UCUCAAUCAGAAAGGUCCUGCUCCGCUUCGCAGUGGAAUGGAACGGAUUUAGAAGCCUGCAGUAGGGGAGUGGGGAGUGGAGAGAG
GGAGCCCAGAGUUACAGACGGCGGCGAGAGGAAGGAGGGGCGUCUUUAUU
104
26
GUGGAAUGGAACGGAUUUAGAAGCCUGCAGUAGGGGAGUGGGGAGUGGAGAGAGGGAGCCCAGAGUUACAGACGGCGGCGAGAGGA
AGGAGGGGCGUCUUUAUU
GUGGAAUGGAACGGAUUUAGAAGCCU
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Name
HOTAIR
-D
GFP-A
GFP-B
GFP-C
GFP-D
Length Sequence
UAUUUUCCAUACAUUGGGUAGGUAUGCACUUUGUGUAUAUAUAAUAAUGUAUUUUCCAUGCAGUUUUAAAAUGUAGAUAUAUUAAU
102
AUCUGGAUGCAUUUUC
GGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAA
GCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGC
ACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGC
GCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGG
645
GCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGA
UCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUG
CCCGACAACCACUACCUGAGCACCCAGUCCGCCCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGU
GACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAG
GGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAA
GCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGC
318
ACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGC
GCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGAC
GGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAA
102
GCUGCCCGUGCCCUGG
GGCCACAAGUUCAGCGUGUCCGGCGAGG
28
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Supplementary methods
Plasmid constructions
PRC2 cDNA: EED, EZH2, RbAp48 and SUZ12 were amplified from HeLa S3 cDNA
using PrimeStar Max (Takara) and the following primers pairs.
EED:
EZH2:
RbAp48:
SUZ12:
EED extF1
EED extR1
EZH2 extF1
EZH2 extR1
RbAp48 extF1
RbAp48 extR1
SUZ12 extF1
SUZ12 extR1
TGGGCGCGATTTGCGACAGT
TGCTCTACGTGCCCTTACTAGCA
TCCGACACCCGGTGGGACTC
GCAGCTGTTTCAGAGGAGGGGG
TCGACCCCAGGATTCCCCCG
GAAAACACCCACGGTTTGGGCT
GGGCGAGCGGTTGGTATTGCA
ACTCAACCACAGTGCTCGGAGT
pCAGEN-SBP-EZH2, -SUZ12 and pCAGEN-SBP-ps-EED, -RbAp48: The PRC2
component sequences were reamplified using a nested PCR strategy and the SBP tag was
amplified from pASW (Iwasaki 2010). The fragments were cloned simultaneously into
the EcoRI site of pCAGEN (Matsuda and Cepko, 2004) using Infusion (Clontech) to
produce pCAGEN-SBP-EZH2, and -SUZ12. The following primers were used:
SBP:
SBP F2
SUZ12:
SBP R2
SUZ12 F1
SUZ12 R1
EZH2:
EZH2 F1
EZH2 R1
TTTTGGCAAAGAATTCCCATGGACGAGAA
GACCACCGGC
GGCCGCGGAGCCTGCTTTTT
GCAGGCTCCGCGGCCATGGCGCCTCAGAA
GCAC
TATCCTCGAGGAATTTGGGGTTAGAGCTTT
TCAGAGT
GCAGGCTCCGCGGCCATGGGCCAGACTGG
GAAG
TATCCTCGAGGAATTGGGGAGGAGGTAGC
AGATGT
EED and RbAp48 were cloned with a cleavage site for HRV3C (PreScission [ps])
protease following the SBP tag. The following oligos containing the recognition site of
the protease were annealed and cloned simultaneously with the SBP tag into the EcoRI
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site of pCAGEN using Infusion (Clontech) to produce pCAGEN-SBP-ps. The following
primers were used.
SBP:
SBP F3
ps:
SBP R2
ps F1
ps R1
TTTTGGCAAAAATTCCCATGGACGAGAAG
ACCACCGGC
GGCCGCGGAGCCTGCTTTTT
GCAGGCTCCGCGGCCCTGGAAGTTCTGTTC
CAGGGGCCCGAATTCCTCGAGGATA
TATCCTCGAGGAATTCGGGCCCCTGGAAC
AGAACTTCCAGGGCCGCGGAGCCTGC
EED and RbAp48 were reamplified and cloned into the EcoRI site of pCAGEN-SBPps. The following primers were used.
