Cell Reports, Volume 28

Supplemental Information

RNA Binding Protein CELF2 Regulates Signal-Induced

Alternative Polyadenylation by Competing

with Enhancers of the Polyadenylation Machinery

Rakesh Chatrikhi, Michael J. Mallory, Matthew R. Gazzara, Laura M. Agosto, Wandi S.Zhu, Adam J. Litterman, K. Mark Ansel, and Kristen W. Lynch

Dox (hr):

CELF2

hnRNPL

0 4 8 12 24

Dox (hr): 0 4 8 12 24

Dox (hr): 0 4 8 12 24

Western Blot

RTPCR

3’RACE

PAS2

PAS3

Figure S1 (related to Fig 1): CELF2 protein directly regulates IR and APA in the CELF2 3’UTR. Representative detection of CELF2 protein (by Western blot, top), CELF2 mRNA 3’UTR IR (by RT-PCR, middle), and switch from PAS2 to PAS3 (by 3’ RACE, bottom) following doxycycline (Dox) induction in Jurkat cells for the indicated time points. hnRNP L is used as a loading control for the Western blot as this has previously been shown not to change in response to PMA (Shankarling et al., 2014).

PMA: - + - +WT KD

CELF2

hnRNPL

%Inc.: 20 72 22 393 66 5s. d.:

RTPCR

WesternBlot

Figure S2 (related to Fig 2): CELF2 drives stimulation-induced retention of additional terminal introns in Jurkat cells. (A) Analysis of CELF2 3’UTR intron retention by RT-PCR in parental wild-type (WT) Jurkat cells versus cells that are depleted of CELF2 by shRNA (KD). Western blot confirms reduced expression of CELF2 protein with hnRNPL as loading control. The gap between WT and KD lanes indicates that the WT and KD samples were run on separate gels. (B-D) RT-PCR analysis of splicing of the indicated introns in wildtype (WT) or CELF2-deficient (KD) cells before (-) or after (+) stimulation with PMA. For each panel the gene name, 3’UTR schematic and location of CELF2 CLIP-peaks (top blue track) are shown. Gray box is exon and line is intron. Percent intron retention or inclusion (%Inc.) and standard deviation (s.d.) represent three independent experiments.

A B

C D

PMA: - + - +WT KD

SEPT6

%Inc.:s. d.:

58 71 16 102 2 32

PMA: - + - +WT KD

DICER1

%Inc.:s. d.:

87 94 76 642 4 3 4

- + - +WT KD

PMA:

PDCD2

%Inc.:s. d.:

89 95 78 772 4 4 5

PMA: - + - +

WT KD

CELF2Western

Blot

PAS2

PAS3

3’RACE

hnRNPL

Figure S3 (related to Fig 3): CELF2 protein is necessary for APA in the CELF2 3’UTR. Analysis of CELF2 polyadenylation site usage by 3’ RACE in parental wild-type (WT) Jurkat cells versus cells that are depleted of CELF2 by shRNA (KD). Western blot confirms reduced expression of CELF2 protein with hnRNPL as loading control.

0 6 12 25 50 100 (ng)CELF2:

CstF64

CFIm25

CELF250 kD

75 kD

25 kD

PAS2 oligo

(150 ng)

(150 ng)

A

C

B

E F G

D

0 6 12 25 50 100

M1

hnRNPK: 100

PAS2 oligo

hnRNPKCstF64

0 6 12 25 50 100

CFIm25

50 kD

75 kD

25 kD

hnRNPK:

(ng)

(ng)

(300 ng)

(300 ng)

PAS2

CFI CstF

PAS3

PAS2

CFI CstF

PAS3

PAS2

CFI CstF

PAS3

PAS2

CFI CstF

PAS3

FLAG-CELF2:

WT

mUE-C2

mDE-C2

mU/D-C2

PAS3

PAS2

CELF2

hnRNPL

*

WTmUE-

C2mDE-

C2 dPAS3- + - + - + - + - +

Lanes: 1 2 3 4 5 6 7 8 9 10

3’RACE

WesternBlot

mU/D-C2

0

10

20

30

40

50

60

70

80

- CELF2

%PA

S2

+ CELF2

WT mUE-C2

mDE-C2

mU/D-C2

WT mUE mDE mU/DHexMut

swPAS3

0

20

40

60

80

100- CELF2+ CELF2

Figure S4 (related to Fig 4): CELF2 competes with CFIm25 and CstF64 to regulate APA of its own 3’UTR. (A) (Upper) UV crosslinking analysis of recombinant CFIm25 and CstF64 proteins co-incubated in combination with radiolabeled PAS2 oligonucleotide. Addition of recombinant CELF2 reduces binding of both CFIm25 and CstF64, in a dose-dependent manner, with a concomitant increase in binding of CELF2. (Lower) CELF2 3‘UTR track showing hnRNPL and hnRNPK CLIP reads from indicated cells. 3’UTR zoomed in to show the CLIP peaks on the PAS2 oligo used for the UV crosslinking experiments. (B) Control UV crosslinking experiment showing no competiton of binding to PAS2 oligonucleotide between recombinant hnRNPK and recombinant CFIm25/CstF64 proteins. hnRNPK protein crosslinked to M1 oligonucleotide is shown as a positive control for hnRNPK binding to RNA. (C) Schematic of reporter constructs. Red boxes indicate location of mutations. The mutations disrupt binding of CFIm25 and CstF64, but maintain binding of CELF2. (D) 3’ RACE analysis of mutant constructs from panel C upon transfection in HeLa cells with (+) or without (-) co-transfected CELF2 cDNA. (E) Quantification of the gel in Fig 4C and (F) Quantification of the gel from panel D. The bar graphs compare PAS2 usage in each reporter construct with (dark gray) or without (light gray) CELF2 cDNA co-transfected in HeLa cells (%PAS2 = [PAS2]/[PAS2+PAS3]). Error bars represent at least two independent experiments. (G) UV crosslinking analysis of recombinant CELF2 protein to show that mutations in mU/D-C2 construct (panel C) maintain CELF2 protein binding.

