cell line cdh1 mrna as-parna s-parna - nature · bs * s parna-790 +48 tss (cdh1) ¶ isomir-4534...
TRANSCRIPT
Cell line CDH1 mRNA AS-paRNA S-paRNA
HCT116 P53 +/+ 390.47 + +
HCT116 P53 -/- 350.36 + +
LNCAP 1562.40 + -
MCF7 C1 1864.76 + -
MCF7 C2 1812.81 + -
a
b
Supplementary Figure 1. Promoter-associated transcripts at the CDH1 gene locus.
(a) Transcriptional landscape in the CDH1 promoter in HCT116 p53 -/- cells based on
GRO-Seq data. Blue and red tracks, S and AS transcription, respectively. Blue and red
boxes, predicted S- and AS-paRNAs. (b) CDH1 mRNA expression (normalized read
counts) and presence of AS- and S-paRNA from GRO-seq data analysis in the indicated
cell lines.
-139
CDH1
Antisense paRNA
+ 1
-261
-456
-10
CDH1
Sense paRNA
+ 1
-280
-476
-234 -161
-107
-54 S AS
b
d
CDH1
-204 -720
Sense paRNA
+ 1
-190
-153
CDH1
-238
- 135
Antisense paRNA
+ 1
-282
-303
e
+48 -373
pGL3-CDH1 Del
promoter Luc
-790
pGL3-CDH1 WT
promoter -720
S paRNA TSS (CDH1)
-153 Fp Rp
Luc
-281
AS paRNA -135 f
0
0.4
0.8
1.2
1.6
po
lyA
+/
tot
PC3
LNCaP
a
500-
100-
500-
100-
500-
100-
siGL3 si Rrp44
S paRNA
Rrp 44
-actin
c
200-
200-
200-
AS paRNA
pGL3-basic pGL3-TSS
PC3
LNCaP
S paRNA
200-
100-
DNA (20ng)
pGL3-CDH1 WT promoter
RNA (100ng)
PC3
-200
-100
S paRNA
AS paRNA
PC3
LNCaP
200-
200-
Supplementary Figure 2. (a) Poly-adenylation of paRNAs in PC3 (S paRNA) and
LNCaP (AS paRNA) cells analysed by differential retro-transcription with random
hexamers (total RNA) and oligo dT (polyA+ RNA). -actin mRNA was used as positive
control for a polyA+ mRNA. Data are presented as ratio of each transcript measured in
the two conditions. (b) 3’RACE of S- and AS-paRNAs in PC3 (top) and LNCaP (bottom)
cells, respectively. Red bars indicate the positions of the transcripts 3’ends. Arrows
indicate position of gene-specific primers used for 3’RACE. (c) S-paRNA level after
knockdown of Rrp44 in PC3 cells. (d) 5’RACE of S- and AS-paRNAs in PC3 (top) and
LNCaP (bottom) cells. Red bars indicate the positions of the transcripts 5’ends. Arrows
indicate position of gene-specific primers used for 5’RACE. (e) paRNA detection by
strand-specific RT-PCR with or without RNase treatment. Left, PC3 and LNCaP cells
were transfected with pGL3 basic and S-TSS or AS-TSS reporters (pGL3-TSS). Right,
PC3 cells were transfected with full length CDH1 promoter reporter. (f) Detection of S and
AS transcripts in PC3 and LNCaP cells transfected with pGL3-basic, wild type (WT) or
5’deleted (Del) promoter reporter (left panel) and analysed by strand-specific RT-PCR
(right panel).
