supplemental fig. 1genesdev.cshlp.org/content/suppl/2012/10/09/gad.204438...2012/10/09 ·...

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


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


SpeckleF2 -MALAT1_3’

B

Genome gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttgctttttggcctttccctagctttaaaaaaaaaaaagcaaaa RACE #1 gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttctcaggttttttttttttttttt RACE #2 gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaaccttttgttttttttttttttt RACE #3 gattcgtcagtagggttgtaaaggtttttcttttcctgagaaaacaacctttttt


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


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


cGFP -MALAT1_3’


Moc

k

WT

Com

p.5

Com

p.6

Com

p.7

70

60

150

190


cGFP -MALAT1_3’


Moc

k

WT

Com

p.8

70

60

150

190


cGFP -MALAT1_3’

A

B


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


cGFP -MALAT1_3’

Supplemental Fig. 5

Supplemental Fig. 6


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


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


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

supplemental fig. 1genesdev.cshlp.org/content/suppl/2012/10/09/gad.204438...2012/10/09 ·...

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