U1: GGTATCTCCCCTGCCAGGTAAGTAT U2:...

Morpholinos:

Control: CCTCTTACCTCAGTTACAATTTATA

U1: GGTATCTCCCCTGCCAGGTAAGTAT

U2: TGATAAGAACAGATACTACACTTGA

U4: TACGATACTGCCACTGCGCAAAGCT

siRNAs:

Control Life technologies silencer negative control #1

Cdk12 Life technologies silencer select s28622

CPSF73 Life technologies silencer select s28533

CstF77 Life technologies silencer select s3687

Primers:

Stable lines:

PromR cttaagtttaaacgctagagtc

βpAF gatattatgaagggccttgag

NS1f atggatccaaacactgtgtca

NS1r tcaaacttctgacctaattgtt

NS1mF tttaaatcgtccgatgaggaccccaa

NS1mR cggtataataacacagtttcgagtc

NR3C1 RACE:

dtTAG cttcttcgtaacctgtacgaa(t)16

NR3C1 PCPA F gttgctcattaacagatatct

TAG cttcttcgtaacctgtacgaa

NR3C1 ChIP

Prom F gggcaatgggagactttctt

Prom R cgcagccgagataaacaact

5’F tgtgttctctaaagattcatgtgc

5’R gttggccaatgggatactga

3’AF tgatggccaaaattgttaagc

3’AR aattcacccctaccaacacg

3’BF ggaaggtgtgtggcaagttt

3’BR ctttgcttggaaccttcagc

RTF tgggcactcttgtcttgttg

RTR ttcaagcttatggccgaaat

TermExF cctacgcagtgaaatgtcaga

TermExR tttggcattgctgtaaatgg

pAF ccatttacagcaatgccaaa

pAR tattagcaggcgaacattgc

MYC Analysis

-2000 F gctggaaacttgttttaagg

-2000 R tactggcagcagagatcat

Myc+Prom F ggagggatcgcgctgagta

Myc+ Prom R tctgcctctcgctggaattac

Ex1F gctgcttagacgctggattt

In1R gtgtcaatagcgcaggaat

Myc ex2F gcaccaagacccctttaactc

Myc ex2R cctcctcgtcgcagtagaaa

Myc e2F tcggattctctgctctcctc

Myc I2r ctgccaatgaaaatgggaaa

Myc e3F gagaggttcgggactgtgg

Myc e3R cctcctcgtcgcagtagaaa

UCPA F ggcaaatatatcattgagccaaa

UCPA R cccagacccatttcaacaga

Myc Flank1F ggaattctgcccagttgatg

Myc Flank1R tctgcgtggctacagataagtt

Myc Flank2F tggctgcttgtgagtacagg

Myc Flank2R aactggcttcttcccaggag

Myc Flank2AF ggcgctcttaaacagctcag

Myc Flank2AR ccaagctccacatccctaaa Supplementary Table 1

NTS

NTSf ggctaatcctctatgggagtctgtc

NTSr ccaggtgctcaaggtcaacatc

TAF7

TAFF ttgttttgctaggtgttcatc

TAFR acgtcatcacaaagcagcaa

Histone H1E

5’F ttcaacatgtccgagactgc

5’R aggcggcaacagctttagta

3’F cgaaggccaaagcagttaaa

3’R tgaaaagagcctttgggttg

UCF caacacaacccaaaggctct

UCR gctccggagacacacaaaat

F1F ggcagtgagggacttgagag

F1R aatccatccaaatgccacat

pA processing analysis

UCPA F ggcaaatatatcattgagccaaa

UCPA R cccagacccatttcaacaga

Myc Flank2F tggctgcttgtgagtacagg

Myc Flank2R aactggcttcttcccaggag

GAPDH UCPAF ccgcaccttgtcatgtacc

GAPDH UCPAR aaatggttctcgaagcaagc

GAPDH F2F agcccctaaggtcttcaagc

GAPDH F2R ttcccctaaggacgatgatg

β-globin ChIP

Ex1F ggcaggttggtatcaaggtt

Ex1R ggtagaccaccagcagccta

Ex2F ctgctggtggtctacccttg

Ex2R agcttgtcacagtgcagctc

3’AF tatgaagggccttgagcatc

3’AR tttgcagcctcaccttcttt

3’BF gctctatggcttctgaggcggaa

3’BR ggcgaacgtggcgagaaaggaa

GAPDH and ACTB ChIP

ActmidF tctgaacagactccccatcc

ActmidR ggggtgttgaaggtctcaaa

ActF1F ccttgagtgggggtgtagtg

ActF1R cccctaccccaacttgactt

ActF2F tgggccacttaatcattcaac

ActF2R cctcacttccagactgacagc

GAPDHIN2F gggacgctttctttcctttc

GAPDHIN2R gctgcccattcatttcctt

GAPDHF1F ctctcctcgctccagtccta

GAPDHF1R aggctgagctccactaacca

GAPDHF2F agcccctaaggtcttcaagc

GAPDHF2R ttcccctaaggacgatgatg

U6 snRNA

U6F acatatactaaaattggaacgatac

U6R ggaacgcttcacgaatttgcgt