a
TSS
-700 -600 -500 -400 -300 -200 -100 +1
Sense paRNA si 304 si217 si63
0
0.2
0.4
0.6
0.8
1
1.2
pa
RN
A
* *
d
h
0
2
4
6
8
10
ce
ll n
um
be
r (
X 1
04)
Time (days)
Ctr
siGL3
si304
si63
*
*
c
0
2
4
6
8
10
12
CD
H1 m
RN
A
* *
siGL3 siAGO2
0
5
10
15
20
25
Rela
tive
lucife
rase
activity
PC3
g
f
S paRNA
WT
T1 T1
-790 +48 TS
TS
200-
200-
200-
200-
b
β-actin
CDH1
CDH1
β-actin
CDH1
Tubulin
130-
55-
LNCaP
siGL3 si217
S paRNA
AS paRNA
CDH1
β-actin
e
200-
200-
100-
100-
S paRNA
200-
CDH1
LNCaP PC3
β-actin
-200
-200
Supplementary Figure 3. siRNA-mediated targeting of promoter-associated RNAs
at the CDH1 Gene. (a) Position of siRNA target sequences in the CDH1 promoter
named according to their 5’ nucleotide relative to the transcription start site (TSS; +1). (b)
CDH1 mRNA in PC3 cells 72 h after transfection with si217 and si304 (top) and si217 and
si63 (middle). E-cadherin protein levels in PC3 cells 72 h after transfection with si304 and
si63 (bottom). (c) CDH1 mRNA in PC3 cells transfected with siAGO2 or siGL3 followed
by si217 or siGL3. (d) paRNA levels in PC3 cells 24 h after transfection with siGL3, si304
and si63. (e) CDH1 mRNA and paRNA levels in LNCaP cells transfected with si217 and
siGL3. (f) Detection of S-paRNA in PC3 cells transfected with pGL3-basic and CDH1
promoter reporter either wild type (WT) or with the insertion of a restriction site (T1) or a
termination site (TS, SV40 polyA cassette) by strand-specific RT-PCR (right panel). Left
panel, schematics of CDH1 promoter reporter constructs. (g) Activity of CDH1 promoter in
luciferase reporter assay (left panel) and CDH1 mRNA level (right panel) in cells
transfected with control vector (EV), sense (S-paRNA) or antisense (AS-paRNA)
expression vector. Full length wild type CDH1 promoter reporter was co-transfected for
the reporter assay. (h) Proliferation of PC3 cells transfected with si63, si304 and siGL3.
Ctr, non-transfected control cells. Data are mean ± SD of three independent
determinations. Student’s t test was used for P value assessment. * P <0.05.
a b e
0.0
0.1
0.2
0.3
0.4
IgG Ago 1
CD
H1 p
aR
NA
0.0
0.1
0.2
0.3
0.4
IgG Ago 1
CD
H1 p
aR
NA
DU145
*
LNCaP
LNCaP
0
5
10
15
20
25
RLA
0
5
10
15
20
25
30
RLA *
f PC3
0
1
2
3
4
5
Fold
enri
chm
ent
*
g ChIP
0
0.2
0.4
0.6
0.8
1
1.2
Fold
en
rich
me
nt
SUV39H1
*
0
0.2
0.4
0.6
0.8
1
1.2
Fold
en
rich
me
nt
H3K9 me2
*
k
i
CDH1
β-actin
200-
200-
d
paRNA 100-
S paRNA
AS paRNA
h
100-
100-
S paRNA AS paRNA -RT
200-
Chaetocin (nM)
0
0.5
1
1.5
2
2.5
3
0 100 200
CD
H1 m
RN
A
*
*
m
0
0.5
1
1.5
2
CD
H1 m
RN
A
*
l
S paRNA
- AGO1
+ AGO1
n
100-
100-
AGO1
β-actin
AGO2
β-actin
AGO3
β-actin
AGO4
β-actin
200-
200-
200-
200-
100-
200-
200-
200-
c
AGO1
Tubulin 55-
100-
β-actin
SUV39H1
j
200-
200-
Supplementary Figure 4. Argonaute 1 is required for transcriptional regulation of
CDH1. (a) Knockdown efficiency of Argonaute 1, 2, 3 and 4 in PC3 cells. Control, non-
transfected cells. (b) CDH1 mRNA in PC3 cells transfected with siRNA targeting
Argonaute 1, 2, 3 and 4. (c) Nuclear and cytoplasmic localization of AGO1 in PC3 cells.
(d) Binding of HA-tagged Argonaute 1 (HA-AGO1) to promoter-associated RNA in PC3
cells by RIP. (e) Binding of AGO1to paRNA in DU145 and LNCaP cells by RNA-ChIP. (f)
CDH1 promoter reporter activity in PC3 and LNCaP cells transfected with wild type (WT)
alone or with HA-AGO1 (WT+AGO1). (g) Binding of HA-AGO1 to wild type (WT) CDH1
promoter reporter or mutated with termination site (TS) insertion. (h) AGO1 binding to S-
and AS-paRNA in LNCaP cells transfected with the respective expression vectors. (i)
Detection of S- and AS- paRNA by strand-specific RT-PCR in PC3 cells transfected with
siGL3 or siAGO1. -RT, control reaction without RT step. (j) Efficiency of SUV39H1
knockdown by siRNA in PC3 cells. (k) SUV39H1 occupancy and H3K9me2 at the CDH1
promoter in PC3 cells transfected with siSUV39H1 or siGL3 and harvested after 72 h. (l)
CDH1 mRNA in PC3 cells transfected with siSUV39H1. (m) CDH1 mRNA in PC3 cells
treated with chaetocin. (n) Binding of SUV39H1 to promoter-associated RNA in PC3 cells
transfected with S paRNA expression vector with or without HA-AGO1 vector. Data are
mean ± SD of three independent determinations. Student’s t test was used for P value
assessment. * P <0.05.