EED:
EED F2
EED R1
RbAp48:
RbAp48 F1
RbAp48 R1
CTGTTCCAGGGGCCCATGTCCGAGAGGGA
AGTGTC
TATCCTCGAGGAATTAGGCAAAAGTATTTT
ATCGAAGTC
CTGTTCCAGGGGCCCATGGCCGACAAGGA
AGCAGCC
TATCCTCGAGGAATTCTAGGACCCTTGTCC
TTCTGG
pEFh-SBP-GFP: GFP was amplified from pMXs-IG (Cell Biolabs) and subcloned
into the EcoRI site of pEFh-SBP (kind gift from Akio Yamashita, Yokohama City
University) using Infusion (Clontech). The following primers were used.
GFP:
pEF-G-GFPF
pEF-GFPR
TTTCAGGGCGAATTCATGGTGAGCAAGGG
CGAGGA
GATTGTCGATGAATTTTACTTGTACAGCTC
GTCCAT
pENTR-hRepA: the A-repeat domain of human Xist was amplified from HEK293T
cDNA using KOD Plus-NEO (Toyobo) and the following primers.
A-repeat
XistTOPOF
CACCAGTGTCTTCTTGACACGTCCTCCA
XistR
AGAGTGCAACAACCCACAAAACCA
The PCR product was cloned into pENTR using pENTR/D-TOPO cloning
(Invitrogen).
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Cryptic RNA-binding by PRC2 components EZH2 and SUZ12
pMK-RQ-8rep_Mut: a mutant 8.5 rep plasmid was custom synthesized by Invitrogen’s
gene synthesis service.
pCRII-2 rep_Mut: the following oligos were annealed and cloned using Zero Blunt
TOPO PCR cloning kit (Invitrogen).
2 rep
2rep mut F
2rep mut R
TCTTCCACTCTCTTTTCTATATTTTAGCAAT
CGGGGCTGCAGATACATAGTTTTATTATTT
TTTCTTTAGCAAACGGGGCCGTAGATACAT
ACC
GGTATGTATCTACGGCCCCGTTTGCTAAAG
AAAAAATAATAAAACTATGTATCTGCAGC
CCCGATTGCTAAAATATAGAAAAGAGAGT
GGAAGA
In vitro transcription
Templates for in vitro transcription were synthesized using KOD Plus-Neo (Toyobo)
and the following primers and plasmid templates.
2 rep Wt
Template
pENTRhRepA
Primer name
hRepA_T7_346372F
hRepA_410-439
2 rep Mut pCRIIhRepA_T7_3462rep_Mut 372mutF
hRepA_410439mutR
4 rep Wt pENTR- hRepA_T7_346hRepA
372F
hRepA_499529R
4 rep Mut pMK-RQ- hRepA_T7_3468rep_Mut 372mutF
hRepA_499529mutR
8.5 rep Wt pENTR- hRepA_T7_346hRepA
372F
hRepA_749-769
8.5 rep
pMK-RQ- hRepA_T7_346Mut
8rep_Mut 372mutF
Sequence
CTTAATACGACTCACTATAGTCTT
CCACTCTCTTTTCTATATTTTGC
GGCAGGTATCCACGGCCCCGTTG
GGCAAAG
CTTAATACGACTCACTATAGTCTT
CCACTCTCTTTTCTATATTTTAG
GGTATGTATCTACGGCCCCGTTTG
CTAAAG
CTTAATACGACTCACTATAGTCTT
CCACTCTCTTTTCTATATTTTGC
AGCAGGTATCCGATACCCCGATG
GGCTAAGG
CTTAATACGACTCACTATAGTCTT
CCACTCTCTTTTCTATATTTTAG
AGTATGTATCTGATACCCCGATTG
CTTAAGG
CTTAATACGACTCACTATAGTCTT
CCACTCTCTTTTCTATATTTTGC
TCCATAAAAAGCACCGATGGG
CTTAATACGACTCACTATAGTCTT
CCACTCTCTTTTCTATATTTTAG
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Cryptic RNA-binding by PRC2 components EZH2 and SUZ12
hRepA_749TCCATAAAAAGCATCGATGTG
769mutR
HOTAIR- pLZRSHotair_T7_1-20F GCGTAATACGACTCACTATAGGA
FL
HOTAIR
CTCGCCTGTGCTCTGGAG
(Gupta et Hotair_2118GAAAATGCATCCAGATATTAATA