gRNA plasmid generation

gMYCF caacgtaggagttttagagctagaaatagcaag

gMYCR cggtcacaccggtgtttcgtcctttccacaag

Supplementary Table 1

0

0.2

0.4

0.6

0.8

1

1.2

Prom Ex2 Ex3 F1 F2

N20

IgG

0

0.5

1

1.5

2

2.5

Prom Ex2 Ex3 F1 F2

Ser7pIgG

0

0.05

0.1

0.15

0.2

0.25

0.3

Prom Ex2 Ex3 F1 F2

Ser5pIgG

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Prom Ex2 Ex3 F1 F2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Prom Ex2 Ex3 F1 F2

CPSF30IgG

CstF77IgG

0

1

2

3

4

5

6

7

Prom Ex2 Ex3 F1 F2

Ser7pIgG

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Prom Ex2 Ex3 F1 F2

N20

IgG

0

0.05

0.1

0.15

0.2

0.25

0.3

Prom Ex2 Ex3 F1 F2

Ser5pIgG

A

B

C

D

E

F

Supplementary Figure 1:ChIP experiments across the MYC gene showing % input for IgG as well as Pol II (A), Ser2p (B), Ser7p (C), Ser5p (D), CPSF30 (E) and CstF77 (F). For each antibody, a representative experiment is shown

for two separate batches of antibody.

% Input

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Prom Ex2 Ex3 F1 F2

Ser2pIgG

% Input

% Input

% Input

% Input

% Input

% Input

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Prom Ex2 Ex3 F1 F2

Ser2pIgG

% Input

% Input

% Input

0

0.5

1

1.5

2

2.5

Prom Ex2 Ex3 F1 F2

CstF77IgG

% Input

0

0.05

0.1

0.15

0.2

0.25

0.3

Prom Ex2 Ex3 F1 F2

CPSF30IgG

% Input

Davidson_Supplementary Fig1

pA

Prom Ex2 Ex3 F1 F2

Re

lative %

Input

Re

lative %

Input

CstF77 Ser2p

A

B C


Supplementary Figure 2

A. Diagram of the MYC gene with primer pair positions indicated and underlined.B. Comparison of CstF77 recruitment using denaturing native conditions or standard

formaldehyde cross-linking. Values are normalised to the maximal value obtained in X-

link ChIP.C. Comparison of Ser2p using denaturing native conditions or standard formaldehyde

cross-linking. Values are normalised to the maximal value obtained in X-link ChIP. Note that while CstF is barely detectable under native conditions, Ser2p ChIP is more

efficient than with formaldehyde cross-linking.

Graphs show average of two side by side replicates.

1 kb

0

0.2

0.4

0.6

0.8

1

1.2

Prom Ex2 Ex3 F1 F2

X-link

Native

0

0.5

1

1.5

2

2.5

Prom Ex2 Ex3 F1 F2

X-link

Native

0

0.2

0.4

0.6

0.8

1

1.2

Prom Ex2 Ex3 F1 F2

siCont

siCdk12

Re

lative %

Input

CPSF30

siRNA C Cdk12

Cdk12

*

A B

Supplementary Figure 3A. Western blot of Cdk12 protein in cells treated with control or Cdk12 siRNA. Top panel

shows Cdk12 and lower panel shows a protein bound non-specifically by the antibody that

acts as a loading control.B. ChIP of CPSF30 recruitment to the MYC gene in control or Cdk12 depleted cells. Values

normalised to the maximum signal in control cells.


Myc In1-Ex2 Myc In1-Ex2

0

0.2

0.4

0.6

0.8

1

0 10 20 40

C AMO

U4 AMO

Re

lative R

NA

le

ve

l

Time (mins)

0

0.2

0.4

0.6

0.8

1

0 10 20 40

siCont

siCdk12

Re

lative R

NA

le

ve

l

Time (mins)

A

C

E

B

D

F

Supplementary Figure 4A. Steady-state analysis of total polyadenylated Myc transcripts in control and Cdk12 depleted cells.