d RNA DNA
300 bp -
200 bp -
100 bp -
-102 -162 -102 -162
+58 +58 +58 +58
Marker
Primer
sets
* *
nc AK098727 TGTGAATGACCCCCTTCCAGAGCCAAAATCACCAGGGATGGAGGAGGGGTCTTGGGTACT
Pre-miR-4534
Pre-miR-4534-566
Pre-miR-4534-416
Pre-miR-4534-162
Pre-miR-4534-102
Pre-miR-4534+58
siRNA pri-miR-4534(-118) +1
c
g
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
miR-4534 isomiR-4534
miR
NA
ex
pre
ss
ion
empty cl3 del -91-55 cl5
del -88-52 cl2 del -91-55 cl2
* *
*
PC3 DU145
LNCaP LHS
DU145 MiR-4534 --CCCCCTTCCAGAGCCAAAATCACCAGGGATGGAGGAGGGGTCTTGGGTACTGGACCCACCAGCCAGGCAGGTTATACTGGGGTATCCGGAAGGA 94
PC3 MiR-4534 -----------AGAGCCAAAATCACCAGGGATGGAGGAGGGGTCTTGGGTACTGGACCCACCAGCCAGGCAGGTTATACTGGGGTATCCGGTA--- 82
LHS MiR-4534 --CCCCCTTCCAGAGCCAAAATCACCAGGGATGGAGGAGGGGTCTTGGGTACTGGACCCACCAGCCAGGCAGGTTATACTGGGGTATCCGGAATNN 94
LNCaP MiR-4534 GACCCCCTTCCAGAGCCAAAATCACCAGGGATGGAGGAGGGGTCTTGGGTACNGGACCCACCAGCCAGGCAGGTTATACTGGGGTATCCGGAANNA 96
Mature miR-4534
b
5’- GGATGGAGGAGGGGTCT miR-4534
5’- GATGGCGGAGCGGTCT isomiR-4534
Number of reads
5’- GATGGCGGAGCGGTCT 112,731
5’- ATGGCGGAGCGGTCT 68,645
5’- GGATGGCGGAGCGGTCT 23,300
5’- AGGGATGGCGGAGCGGTCT 1,328
5’- GATGGCGGAGCGGTC 1,046
5’- GGGATGGCGGAGCGGTCT 888
5’- CAGGGATGGCGGAGCGGTCT 745
5’- GATGGCGGAGTGGTC 607
Number of reads
5’- AGGGATGGCGGAGCGGTCT 1,380
5’- GGGATGGCGGAGCGGTCT 938
5’- CAGGGATGGCGGAGCGGTCT 756
5’- AGATGGAGGAGCGGTCT 316
5’- AGATGGCGGAGGGGTCT 169
5’- AGATGGAGGAGAGGTCT 17
5’- GGGATGGTGGAGCGGTCT 15
5’- AGGGATGGCGGAGCGGTC 14
a
CAGCCTGGGTGACAGAGTAAGACTCTGTCTCATAAAAACAAACAACAACAACAACAAAAAAACATACTGGAGCCTAGATCCCACCCCAGACTGGTTGATTCTGAATTTCTGGGGC
CAGCCTGGGTGACAGAGTAAGACTCTGTCTCATAAAAACAAACAACAACAACAACAAAAAA--CATACTGGAGCCTAGATCCCACCCCAGACTGGTTGATTCTGAATTTCTGGGGC
CAGCCTGGGTGACAGAGTAAGACTCTGTCTCATAAAAACAAACAACAACAACAACAAAAAAACATACTGGAGCCTAGATCCCACCCCAGACTGGTTGATTCTGA------------GGGC
CAGCCTGGGTGACAGAGTAAGACTCTGTCTCATAAAAACAAACAACAACAACAACAAAAAAACATACTGGAGCCTAGATCCCACCCCAGACTGGTTGATTCTGA--------------------
TGAGGTACAAGCATAAAGTTCCCTGGGAGGTCTTCACAGAGTCTCTGGATCCCTGAGGCTGTGAATGACCCCCTTCCAGAGCCAAAATCACCAGGGATGGAGGAGGGGTCTTGG
TGAGGTACAAGCATAAAGTTCCCTGGGAGGTCTTCACAGAGTCTCTGGATCCCTGAGGCTGTGAATGACCCCCTTCCAGAGCCAAAATCACCAGGGATGGAGGAGGGGTCTTGG
TGAGGTACAAGCATAAAGTTCCCTGGGAGGTCTTCACAGAGTCTCTGGATCCCTGAGGCTGTGAATGACCCCCTTCCAGAGCCAAAATCACCAGGGATGGAGGAGGGGTCTTGG
XXAGGTACAAGCATAAAGTTCCCTGGGAGGTCTTCACAGAGTCTCTGGATCCCTGAGGCTGTGAATGACCCCCTTCCAGAGCCAAAATCACCAGGGATGGAGGAGGGGTCTTGG