al., 2010) 2148R
TATCTACA
HOTAIR- (Addgene Hotair_T7_1-20F GCGTAATACGACTCACTATAGGA
plasmid
A
CTCGCCTGTGCTCTGGAG
26110)
Hotair_289-308R AATAAAGACGCCCCTCCTTC
HOTAIRHotair_T7_205- GCGTAATACGACTCACTATAGGT
B
224
GGAATGGAACGGATTTAG
Hotair_289-308R AATAAAGACGCCCCTCCTTC
HOTAIRHotair_T7_2044- CTTAATACGACTCACTATAGTATT
D
2069
TTCCATACATTGGGTAGGTATG
Hotair_2118GAAAATGCATCCAGATATTAATA
2148R
TATCTACA
GFP-A
pMXs-IG GFP_T7_2650CTTAATACGACTCACTATAGGGCC
(Cell
2670F
ACAAGTTCAGCGTGTCC
Biolabs)
GFP_3053GTTCACCTTGATGCCGTTCT
3072R
GFP-B
GFP_T7_2650CTTAATACGACTCACTATAGGGCC
2670F
ACAAGTTCAGCGTGTCC
GFP_2950GTCGATGCCCTTCAGCTC
2967R
GFP-C
GFP_T7_2650CTTAATACGACTCACTATAGGGCC
2670F
ACAAGTTCAGCGTGTCC
GFP_2732CCAGGGCACGGGCAGCTTGC
2751R
PCR products were gel purified with PCR and Gel Clean-up kit (Macherey Nagel) and
in vitro transcribed using T7-scribe standard RNA IVT kit (Cellscript). The full
sequences of all RNAs are shown in Supplementary Table 2.
Other RNAs were chemically synthesized by Gene Design. Sequences are indicated in
Supplementary Table 2.
Protein expression and purification
Typically 8 x 106 HEK293T cells were seeded per 15 cm dish in 15 ml D-MEM
supplemented with 10 % FBS. Around 18 h later cells were transfected using
polyethyleneimine (PEI). Cells were harvested 24 h post transfection and lysed
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immediately for protein purification. The pelleted cell weight (PCW) was estimated
(typically ~6 g from sixty 15cm dishes) and cells were resuspended in 1× PCW volume
0.5 M high salt buffer (20 mM HEPES-KOH pH 8.0 at 4 °C, 1.5 mM MgCl2, 0.5 M KCl,
0.2 mM EDTA, 25 % glycerol, 1 mM DTT, 1× complete protease inhibitor cocktail) and
lysed in a douncer. The lysate was then centrifuged at 100000g for 1 h. The supernatant
was collected and diluted with 1× PCW volume of low salt buffer (high salt buffer
containing 20 mM KCl) (Abmayr et al., 2006). The lysate was mixed with 0.1× PCW
volume of Streptavidin Sepharose High Performance beads (GE) and rotated at 4 °C for
10 min. The beads were precipitated and washed 3 times with 1 ml 0.5 M NaCl wash
buffer (1× lysis buffer [30mM HEPES-potassium hydroxide pH 7.4, 100mM potassium
acetate, 2mM magnesium acetate], 0.1 % Tween-20, 0.5 M NaCl, 1 mM DTT, 1 mM
PMSF) and rinsed twice with wash buffer (without NaCl). Proteins were eluted in 500 μl
elution buffer (1× lysis buffer, 2.5 mM biotin, 10 % glycerol, 1 mM DTT, 1 mM PMSF)
and concentrated using VivaSpin 2 ml columns (Sartorius). Quantification was done by
SDS-PAGE followed by CBB staining using BSA standard curves.
