A +CMV-cGFP-mMALAT1_3’
200
180
70
60
50
kcoM W
T
Mut
10
Mut
7
70
60
50
mascRNA(All mascRNA Probe)
cGFP -MALAT1_3’
mascRNA(Mouse mascRNA Only Probe)
mascRNAMut 10
GACGCUG
G U GGC
UG G
C A CU
CCUGG
UU
U
CCAGG A
CGGGG
U UC
AAG
C C C UU
GCGGUGUC
5’
3’U
GA
C
G
mascRNAMut 7
GACGCUG
G U GGC
UG G
C A CU
CCUGG
UU
U
CCAGG A
CGGGG
U UC
AAG
C C C UU
GCGGUGUC
5’
3’U
A
B
C Mut 10 Vector TTCAAGTCCCTGCGGTACCGttgctt 6 Clones TTCAAGTCCCTGCGGTACCGCCACCA 4 Clones TTCAAGTCCCTGCGGTACCGCCACC 2 Clones TTCAGGTCCCTGCGGTACCGCCACCA 1 Clone TTCAAGTCCCTGCGGTACCGCCACCAG
D Mut 7 Vector TTCAAGTCCCTGCGGTATCTttgctt Clone #1 TTCAAGTCCCTGCGGTAAAACTCTTTTTTT Clone #2 TTCAAGTCCCTGCGGTTTAAATTTTTTTT Clone #3 TTCAAGTCCCTGCGGTATCTTTTTTTTTCTT Clone #4 TTCAAGTCCCTGCGGTATTTTTATTTTTTTTATTTCTT Clone #5 TTCAAGTCCCTGCGGTATCTC Clone #6 TTCAAGTCCCTGCGGTATCTCCA
Supplemental Fig. 1
AAAAAAAGCAAAA
CMV Promoter
cGFP ORF
U U6581 6754
AAAAAAAGCAAAA
CMV Promoter
U U6581 6754
GGACAAAAACGA
CMV Promoter
6754
CMV-cGFP-mMALAT1_3’ Sense
CMV-mMALAT1_3’ Sense
CMV-Leader-mmascRNA Sense
Moc
k
Sens
e
Ant
isen
se
Sens
e
Ant
isen
se
Sens
e
Ant
isen
se
CMV-cGFP-
mMALAT1_3’
CMV-mMALAT1_3’
CMV-Leader-
mmascRNA
HeLa
mascRNA
U6
(All mascRNA probe)
70
60
50
80
A B
Supplemental Fig. 2
mMALAT1_3’
mMALAT1_3’
Moc
k
CM
V-cG
FP-m
MA
LAT1
_3’
CM
V-cG
FP-S
V40
Poly
(A)
CM
V-cG
FP-b
GH
Pol
y(A
)
CM
V-cG
FP-m
MEN
β_3
’
70
60
180
200
120
140
160
mascRNA(All mascRNA Probe)
cGFPmRNA
A
6581 6754
CMV
cGFP ORF
AAAAAGCAAAAU U
cGFP ORF
AAUAAA
CMV
CMV-cGFP-mMALAT1_3’
CMV-cGFP-bGH/SV40 Poly(A)
20661 20834
CMV
cGFP ORF
AAAAAGCAAAAU U
CMV-cGFP-mMEN β_3’
B
Supplemental Fig. 3
Moc
k
CM
V-cG
FP-m
MA
LAT1
_3’
CM
V-cG
FP-m
MEN
β_3
’
25 kDa
20 kDa
100 kDa
150 kDa
cGFP
Vinculin
C
mMALAT1_3’
mMEN β_3’
AWT gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaaMut U1 gattcgtcagtagggttgtaaaggtttAAAAAttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaa Mut U2-CGAAAA gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttCGAAAAtggcctttccctagctttaaaaaaaaaaaagcaaaa Mut A-CGAAAA gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaTTTTCGaaaa
Conserved Stem LoopA-rich Tract
U-rich Motif 2
U-rich Motif 1
Supplemental Fig. 4
+CMV-SpeckleF2-mMALAT1_3’
Moc
k
CM
V-cG
FP-m
MA
LAT1
_3’
WT
Mut
U1
Mut
U2-
CG
AA
AA
Mut
A-C
GA
AA
A
70
60
180
200
mascRNA(All mascRNA Probe)
SpeckleF2 -MALAT1_3’
B
Genome gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaa RACE #1 gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttttttttttttttt RACE #2 gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttttttttttttt RACE #3 gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaacctttttt
Conserved Stem LoopA-rich Tract
U-rich Motif 2
U-rich Motif 1
C
WT gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaa Yes
Comp.1 *******************aaaggtttttcttttcctga********************tcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaa YesComp.2 *******************aaaggtttttcttttcctga********************tcaggttttgcttttt*****************aaaaaaaaaaaagcaaaa NoComp.3 *******************aaaggtttttcttttcctga********************tcaggttttgctttttggcctttccctagcttt*******aaaaagcaaaa NoComp.4 *******************aaaggtttttcttttcctga********************tcaggttttgcttttt**********tagcttt*******aaaaagcaaaa NoComp.5 gattcgtcagtagggttgtaaaggtttttcttttcctga********************tcaggttttgcttttt*****************aaaaaaaaaaaagcaaaa NoComp.6 *******************aaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgcttttt*****************aaaaaaaaaaaagcaaaa YesComp.7 *******************aaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagcttt*******aaaaagcaaaa YesComp.8 *******************aaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgcttttt************************aaaaagcaaaa NoComp.9 *******************aaaggtttttcttttcctgagaaaacaa****ttgttttctcaggttttgcttttt*****************aaaaaaaaaaaagcaaaa Yes Comp.10 *******************aaaggtttttcttttcctgagaaaac********gttttctcaggttttgcttttt*****************aaaaaaaaaaaagcaaaa YesComp.11 *******************aaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgcttttt**********tagcttt*******aaaaagcaaaa YesComp.12 *******************aaaggtttttcttttcctgagaaa************tttctcaggttttgcttttt*****************aaaaaaaaaaaagcaaaa YesComp.13 *******************aaaggtttttcttttcctgaga****************tctcaggttttgcttttt*****************aaaaaaaaaaaagcaaaa NoComp.14 *******************aaaggtttttcttttcctgagaaa************tttctcaggttttgcttttt********************aaaaaaaaagcaaaa Yes
Conserved Stem LoopA-rich Tract
U-rich Motif 2
U-rich Motif 1
Stable?
+ CMV-cGFP-mMALAT1_3’
Moc
k
WT
Com
p.1
Com
p.2
Com
p.3
Com
p.4
70
60
150
190
mascRNA(All mascRNA Probe)
cGFP -MALAT1_3’
+ CMV-cGFP-mMALAT1_3’
Moc
k
WT
Com
p.5
Com
p.6
Com
p.7
70
60
150
190
mascRNA(All mascRNA Probe)
cGFP -MALAT1_3’
+ CMV-cGFP-mMALAT1_3’
Moc
k
WT
Com
p.8
70
60
150
190
mascRNA(All mascRNA Probe)
cGFP -MALAT1_3’
A
B
+ CMV-cGFP-mMALAT1_3’
Moc
k
WT
Com
p.6
Com
p.11
Com
p.9
Com
p.10
Com
p.12
Com
p.13
70
60
140
190
mascRNA(All mascRNA Probe)
cGFP -MALAT1_3’
Supplemental Fig. 5
Supplemental Fig. 6
+ CMV-cGFP-mMALAT1_3’
Moc
k
WT
Mut
U2
Mut
U2.
3
Mut
U2.
2
Mut
U2.
1
70
60
180
200
Mut
U2-
AA
Mut
U2.
4
Mut
U2.