gRNA-91
gRNA-88
gRNA-55
gRNA-52
-162
+55
----
WT
del-70/-64 (n=2)
del-113 (n=9)
del-70/-57(n=1)
WT
del-70/-64 (n=2)
del-113 (n=9)
del-70/-57(n=1)
f
e
GAPDH
primiR-4534
(-102; +58)
siGL3 siprimiR
GAPDH
primiR-4534
(-102; +58)
200-
300-
200-
300-
Supplementary Figure 5. Origin of isomiR-4534. (a) Alignment of miR-4534, isomiR-4534 and
sequences retrieved from miRGator (top) and YM500 (bottom) small RNA-seq databases. (b)
Genomic sequencing of the miR-4534 locus and sequence alignment in prostate epithelial cell lines.
Circles, base changes in isomiR-4534. (c) Schematic representation of the pre-miR-4534 locus on
chromosome 22 with position of PCR primer sets and siRNA target site. (d) Pri-miR-4534 transcript
assessed by RT-PCR with the indicated primers sets. Genomic DNA (right panel) is shown as
positive control of primer efficiency and amplicon size. (e) Pri-miR-4534 level in PC3 (top) and DU145
(bottom) cells transfected with siGL3 or pri-miR targeting siRNA (sipri-miR-4534). (f) Map of pre-miR-
4534 locus and alignment of WT and targeted deleted sequences (delA -113 clones, n=9, and
delATTTCTG -70-64 clones, n=2). Red circles indicate positions of deletions. Red and orange lines
indicate the positions of the gRNA pair encoded by the two (del91-55 and del88-52) targeting
constructs, respectively. Dark and light green boxes represent pre-miR-4534 and mature miR-4534
sequence, respectively. (g) miR-4534 and isomi-R453 levels in three targeted clones of DU145 cells
obtained by transfection with del91-55 and del88-52 constructs and a control clones (empty vector).
Data are mean ± SD of three independent determinations. Student’s t test was used for P value
assessment. * P <0.05.
Input Target database Input sequenceNumberof sites
Conservedsites
Numberof target
genes
hsa-miR-4534 Target ScanGAUGGAG
(nt 2-8; seed region)157 157 155
hsa-isomiR-4534 Target ScanAUGGCGG
(nt 2-8; seed region)7 7 7
hsa-isomiR-4534 Target ScanGGCGGAG
(nt 4-10; alt seed region) 9 9 9
hsa-miR-4534 DIANA MicroT v3GGAUGGAGGAGGGGUCU
(full miRNA sequence)405 132 83
hsa-isomiR-4534 DIANA MicroT v3GAUGGCGGAGCGGUCU
(full miRNA sequence)7 6 2
miR-4534
S paRNA-134 -117
* *
a
b
0 20 40
RLA
WT
BS
*
S paRNA
-790 +48
TSS (CDH1)3’
5’isomiR-4534
-790 +48
WT
BS
S paRNA
TSS (CDH1)3’
5’isomiR-4534
0 10 20 30
RLA
WT
TS
TS/BS
*
n.s
.
