Supplementary Figure S1 (A) Change of reporter activity levels after actinomycin D treatment....

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Supplementary Figure S1 (A) Change of reporter activity levels after actinomyci n D treatment. HEK293T cells were transiently transf ected with the reporter and incubated with actinomyc in D for the indicated times. (B) Schematic presentation of deletion constructs of mc ry1 3’UTR and their reporter activity. (C) Change of reporter mRNA levels after actinomycin D treatment. NIH3T3 cells were transiently transfected with the reporter and incubated with actinomycin D f or the indicated times. (D) Each 3′UTR in vitro transcribed with 32- α P-rUTP was su bjected to in vitro binding and UV cross-linking assay with HEK 293T nuclear extract. The arrow indicates the proteins that shows differential binding. (E) wt 3′UTR in vitro transcribed with 32- α P-rUTP was sub jected to in vitro binding and UV cross-linking assay w ith HEK 293T cytoplasmic extract and unlabeled cold wtUTR. The arrow indicates the proteins that shows differential binding. (F) Each 3′UTR in vitro transcribed with 32- α P-rUTP was su bjected to in vitro binding and UV cross-linking assay with NIH-3T3 cytoplasmic extract. The arrow indicat es the proteins that shows differential binding. (G) wt 3′UTR in vitro transcribed with 32- α P-rUTP was subj ected to in vitro binding and UV cross-linking assay wi th HEK 293T cytoplasmic extract and homo-oligoribonu cleotide A, C, and U. The arrow indicates the prote ins that shows differential binding. (H) Alignment of conserved U-tract sequences among vari ous species compared with between 252 and 280 in mcr y1 3'UTR.

Transcript of Supplementary Figure S1 (A) Change of reporter activity levels after actinomycin D treatment....

Page 1: Supplementary Figure S1 (A) Change of reporter activity levels after actinomycin D treatment. HEK293T cells were transiently transfected with the reporter.

• Supplementary Figure S1

(A) Change of reporter activity levels after actinomycin D treatment. HEK293T cells were transiently transfected with the reporter and incubated with actinomycin D for the indicated times.

(B) Schematic presentation of deletion constructs of mcry1 3’UTR and their reporter activity.

(C) Change of reporter mRNA levels after actinomycin D treatment. NIH3T3 cells were transiently transfected with the reporter and incubated with actinomycin D for the indicated times.

(D) Each 3′UTR in vitro transcribed with 32- α P-rUTP was subjected to in vitro binding and UV cross-linking assay with HEK 293T nuclear extract. The arrow indicates the proteins that shows differential binding.

(E) wt 3′UTR in vitro transcribed with 32- α P-rUTP was subjected to in vitro binding and UV cross-linking assay with HEK 293T cytoplasmic extract and unlabeled cold wtUTR. The arrow indicates the proteins that shows differential binding.

(F) Each 3′UTR in vitro transcribed with 32- α P-rUTP was subjected to in vitro binding and UV cross-linking assay with NIH-3T3 cytoplasmic extract. The arrow indicates the proteins that shows differential binding.

(G) wt 3′UTR in vitro transcribed with 32- α P-rUTP was subjected to in vitro binding and UV cross-linking assay with HEK 293T cytoplasmic extract and homo-oligoribonucleotide A, C, and U. The arrow indicates the proteins that shows differential binding.

(H) Alignment of conserved U-tract sequences among various species compared with between 252 and 280 in mcry1 3'UTR.

Page 2: Supplementary Figure S1 (A) Change of reporter activity levels after actinomycin D treatment. HEK293T cells were transiently transfected with the reporter.

0 20 40 60 80 100 120NAT activity

pcNAT

pcNAT_wt610

pcNAT_N201

pcNAT_N401

610mcry1_3′UTR

201

401

1

CMV NAT

Woo et al_Supplementary Fig. S1

B

A

C

0

20

40

60

80

100

120

140

Act.D 0h Act.D 5h

AA

NA

T a

ctiv

ity

(%o

f A

ct.D

0h

) pcNATpcNAT_wt610

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 2 4 6Time after Act.D [h]

pcNAT-N401pcNAT-N201

Rel

ativ

e m

RN

A l

evel

(aan

at/r

pl3

2)

Page 3: Supplementary Figure S1 (A) Change of reporter activity levels after actinomycin D treatment. HEK293T cells were transiently transfected with the reporter.

homopolymer

HEK_cyto + +

A C U

+ +

mCry1_3′UTR

115

90.5

61.5

46.2

37.5

F G

Woo et al_Supplementary Fig. S1

H

N401

wt610

N201

NIH_cyto + + +

++

+

115

90.5

61.5

46.2

37.8

37.5

N401

wt610N201

HEK_nuc +

++

+

+ +

115

90.5

61.5

46.2

+

+ + +

+ +

Cold

37.5

wt610

HEK_cyto

115

90.5

61.5

46.2

D E

Page 4: Supplementary Figure S1 (A) Change of reporter activity levels after actinomycin D treatment. HEK293T cells were transiently transfected with the reporter.

Sequence(5′=>3′)

Endo-mcry1 F CCT TGA AAA GCC TGG GAA AT

Endo-mcry1 R TCC GCT GCG TCT ATA TCC TC

Endo-mper2 F GCC TTC AGA CTC ATG ATG ACA GA

Endo-mper2 R TTT GTG TGC GTC AGC TTT GG

mRPL32 F AAC CCA GAG GCA TTG ACA AC

mRPL32 R CAC CTC CAG CTC CTT GAC AT

aanat F TTT GAG ATT GAG CGC GAA G

aanat R TCG AAC CAG CCC AGT GAC

mGAPDH F GCC ATC AAT GAC CCC TTC ATT

mGAPDH R GCT CCT GGA AGA TGG TGA TGG

mTBP F CAG CCT TCC ACC TTA TGC TC

mTBP R TTG CTG CTG CTG TCT TTG TT

Supplementary Table primer sequences for real time PCR