5
mascRNA(All mascRNA Probe)
cGFP -MALAT1_3’
1 2 3 4 5 6 7 8 9
aaagg
ccugaga
aa u
u
ucucagg
u u u u g c u u u u uaaaaagcaaaa
5ʼ
3ʼ
u u u u c u u u u u
U-rich Motif 1
aaa
a
4
35
67
89
WT ttttgcttttt 2Mut U2 tttAAAAAttt 3Mut U2.3 AAttgcttttt 4Mut U2.2 ttAAgcttttt 5Mut U2.1 ttttAAttttt 6Mut U2-AA ttttgcAAttt 7Mut U2.4 ttttgcttAAt 8Mut U2.5 ttttgctttAA 9
U-rich Motif 2
LaneA B
5’
Supplemental Fig. 7
A-1 A-2
A-3
G-4
G-5
U-6
A-45
A B
RMSD to Best-scoring Model
Ros
etta
All-
atom
Sco
re
C
Supplemental Fig. 8
AMock Transfected
log
10(m
Che
rry)
log 10(eYFP)0
1
2
1.5
0.5
3
4
3.5
2.5
5
5.5
4.5
1 2 3 4 5
SV40 Poly(A)
log
10(m
Che
rry)
log 10(eYFP)0
1
2
1.5
0.5
3
4
3.5
2.5
5
5.5
4.5
1 2 3 4 5
mMALAT1_3’ WT
log
10(m
Che
rry)
log 10(eYFP)0
1
2
1.5
0.5
3
4
3.5
2.5
5
5.5
4.5
1 2 3 4 5
mMALAT1_3’ Comp.14
log
10(m
Che
rry)
log 10(eYFP)0
1
2
1.5
0.5
3
4
3.5
2.5
5
5.5
4.5
1 2 3 4 5
mMALAT1_3’ Comp.15
log
10(m
Che
rry)
log 10(eYFP)0
1
2
1.5
0.5
3
4
3.5
2.5
5
5.5
4.5
1 2 3 4 5
B C D
2
1.5
3
4
3.5
2.5
5
5.5
4.5
2 3 4 5
log 10(eYFP)
log
10(m
Che
rry)
SV40 Poly(A)mMALAT1_3’ WT
2
1.5
3
4
3.5
2.5
5
5.5
4.5
2 3 4 5
log 10(eYFP)
log
10(m
Che
rry)
mMALAT1_3’ WTmMALAT1_3’ Comp.14
2
1.5
3
4
3.5
2.5
5
5.5
4.5
2 3 4 5
log 10(eYFP)
log
10(m
Che
rry)
mMALAT1_3’ WTmMALAT1_3’ Comp.15
E
Moc
k
SV40
Pol
y(A
)
mM
ALA
T1_3
’ WT
250
130
200
160
mCherry mRNA
Supplemental Fig. 9
log 10(eYFP)
2
1.5
3
4
3.5
2.5
5
5.5
4.5
log
10(m
Che
rry)
6
2 3 4 53.52.5 4.5 5.5
0x - SV40 Poly(A)
log 10(eYFP)
2
1.5
3
4
3.5
2.5
5
5.5
4.5
log
10(m
Che
rry)
2 3 4 53.52.5 4.5 5.5
let-7 bg - SV40 Poly(A)
log 10(eYFP)
2
1.5
3
4
3.5
2.5
5
5.5
4.5
log
10(m
Che
rry)
2 3 4 53.52.5 4.5 5.5
let-7 pf - SV40 Poly(A)
A
log 10(eYFP)
2
1.5
3
4
3.5
2.5
5
5.5
4.5
log
10(m
Che
rry)
2 3 4 53.52.5 4.5 5.5
0x-SV40 Poly(A)let-7 bg-SV40 Poly(A)let-7 pf-SV40 Poly(A)
B
log 10(eYFP)
2
1.5
3
4
3.5
2.5
5
5.5
4.5
log
10(m
Che
rry)
2 3 4 53.52.5 4.5 5.5
0x - mMALAT1_3’ WT
log 10(eYFP)
2
1.5
3
4
3.5
2.5
5
5.5
4.5
log
10(m
Che
rry)
2 3 4 53.52.5 4.5 5.5
let-7 bg - mMALAT1_3’ WT
log 10(eYFP)
2
1.5
3
4
3.5
2.5
5
5.5
4.5
log
10(m
Che
rry)
2 3 4 53.52.5 4.5 5.5
let-7 pf - mMALAT1_3’ WT
log 10(eYFP)
2
1.5
3
4
3.5
2.5
5
5.5
4.5
log
10(m
Che
rry)
2 3 4 53.52.5 4.5 5.5
0x-mMALAT1_3’ WTlet-7 bg-mMALAT1_3’ WTlet-7 pf-mMALAT1_3’ WT
Moc
k
mM
ALA
T1_3
’
V. v
ul. a
dd-m
asc
B. s
ub. x
pt-m
asc
V. c
hol.