0 10 20 30
RLA
WT
BS
S paRNA
-790 +48
TSS (CDH1)3’
5’isomiR-4534
*
PC3
PC3
DU145
c
d
Supplementary Figure 6. IsomiR-4534 selective targeting of S-paRNA. (a) Predicted
base pairing of miR-4534 with the isomiR-4534 target sequence in S-paRNA. Asterisks
mark edited bases in isomiR-4534. (b) Number of predicted targets of miR-4534 and
isomiR-4534 in 3’ UTRs of human mRNAs retrieved using TargetScan and DIANA
MicroTv3, taking into account seed regions or full length sequences of the miRNAs. (c)
Luciferase reporter activity of wild-type (WT) and mutated (BS) promoter with altered
isomiR-4534 binding site in PC3 (top) and DU145 (bottom) cells. (d) Luciferase reporter
activity of wild-type (WT) and the reporter construct (TS) with termination site insertion
and with or without the BS mutation tested in PC3 cells. Data are mean ± SD of three
independent determinations. Student’s t test was used for P value assessment. * P <0.05.
e
Cell line Genotype
Sanger seq
Genotype
RFLP
CDH1 mRNA
PC3 C/C C/C - -
DU145 A/C A/C -
LNCaP A/C A/C ++
PrEC A/C A/C ++
Genotype Rs16260
Number of samples
A/A 4
C/C 24
A/C 19
Total 47
- 190 bp
A/A C/C A/C
- 110 bp
- 80 bp
1 2 3
a
0.00
0.25
0.50
0.75
1.00
Recu
rre
nce
-fre
e s
urv
iva
l
0 2 4 6 8
Time (years)
p-val<0.05
non A/A
A/A
b
DU145 S-paRNA
LNCaP AS-paRNA
PC3 S-paRNA
g
c PC3 LNCaP
0
10
20
30
40
50
60
RLA
0
40
80
120
RLA
* S paRNA
f
-160 (A)
-160 (C)
200-
200-
d
PC3
S paRNA
AS paRNA
200-
200-
Supplementary Figure 7. SNP rs16260 in the CDH1 promoter. (a) SNP rs16260
genotype in human prostate tumors determined by RLFP analysis. Top, representative
gel image. Bottom, genotype distribution in human tumors (n=47). (b) Relapse-free
survival of prostate cancer patients (n= 47) grouped according to rs16260 genotype. (c)
Promoter activity of the -160A and -160C promoter reporters in PC3 and LNCaP cells. (d)
Synthesis of S and AS transcripts in PC3 cells transfected with the -160A or -160C CDH1
promoter reporters. (e) SNP rs16260 genotype in human prostate cancer cell lines
determined by RFLP analysis and Sanger sequencing and comparative levels of CDH1
expression. (f) Allele-specific binding of SUV39H1 to S-paRNA in the presence of AGO1.
(g) Rs16260 allele representation in S and AS promoter-associated RNAs in
heterozygous (C/A) DU145 and LNCaP cells and homozygous (C/C) PC3 cells. S and AS
transcripts were amplified by directional RT-PCR and the resulting amplicon sequenced.
Circles indicate the position of the polymorphic site.
-161
SNP -160A
-57 -281
-81
-61
-101
-121
-141
-201
-181
-221
-241
-261
SNP -160C
-57 -281
-81
-61
-101
-121
-141
-201
-181
-221
-241
-261
-161
a b
S paRNA CDH1rs16260TSS
-160-720
5’-TAGAGGGTCA[A/C]CGCGTCTATG-3’
SNP -160 C
-280 -230 -180 -130 -80
Nucleotide position
Pro
bability
SNP -160 A
isomiR
c
Supplementary Figure 8. Differential folding of sense promoter-associated RNA
polymorphic variants. (a,b) In silico predicted folding of the S-paRNA (-281/-57)
carrying either the -160 C (a) or -160A (b) alleles using Mfold. (c) Hairpin probability
profiles of the S-paRNA region encompassing SNP rs16260 and isomiR-4534 binding
sequence for the -160A and -160C allelic variants using Sfold.
SNP Non-forced G (kcal/mol)
Forced G (kcal/mol)
G (kcal/mol)
160A -87.44 -78.19 -9.21
160C -92.94 -79.61 -13.33
Forced single-strand
structure
SNP -160A
Non-forced single-
strand structure
SNP -160C
Forced single-strand
structure
Non-forced single-
strand structure
a
b
c
Supplementary Figure 9. Differential unwinding of sense promoter-associated RNA
polymorphic variants. (a,b) Predicted structures of the isomiR-4534 binding region in -
160 A (a) or -160C (b) S-paRNA allelic variants generated using Mfold with (right) or
without (left) forcing the hairpin sequence (-138 to -117) in single-strand conformation. (c)
Estimated differences in Gibbs free energy between the two allelic variants.