Antibodies
The following antibodies were used for western blot: anti-EZH2 (AC22, Cell Signaling),
anti-SUZ12 (D39F6, Cell Signaling), anti-EED (ab96801, Abcam) and anti-RbAp48 (N19, Santa Cruz).
RNA labeling and quantification
Two picomoles of in vitro transcribed RNAs were radiolabeled at their 3’ ends using
yeast poly A polymerase (Afymetrix) and 32P-αATP in a final volume of 10 μl for 20 min
at 37 °C. One microliter aliquots were taken from each reaction for the generation of
standard curves. The remaining sample was run on Urea-PAGE and purified.
Radiolabeled RNAs were resuspended in 1× lysis buffer, heated at 95 °C for 2 min and
incubated at room temperature for 30 min to allow spontaneous refolding. The
concentration of the purified RNA was estimated by running the sample in Urea-PAGE
along with a standard curve.
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Gel shift and crosslinking assays
Typical RNA-binding reactions contained 5 μl recombinant protein (~50 nM), 3 μl
40× reaction mix (133 mM potassium acetate, 9.33 mM magnesium acetate, 1.7 mM
DTT, 3.33 mM ATP, 0.33 U/μl, 83.33 mM creatine monophosphate, 0.1 U/μl creatine
phosphokinase in ultrapure water, in a final volume of 120 μl), 1μl radiolabeled RNA
(10nM) and 1 μl 1× lysis buffer or cold competitor RNA (final volume = 10μl). The
samples were incubated at 25 °C for 15 minutes, and then transferred to ice. For gel shift
assays, 10 μl of sucrose loading buffer (400 mg/ml sucrose, 2.5 mg/ml xylencyanol and
2.5 mg/ml bromophenol blue) were added and 10 μl were run in a 1× TBE 4.5%
polyacrilamide gel (acrylamide/bisacrylamide 29:1) containing 2 mM MgCl2, at 4 °C.
For crosslinking, after incubation at the samples were transferred to terasaki dishes
placed on ice in 10 μl aliquots, and were irradiated for 7 min with 254 nm UV light. After
addition of SDS-PAGE loading buffer samples were heated at 95 °C for 2 min and run in
an 8% wide range SDS-PAGE.
Filter-binding assay
A Protran BA85 nitrocellulose membrane (GE Healthcare), a Hybond N+ nylon
membrane (GE Healthcare) and a filter paper were pre-rinsed in 1× lysis buffer and
assembled in a dot-blotter. RNA-binding reactions were prepared as above in a final
volume of 6 μl using RNA stocks set to 10nM. After incubation for 15 min at 25 °C
samples were placed on iced and 5 μl were loaded per well. Each well was washed twice
with 10 μl of 1× lysis buffer. Membranes were air dried and exposed to phosphorimaging
plates. The intensity per sample was measured using Multi Gauge (Fujifilm) and the
fraction of bound RNA was calculated [fraction bound = bound RNA/(bound RNA +
unbound RNA)]. Graphs were generated using IgorPro software (Wavemetrics) and data
was fitted to Hill curves.
Supplementary references
Abmayr, S.M., Yao, T., Parmely, T., and Workman, J.L. (2006). Preparation of nuclear
and cytoplasmic extracts from mammalian cells. Curr Protoc Pharmacol Chapter 12,
Unit12 13.
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Cryptic RNA-binding by PRC2 components EZH2 and SUZ12
Gruber, A.R., Lorenz, R., Bernhart, S.H., Neubock, R., and Hofacker, I.L. (2008). The
Vienna RNA websuite. Nucleic Acids Res 36, W70-74.
Gupta, R.A., Shah, N., Wang, K.C., Kim, J., Horlings, H.M., Wong, D.J., Tsai, M.C.,
Hung, T., Argani, P., Rinn, J.L., et al. (2010). Long non-coding RNA HOTAIR
reprograms chromatin state to promote cancer metastasis. Nature 464, 1071-1076.
Matsuda, T., and Cepko, C.L. (2004). Electroporation and RNA interference in the rodent
retina in vivo and in vitro. Proc Natl Acad Sci U S A 101, 16-22.
13