Vc2-
mas
c
T. te
ng. S
AM
-mas
c
T. te
ng. g
lmS-
mas
c
25 kDa
20 kDa
100 kDa
150 kDa
+ CMV-cGFP Plasmid
cGFP
Vinculin
A
6581 6754
CMV
cGFP ORF
AAAAAGCAAAAU U
CMV-cGFP-mMALAT1_3’
6754
CMV
cGFP ORF
CMV-cGFP-Riboswitch-masc
Riboswitch
cGFP ORF
Riboswitch Senses Size (nt)V. vulnificus add Adenine 71B. subtilis xpt Guanine 68V. cholera Vc2 Cyclic di-GMP 98T. tengcongensis SAM S-adenosylmethionine (SAM) 132T. tengcongensis glmS Glucosamine-6 phosphate (GlcN6P) 125
B C
Supplemental Fig. 10
mMALAT1_3’
70
60
180
200
100
+ CMV-cGFP Plasmid
Moc
k
mM
ALA
T1_3
’
V. v
ul. a
dd-m
asc
B. s
ub. x
pt-m
asc
V. c
hol.
Vc2-
mas
c
T. te
ng. S
AM
-mas
c
T. te
ng. g
lmS-
mas
c
cGFPmRNA
mascRNA(All mascRNA Probe)
70
60
180
200
100
Supplementary Table 1
Figures where plasmid is usedcGFP Plasmids Sequence inserted downstream of cGFP Open Reading Frame (unless ORF not present as noted by * in plasmid name) Main Figures Supp. FigurespCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' WT Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 1E; 2B-‐G; 3C-‐E; 4F; 5A,F,G 1B; 2B; 3B,C; 4B; 5B; 6B; 10B,C
pCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut 7 Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtAtctttgctt 1B,DpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut 10 Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagGcgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtACcGttgctt 1B,C
pCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U1 Sense gattcgtcagtagggttgtaaaggtttAAAAAttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 2E,FpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U2 Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggtttAAAAAtttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 2E,F 6BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U1/U2 Sense gattcgtcagtagggttgtaaaggtttAAAAAttcctgagaaaacaaccttttgttttctcaggtttAAAAAtttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 2E
pCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U1.1 Sense gattcgtcagtagggttgtaaaggtttttAttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 4FpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U1.2 Sense gattcgtcagtagggttgtaaaggtttAActtttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 4FpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U1.3 Sense gattcgtcagtagggttgtaaaggtttttcAAttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 4FpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U1.4 Sense gattcgtcagtagggttgtaaaggtttttcttAAcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 4FpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U1.5 Sense gattcgtcagtagggttgtaaaggtAAttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 4F
pCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U2.1 Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttAAtttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 6BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U2.2 Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttAAgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 6BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U2.3 Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggAAttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 6BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U2.4 Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgcttAAtggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 6BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U2.5 Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttAAggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 6B
pCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U2-‐CG Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttCGtttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 3CpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut A-‐CG Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaaCGaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 3CpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U2/A-‐CG Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttCGtttttggcctttccctagctttaaaaaaaaaaaaCGaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 3C
pCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U2-‐AA Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgcAAtttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 3D 6BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut A-‐AA Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaTTgcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 3DpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U2/A-‐AA Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgcAAtttggcctttccctagctttaaaaaaaaaaTTgcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 3D
pCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U2-‐CGAAAA Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttCGAAAAtggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 3EpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut A-‐CGAAAA Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaTTTTCGaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 3EpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Mut U2/A-‐CGAAAA Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttCGAAAAtggcctttccctagctttaaaaaaaaTTTTCGaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 3E
pCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.1 Sense *******************aaaggtttttcttttcctga********************tcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.2 Sense *******************aaaggtttttcttttcctga********************tcaggttttgcttttt*****************aaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.3 Sense *******************aaaggtttttcttttcctga********************tcaggttttgctttttggcctttccctagcttt*******aaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.4 Sense *******************aaaggtttttcttttcctga********************tcaggttttgcttttt**********tagcttt*******aaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.5 Sense gattcgtcagtagggttgtaaaggtttttcttttcctga********************tcaggttttgcttttt*****************aaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.6 Sense *******************aaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgcttttt*****************aaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 4E 5BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.7 Sense *******************aaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagcttt*******aaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.8 Sense *******************aaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgcttttt************************aaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.9 Sense *******************aaaggtttttcttttcctgagaaaacaa****ttgttttctcaggttttgcttttt*****************aaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.10 Sense *******************aaaggtttttcttttcctgagaaaac********gttttctcaggttttgcttttt*****************aaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.11 Sense *******************aaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgcttttt**********tagcttt*******aaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.12 Sense *******************aaaggtttttcttttcctgagaaa************tttctcaggttttgcttttt*****************aaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.13 Sense *******************aaaggtttttcttttcctgaga****************tctcaggttttgcttttt*****************aaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5BpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.14 Sense *******************aaaggtttttcttttcctgagaaa************tttctcaggttttgcttttt********************aaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 2G; 5F-‐HpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.15 Sense *******************CCCAAtttttcttttGAATTCTCT************AGAGAATTCttttgcttttt********************CTTCaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5F-‐HpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.25 Sense *******************CCCAAtttttcttttGAAgagaaa************tttctcTTCttttgcttttt********************CTTCaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5F,GpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.26 Sense *******************CCCAAtttttcttttGAAgagaaa************tttctcTTCttttgcttttt********************aaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5F,GpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' Comp.27 Sense *******************aaaggtttttcttttGAAgagaaa************tttctcTTCttttgcttttt********************CTTCaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5F,G
pCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' U*A-‐C Sense *******************aaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgcCCCCt*****************aaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 4EpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' U*G-‐U Sense *******************aaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgcttttt*****************aaaaaaaaGGGGgcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 4EpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' U*G-‐C Sense *******************aaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgcCCCCt*****************aaaaaaaaGGGGgcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 4EpCRII-‐TOPO CMV-‐cGFP-‐mMALAT1_3' C*G-‐C Sense *******************aaaggtCCCCcttttcctgagaaaacaaccttttgttttctcaggttttgcCCCCt*****************aaaaaaaaGGGGgcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 4E