95-90%
-201
-191
-181
-171
-111
-131
isomiR-4534 binding site
-141
-151
-161
3’
5’
-121SNP -160A
-131
-121
isomiR-4534 binding site
>99%
-201
-191
-181
SNP -160C
-171-111
-151
-161
3’5’
3’
-141
a b
Supplementary Figure 10. Base pairing probability in the isomiR-4534 binding
hairpin of sense promoter-associated RNA. (a,b) Estimates of pairing probability in the
SHAPE based structures of the -160C (a) or -160A (b) S-paRNA allelic variants. Colour
code indicates base pairing probability ranges.
Sequence: -280/-57
SNP Opening energy G (kcal/mol)
Free energy of binding G (kcal/mol)
-160A 9.64 -7.78
-160C 12.02 -5.40
SNP -160A SNP -160C Sequence: -280/-57
a b
c
G
i (k
cal/m
ol)
G
i (k
cal/m
ol)
Supplementary Figure 11. Accessibility and binding capability of sense promoter-
associated RNA to isomiR-4534. (a,b) Unwinding and RNA:RNA interactions examined
for -160A (a) and -160C (b) S-paRNA allelic variants binding to isomiR-4534 using
RNAup (Vienna Web package). Plots show unwinding energy for opening existing
structures (black line) and interaction free energy (red line) for the two allelic variants. (c)
Estimated unwinding energy and total binding energy for the two allelic variants.
-141
-160 -160
-141
a b
Supplementary Figure 12. Modeling the effect of single base mutation on the
secondary structure of the AGO1/isomiR-4534 binding region. Predicted folding of
the -160A (a) S-paRNA and the -160A/G141U (b) S-paRNA using Mfold.
Fig. 1d
Fig. 1e
Fig. 1g
LNCaP
DU145
PC3
PC3
S
AS
S
AS
DU145 LNCaP
S
AS
Fig. 1i
Fig. 1k
PC3 LNCaP
S AS
PC3 LNCaP
Supplementary Figure 13. Uncropped images related to Figure 1, panels 1d, 1e, 1g, 1i and 1k
Actin
CDH1 AGO1
LNCaP CDH1
PrEC
AGO1
CDH1
Actin
DU145
Actin
AGO1
Fig. 3b
Fig. 2a
Fig. 3a
CDH1 AGO1 Tubulin
DU145
CDH1
Actin
LNCaP
CDH1
Actin
PC3
CDH1
Actin
100-
200-
200-
200-
Supplementary Figure 14. Uncropped images related to Figure 2 and 3, panels 2a, 3a and 3b
DU145 LNCaP
130-
-40
-55
PC3
Fig. 3e
S
AS
CD44 CD44
S AS
Fig. 3f
-281,-57 CD44 CD44 -668,-261
CD44 -476,-57 CD44 -668,-57
Supplementary Figure 15. Uncropped images related to Figure 3, panels 3e and 3f
Fig. 4i
Tubulin
CDH1
Fig. 5e
CD44
WT BS
WT BS
S
Fig. 6b
C
A
CD44
A C
Fig. 7d
A G141U
Supplementary Figure 16. Uncropped images related to Figure 4, 5, 6 and 7
Fig. S2c
Rrp44
Actin
S paRNA
Fig. S2f
LNCaP PC3
Fig. S3g
CDH1
Actin
PC3
CDH1
Actin
LNCaP
Fig. S4h
S
AS
Fig. S4n
+AGO1 -AGO1
Fig. S7d
S
AS
Fig. S7f