Data are normalised to U6 snRNA levels.B. Actinomycin D time course of polyadenylated Myc RNA decay in control or Cdk12 depleted cells.

Data are normalised to the amount recovered at time zero. Note that these transcripts are more stable

when Cdk12 is depleted implying a greater effect on their levels at steady-state than seen in A.C. Actinomycin D time course of non-pA cleaved Myc RNA in control and exosome (Rrp6 and Dis3)

depleted cells. Levels were normalised to the amount recovered at time zero.D. Comparison of the level of two confirmed exosome substrates (Myc and 40-33 PROMPT) in control

and Rrp6/Dis3 depleted cells to validate that exosome depletion in C is functional. Graph shows fold

change compared to control cells, which were given a value of 1.E. Actinomycin D time course of Myc intron 1 splicing in control and Cdk12 depleted cells. Levels were

quantitated and, in each case, data are normalised to the amount recovered at time zero.F. As with E but in cells treated with a control or U4 snRNA directed AMO. The signal is depleted in

control AMO treated but not in U4 AMO treated cells confirming that its loss is due to splicing. Graph shows average of two biological repeats.


Re

lative R

NA

le

ve

l

Time (mins)

0

0.2

0.4

0.6

0.8

1

siCont siCdk12

Ex3 (dT)

0

0.2

0.4

0.6

0.8

1

0 10 20 40 60

siCont

siCdk12

0

0.2

0.4

0.6

0.8

1

siCont

siR6/D3

0 10 20 40

Re

lative R

NA

le

ve

l

Time (mins)

0

1

2

3

4

5

6

7

8

Myc PROMPT 40-33 PROMPT

Fold

chang

e/s

iCont

**

Ex3 (dT reverse transcription) Ex3 (dT reverse transcription)R

ela

tive R

NA

le

ve

l

A

B C

D

0

1

2

3

4

5

5' pA RT Term Ex UCPA

C AMO

U1 AMO

0

2

4

6

8

10

12

5' pA Term Ex

C AMO

U1 AMO

NR3C1

PCPA pA

Fold

Chang

e

Fold

Chang

e

0

0.2

0.4

0.6

0.8

1

1.2

RT Term Ex

C AMO

U1 AMO

Fold

Chang

e

Pol II ChIP

5’pA RT Term Ex


A. Diagram of the NR3C1 gene with upstream and terminal regions showing primer pair positions (underlined).

B. Real time PCR analysis of the levels of NR3C1 RNA upstream (5’pA) and downstream (RT) of the PCPA site as well as over the terminal exon (Term Ex) and across the

native pA site (UCPA) in cells treated with C and U1 AMO. Reverse transcription was with random hexamers.

C. Real time PCR analysis of the levels of NR3C1 RNA upstream of the PCPA site (5’pA)

or native pA site (Term Ex) in cells treated with C and U1 AMO. Reverse transcription was with oligo-dT to detect cleaved and polyadenylated transcripts. PCPA product

increases following U1 inhibition whereas the native cleaved and polyadenylated species is reduced.

D. ChIP showing Pol II density over regions downstream of those in main text figure 3.

Pol II occupancy is reduced in U1 AMO treated samples compared to control (given a value of 1) indicative of Pol II termination following PCPA.

Hexamer RT dT RT


UCPA

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

C

U1

U2

0

1

2

3

4

5

6

7

C

U1

U2

Re

lative %

Input

PCPA

Prom 5’ 3’A 3’B

1 kb

Pol II Ser2p

A

B


A. Diagram of the 5’ portion of NR3C1, with the position of ChIP primers indicated and underlined. The PCPA site is indicated.B. ChIP analysis of Pol II or Ser2p across the 5’ portion of NR3C1 in cells treated with control,

U1 or U2 AMOs. Note that only the U1 AMO, which specifically induces PCPA, results in Ser2p accumulation. There is no effect of the U2 AMO, which inhibits splicing but does not cause

PCPA.


Prom 5' 3'A 3‘B Prom 5' 3'A 3‘B

A B

C D

0

1

2

3

4

5

6

GAPDH

IN2

GAPDH

F1

GAPDH

F2

ACTB

IN1

ACTB F1 ACTB F2

siCont

siCPSF73

Re

lative S

er2

p/P

ol II

Supplementary Figure 7A. Steady state analysis of polyadenylated exon 3 containing Myc RNA in control cells or cells

depleted of CPSF73. Quantitation shows fold change in CPSF73 depleted cells as compared to control cells (given a value of 1after normalising to U6 snRNA).