pCRII-‐TOPO CMV-‐cGFP-‐SV40 Poly(A) Sense aacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatctta 2B; 5A 3BpCRII-‐TOPO CMV-‐cGFP-‐bGH Poly(A) Sense cgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg 2B; 5A 3B
pCRII-‐TOPO CMV-‐cGFP-‐mMEN β_3' Sense ggcacggagccgccgcaggtgtttcttttcctgaccgcggctcatggccgcgctcaggttttgcttttcacctttgtctgagagaacgaacgtgagcaggaaaaagcaaaaggcactggtggcggcacgcccgcacctcgggccagggttcgagtccctgcagtaccgtgcttc 3B,C
* pCRII-‐TOPO CMV-‐mMALAT1_3' Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 2B* pCRII-‐TOPO CMV-‐mmascRNA Sense GGACAAAAACGAgacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 2B
pCRII-‐TOPO CMV-‐SpeckleF2-‐mMALAT1_3' WT Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 2C 4B,CpCRII-‐TOPO CMV-‐SpeckleF2-‐mMALAT1_3' Mut U1 Sense gattcgtcagtagggttgtaaaggtttAAAAAttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 4BpCRII-‐TOPO CMV-‐SpeckleF2-‐mMALAT1_3' Mut U2-‐CGAAAA Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttCGAAAAtggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 4BpCRII-‐TOPO CMV-‐SpeckleF2-‐mMALAT1_3' Mut A-‐CGAAAA Sense gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaTTTTCGaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 4B
pCRII-‐TOPO CMV-‐cGFP-‐V. vulnificus add-‐masc Sense Ggcttcatataatcctaatgatatggtttgggagtttctaccaagagccttaaactcttgattatgaagtcgacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 10B,CpCRII-‐TOPO CMV-‐cGFP-‐B. subtilis xpt-‐masc Sense Ggatcatataatcgcgtggatatggcacgcaagtttctaccgggcaccgtaaatgtccgactatggtcgacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 10B,CpCRII-‐TOPO CMV-‐cGFP-‐V. cholera Vc2-‐masc Sense ggaaaaatgtcacgcacagggcaaaccattcgaaagagtgggacgcaaagcctccggcctaaaccagaagacatggtaggtagcggggttaccgatgggacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 10B,CpCRII-‐TOPO CMV-‐cGFP-‐T. tengcongensis SAM-‐masc Sense ttaaaatctcttatcaagagaggtggagggactggcccgatgaaacccggcaaccagccttagggcatggtgccaattcctgcagcggtttcgctgaaagatgagagattcttgtagtctcttcttttagcggacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 10B,CpCRII-‐TOPO CMV-‐cGFP-‐T. tengcongensis glm-‐masc Sense ccggctttaagttgacgagggcagggtttatcgagacatcggcgggtgccctgcggtcttcctgcgaccgttagaggactggtaaaaccacaggcgactgtggcatagagcagtccgggcaggaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 10B,C
Figures where plasmid is usedDual-‐Color Fluorescent Reporters Sequence inserted downstream of mCherry (Between HindIII and AatII sites) Main Figures Supp. FiguresRed-‐Yellow Reporter 0x-‐SV40 Poly(A) GTCGACGATATCtccagaggatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcc 5C,D; 7B,C 8A,B,E; 9ARed-‐Yellow Reporter 0x-‐mMALAT1_3' WT GTCGACGATATCgattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5C,D; 7B,C 8A-‐E; 9BRed-‐Yellow Reporter 0x-‐mMALAT1_3' Comp.14 GTCGACGATATC*******************aaaggtttttcttttcctgagaaa************tttctcaggttttgcttttt********************aaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5C,D 8A,CRed-‐Yellow Reporter 0x-‐mMALAT1_3' Comp.15 GTCGACGATATC*******************CCCAAtttttcttttGAATTCTCT************AGAGAATTCttttgcttttt********************CTTCaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 5C,D 8A,D
Red-‐Yellow Reporter let-‐7 bg-‐SV40 Poly(A) AACCATACAACGATCTACCTCAGAATTCAACCATACAACGATCTACCTCAGTCGACGATATCtccagaggatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcc 7B,C 9ARed-‐Yellow Reporter let-‐7 pf-‐SV40 Poly(A) AACCATACAACCTACTACCTCAGTCGACGATATCtccagaggatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcc 7B,C 9A
Red-‐Yellow Reporter let-‐7 bg-‐mMALAT1_3' WT AACCATACAACGATCTACCTCAGAATTCAACCATACAACGATCTACCTCAGTCGACGATATCgattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 7B,C 9BRed-‐Yellow Reporter let-‐7 pf-‐mMALAT1_3' WT AACCATACAACCTACTACCTCAGTCGACGATATCgattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaagacgctggtggctggcactcctggtttccaggacggggttcaagtccctgcggtgtctttgctt 7B,C 9B
Supplementary Table 2
Northern Probe Sequence Main Figures Supplemental FiguresmascRNA (Mouse mascRNA Only Probe) gcaaagacaccgcagggacttgaac 1E 1BmascRNA (All mascRNA Probe) gcaaagacaccgcagggatttgaaccccgtcctggaaaccaggagtgcca 1E; 2E,G; 3C-‐E; 4F; 5F 1B; 2B; 3B; 4B; 5B; 6B; 9BcGFP Probe tccatgccgtgggtgatgcc 1E; 2B,C,E,G; 3C-‐E; 4F; 5F,H 1B; 3B; 4B; 5B; 6B; 9BEndogenous MALAT1 ctaagatgctagcttggccaagtctgttatg 2B; 5HU6 snRNA gctaatcttctctgtatcgttccaattttagtatatgtgctgccg 2BmCherry Probe gccggtggagtggcggccctc 8E
RNase H Oligo for cGFP Transcript cgcgcttctcgttggggtcc 1E; 2C,E,G; 3C-‐E; 4F; 5F 1B; 3B; 4B; 5B; 6BRNase H Oligo for mCherry Transcript ttcgtactgttccacgatgg 8E
Figures where plasmid is used