A C
CDH1
Actin
Fig. S4b
Supplementary Figure 17. Uncropped images related to Figure S2, S3, S4 and S7
Supplementary Table 1. Sequences of small interfering RNAs
siRNA ID Sequence of forward and reverse strand
CDH1 si217 5’ AACCGUGCAGGUCCCAUAA 3’
5’ UUAUGGGACCUGCACGGUU 3’
CDH1 si63 5’ AAUCAGCGGUACGGGGGGC 3’
5’ GCCCCCCGUACCGCUGAUU 3’
CDH1 si304 5’ AGAACUCAGCCAAGUGUAA 3’
5’ UUACACUUGGCUGAGUUCU 3’
siSUV39H1 5’ AGAACAGCUUCGUCAUGGA 3’
5’ UCCAUGACGAAGCUGUUCU 3’
siAGO1 5’ GAGAAGAGGUGCUCAAGAAUU 3’
5’ UUCUUGAGCACCUCUUCUCUU 3’
siAGO2 5’ GCACGGAAGUCCAUCUGAAUU 3’
5’ UUCAGAUGGACUUCCGUGCUU 3’
siAGO3 5’ GAAAUUAGCAGAUUGGUAAUU 3’
5’ UUACCAAUCUGCUAAUUUCUU 3’
siAGO4 5’ GGCCAGAACUAAUAGCAAUUU 3’
5’ AUUGCUAUUAGUUCUGGCCUU 3’
siGL3 5’ CUUACGCUGAGUACUUCGA 3’
5’ UCGAAGUACUCAGCGUAAG 3’
sipri-miR-4534 5’ GAGGUACAAGCAUAAAGUU 3’
5’ AACUUUAUGCUUGUACCUC 3’
siRrp44 5’ GAAAGAGACUGAAACAGAA 3’
5’ UUCUGUUUCAGUCUCUUUC 3’
Supplementary Table 2. PCR, mutagenesis and sequencing primer sets
Primer ID Forward (F) and Reverse (R) primer sequence
CDH1
CDH1 +144 R 5' CTGCGGCTCCAAGGGCCCA 3’
CDH1 +610 F 5' TGCCCAGAAAATGAAAAAGG 3’
CDH1 +810 R 5' GTGTATGTGGCAATGCGTTC 3’
CDH1 +2497 F 5’ ATGAGTGTCCCCCGGTATCT 3’
CDH1 +2619 R 5’ ACGAGCAGAGAATCATAAGGGGCG 3’
CDH1 -1919 F 5’ CCAACATGATGAAACCCTGTC 3’
CDH1 -1759 R 5’ CTGGAGTGCAATGGTGTGTT 3’
CDH1 -476 F 5’ TGGTGGTGTGCACCTGTACT 3’
CDH1 -303 F 5’ GAACTCAGCCAAGTGTAAAAGC 3’
CDH1 -261 R 5’ AAGACCTGGGATCAGAAAGG 3’
CDH1 -238 F 5’ ATTCGAACCCAGTGGAATCA 3’
CDH1 -282 F 5’ CCCTTTCTGATCCCAGGTCT 3’
CDH1 -281 F 5’ CCTTTCTGATCCCAGGTCTT 3’
CDH1 -272 F 5’ TCCCAGGTCTTAGTGAGCCA 3’
CDH1 -204 R 5’ GACCTGCACGGTTCTGATTC 3’
CDH1 -190 R 5’ TAGGTGGGTTATGGGACCTG 3’
CDH1 -171 R 5’ GCCTGGAGTTGCTAGGGTCT 3’
CDH1 -153 R 5’ AGACGCGGTGACCCTCTA 3’
CDH1 -139 R 5’ CACCCGGCCTCGCATAGA 3’
CDH1 -82 R 5’ GGCCACAGCCAATCAGCA 3’
CDH1 -57 R 5’ CTGATTGGCTGAGGGTTCAC 3’
CDH1 -180_C F 5’ ACTCCAGGCTAGAGGGTTAC 3’
CDH1 -180_A F 5’ ACTCCAGGCTAGAGGGTTAA 3’
CDH1 EX3 F 5’ CTCGACACCCGATTCAAAGT 3’
CDH1 EX3 R 5’ GGTGGTGCCCCACTGTATT 3’
CDH1 EX8 F 5’ CGTATACCCTGGTGGTTCAAG 3’
CDH1 EX8 R 5’ GGAGGATTATCGTTGGTGTCA 3’
ACTIN
ACT +221 F 5' AAGAGAGGCATCCTCACCCT 3’
ACT +439 R 5' TACATGGCTGGGGTGTTGAA 3’
ACT +778 F 5’ ATTGGCAATGAGCGGTTC 3’
ACT +864 R 5’ GGATGCCACAGGACTCCAT 3’
CD44
CD44 F 5’ GACACCATGGACAAGTTTTGG 3’
CD44 R 5’ CGGCAGGTTATATTCAAATCG 3’
Rrp44
Rrp44 F 5’ GTGGCATGCTTTCCAAGTCT 3’
Rrp44 R 5’ GCCAACCATCAATAGCAACA 3’
ARGONAUTE 1
AGO1 F 5’ GCACTGCCCATTGGCAACGAA 3’