B. Act D time course analysis of polyadenylated exon 3 containing Myc RNA in control cells or

cells depleted of CPSF73. Quantitation for each condition is shown relative to time zero (given a value of 1). Graph shows an average of two biological repeats. Note that these

transcripts are more stable when CPSF73 is depleted implying a greater effect on their levels at steady-state in A.

C. ChIP analysis of Ser2p and Pol II over GAPDH and ACTB genes in control and CPSF73

depleted cells. Graph shows relative Ser2p signal normalised to Pol II. When compared to primers upstream of each pA signal (GAPDH IN2 and ACTB IN1) there is a higher level of

Ser2p in flank (F1 and F2 in both cases) regions beyond the pA signal. However, the Ser2p level is diminished upon depletion of CPSF73.

D. Western blot displaying levels of Cdk12 protein in cells treated with control or CPSF73 siRNA. * is a non-specifically bound protein acting as a loading control.


Re

lative R

NA

le

ve

l

Time (mins)

Ex3 (dT reverse transcription)

0

0.2

0.4

0.6

0.8

1

siCont siCPSF73

Re

lative R

NA

le

ve

l

siRNA: C CPSF73

Cdk12

*

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 40 60

siCont

siCPSF73

Ex3 (dT reverse transcription)

Re

lative S

er2

p s

ignal/P

ol II s

ignal

Supplementary Figure 8A. ChIP analysis across the Histone H1E gene using the N20 antibody. % input for N20 and IgG

controls are shown for two individual repeats.

B. ChIP analysis across the Histone H1E gene using the Ser2p antibody. % input for Ser2p and IgG controls are shown for two individual repeats.

C. Comparison of Ser2p phosphorylation density over the Histone H1E gene (UC amplicon) in comparison to the MYC Flank 1 region. Quantitation is expressed as the relative Ser2p signal

over that obtained for N20 (ratio given a value of 1 for MYC F1). It is clear that Ser2p levels are generally much lower over Histone H1E than beyond the MYC pA site.


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

MYC F1 H1E UC

Ser2p/Pol II

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

5' 3' UC F1

Ser2p

IgG

0

0.05

0.1

0.15

0.2

0.25

5' 3' UC F1

Ser2p

IgG

0

0.5

1

1.5

2

2.5

3

5' 3' UC F1

N20

IgG

0

0.2

0.4

0.6

0.8

1

1.2

1.4

5' 3' UC F1

N20

IgG

% Input

% Input

% Input

% Input

A

B

C

Re

lative R

NA

le

ve

l

Time (mins)


A. Steady-state analysis of total polyadenylated Myc transcripts in control and CstF77 depleted cells. Data are normalised to U6 snRNA levels.

B. Actinomycin D time course of polyadenylated Myc mRNA decay in control or CstF77

depleted cells. Levels are normalised to the amount recovered at time zero. Graph is an average of two biological repeats. Note that these transcripts are more stable when CstF77

is depleted implying a greater effect on their levels at steady-state in A.


0

0.2

0.4

0.6

0.8

1

siCont siCstF77

A B

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 40 60

siCont

siCstF77

Ex3 (dT reverse transcription) Ex3 (dT reverse transcription)

Re

lative R

NA

le

ve

l

NS1

CPSF30

Tubulin

TET: - + - +

wtNS1 mutNS1

wt/mut NS1A βpA

TETA

B

0

0.2

0.4

0.6

0.8

1

Prom Ex2 Ex3 F1 F2

Mut

WT

Supplementary Figure 10A. Western blotting analysis of NS1 (top panel), CPS30 (middle panel) and, as a control, Tubulin

(lower panel), in cell lines containing single-copy integrates of wt or mut NS1A genes.

Diagram shows both genes where expression is driven by a tetracycline-inducible promoter and CPA mediated by the human β-globin pA site. A tetracycline dependent band is

observed for both wt and mut NS1A and the expression of CPSF30 is not impaired under these conditions.

B. ChIP analysis of CPSF30 and CstF77 recruitment to the MYC gene in wt or mut NS1

expressing cells. Recruitment is reduced in wt NS1A expressing cells. Values are normalised to the maximal value obtained in mut NS1A cells.

Re

lative %

Input

CPSF30 CstF77


0

0.2

0.4

0.6

0.8

1

Prom Ex2 Ex3 F1 F2

Mut

WT

Re

lative %

Input

U1: GGTATCTCCCCTGCCAGGTAAGTAT U2:...

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Transcript of U1: GGTATCTCCCCTGCCAGGTAAGTAT U2:...