AGO1 R 5’ CATTCGCCAGCTCACAATGGCT 3’
ARGONAUTE 2
AGO2 F 5’CGCGTCCGAAGGCTGCTCTA 3’
AGO2 R 5’TGGCTGTGCCTTGTAAAACGCT 3’
ARGONAUTE 3
AGO3 F 5’GGAATTAGACAAGCCAATCAGCA 3’
AGO3 R 5’AGGGTGGTCATATCCTTCTGGA 3’
ARGONAUTE 4
AGO4 F 5’CTAACAGACTCCCAGCGTGTCA 3’
AGO4 R 5’GACTGGCTGGCCGTCTAGTCA 3’
SUV39H1
SUV39H1 F 5’ GGCAACATCTCCCACTTTGT 3’
SUV39H1 R 5’CAATACGGACCCGCTTCTTA 3’
Pri-miR-4534
Pri-miR-4534 -566 F 5’CCTAGATCCCACCCCAGACT 3’
Pri-miR-4534 -416 R 5’ACGCCTGGCTCAGTATGTTT 3’
Pri-miR-4534 -162 F 5’CAGCCTGGGTGACAGAGTAAG 3’
Pri-miR-4534 -102 F 5’CCTAGATCCCACCCCAGACT 3’
Pri-miR-4534 +58 R 5’TACCCAAGACCCCTCCT 3’
Pre-miR-4534
Pre-miR-4534 -104 F 5’AAGTTCCCTGGGAGGTCTTC 3’
Pre-miR-4534 +99 R 5’TCCGGATACCCCAGTATAACC 3’
Mutagenesis primers
CDH1-160SNP F 5’AGGCTAGAGGGTCACCGCGTCTATGCGAGG 3’
CDH1-160SNP R 5’CCTCGCATAGACGCGGTGACCCTCTAGCCT 3’
CDH1-PROMOTER-T1 F 5’CTGCTAGCTCAGTGGGCCCTGGCGAATTCCTGAAATCCTAGCAC 3’
CDH1-PROMOTER-T1 R 5’GTGCTAGGATTTCAGGAATTCGCCAGGGCCCACTGAGCTAGCAGCCT 3’
CDH1-PROMOTER-BS F 5’GGGCGGGCCGTCAGCACGGGCCTGGGGAGGGGTC 3’
CDH1-PROMOTER-BS R 5’GACCCCTCCCCAGGCCCGTGCTGACGGCCCGCCC 3’
S_TSS
S_F 5’ CAAACTAGCAAAATAGGCTGTCC 3’
S_R 5’ AAGCTTCGGAGTCTCGCTCTGTCTTG 3’
AS_TSS
AS_F 5’ AAGCTTCCTTTCTGATCCCAGGTCTT 3’
AS_R 5’ GAGCTCCTGATTGGCTGAGGGTTCAC 3’
S-AS transcript chimera
S-AS_F1 5’ AGACTCCGAAGCTTGGCAT 3’
S-AS_F2 5’ TAGGTGGGTTATGGGACCTG 3’
S-AS_R 5’ GGAACCAGGGCGTATCTCTT 3’
SHAPE primers
pcDNA-120 – paRNA F 5’ CCACTGCTTACTGGCTTATCG 3’
pcDNA-paRNA +61 polyA R 5’ TTT TTT TTT TTT TTT CAACAGATGGCTGGCAACTA 3’
extREV9 5’ CAACAGATGGCTGGCAACTA 3’
extREV8 5’ TCTAGACTCGAGCGGCCGCCA 3’
extREV5_A 5’ AGACGCGTTGACCCTCTA 3’
extREV5_C 5’ AGACGCGGTGACCCTCTAGC 3’
Supplementary Table 3. Predicted paRNA in the CDH1 locus
paRNA Cell line Chrom Start End Strand HOMER annotation Close gene Score
AS lncap chr16 68770846 68771078 - Promoter-antisense CDH1 24
AS hct p53 -/- chr16 68770844 68771044 - Promoter-antisense CDH1 23
AS hct p53 +/+ chr16 68770865 68771045 - Promoter-antisense CDH1 13
AS mcf7 c1 chr16 68770810 68771042 - Promoter-antisense CDH1 18
AS h1esc t1 chr16 68770852 68771039 - Promoter-antisense CDH1 11
S hct p53 -/- chr16 68770887 68771056 + Promoter-sense CDH1 22
S hct p53 -/- chr16 68770523 68770676 + Not annotated for score filter CDH1 0.4S hct p53 -/- chr16 68770731 68770881 + Not annotated for score filter CDH1 0.666667S hct p53 +/+ chr16 68770512 68770729 + Not annotated for score filter CDH1 0.56
.