Scalechr10:

PAS2oligo

100 bases hg1911,376,250 11,376,300 11,376,350 11,376,400 11,376,450

CELF2CELF2CELF2CELF2

JSL1 stim HNRNPL

4 -

0 _

JSL1 unstim HNRNPL

7 -

0 _

K562 HNRNPK

1 -

0 _

100 Vert. Cons

4.88 -

-4.5 _

0 -

Scalechr10:

PAS2oligo

2 kb hg1911,371,500 11,372,000 11,372,500 11,373,000 11,373,500 11,374,000 11,374,500 11,375,000 11,375,500 11,376,000 11,376,500 11,377,000 11,377,500 11,378,000 11,378,500

CELF2CELF2CELF2CELF2CELF2CELF2CELF2CELF2CELF2CELF2

JSL1 stim HNRNPL

57 -

1 _

JSL1 unstim HNRNPL

18 -

1 _

K562 HNRNPK

6 -

1 _

100 Vert. Cons

4.88 -

-4.5 _

0 -

CELF250 ng50 kD

WTmU/D-C2

hnRNPL CLIPStim JSL1 cells

hnRNPL CLIPUnstim JSL1 cells

hnRNPK CLIPK562 cells

CELF2 3’UTR

PAS2 oligo

hnRNPL CLIP (stim)

hnRNPL CLIP (unstim)

hnRNPK CLIP

PMA: - + - +WT KD

PMA: - + - +WT KD

PMA: - + - +WT KD

PMA: - + - +WT KD

Distal site

Proximal site

Distal site

Proximal site

Distal site

Proximal site

Distal site

Proximal site

PMA: - + - +WT KD

Distal site

Proximal site

PRDM8

TMEM201

IGSF3

USP54 HAPLN3

PMA: - + - +WT KD

Distal site

Proximal site

HIVEP3

PMA: - + - +WT KD

CSK

Distal site

Proximal site

PMA: - + - +WT KD

PHF12

Distal site

Proximal site

Figure S5 (related to Fig 6): CELF2 protein drives stimulation-induced APA. 3’RACE validations of CELF2 dependent APA of genes listed in Table S2. In each case, the agarose gel shows comaprison of proximal to distal polyadenylation site usage by 3’ RACE in parental wild-type (WT) Jurkat cells versus cells that are depleted of CELF2 by shRNA (KD).

Table S4 Primers used for indicated experiments (related to STAR Methods)

Experiment Forward (5’ > 3’) Reverse (5’ > 3’) RTPCR

CELF2 3’UTR intron retention

CACAGGAATTTGGAGACCAGG AAGTTGGCAATGTGGTCCTCCTCTGC

CELF2 3’UTR intron splice

CACAGGAATTTGGAGACCAGG GCTAGGCAAACGATGAACTAACGGGC

SEPT6 intron retention

GGATGATGAAGTGAATGCTTTCAAGC TGGACCTTGAGTTTGCAGGGCTTTCC

SEPT6 intron splice

GGATGATGAAGTGAATGCTTTCAAGC GTTAAAATAGGAACCTCGGCTTAAAAGGC

DICER1 intron retention

AGTCACTGTGGAAGTAGTAGGAAAGG AACCTGTCAATTATTTTGAGCACTTGC

DICER1 intron splice

AGTCACTGTGGAAGTAGTAGGAAAGG GGAAATATGAGACACCTCTGCTCAGC

PDCD2 intron retention

GACCATATAATTCCAGACCACAACTTCC CAGAGGAACTTACCAAGCCTCCAGC

PDCD2 intron splice

GACCATATAATTCCAGACCACAACTTCC ATCAGTCATTATATGGACTTTAACTTCC

3’RACE CELF2 3’UTR Intron* CTTCGGGCCAGCGACGATCTGC

CELF2 3’UTR PAS

GGCTTGATTTCTTTTTTCCCTTTGCTTATATCTAGC

LRCH4

CCTTCCTCTCTATTTATAAGGTCCCTGC

RCCD1 CATCACAGTCCTGCCCTTCACCCTC

PNPO GAGCTAGGGCTAGGTGTCAAGAGAGG

USP54

TCCTTTCCTTTCCTGGAGCTACACC

TNKS

AGCTATAGACCTTACTAATTTGGCAGG

PRDM8 GCAAGCACAGTTAAGCCACCTGCAGG

IGSF3 GTCTTGGTTGGTTAGCTATTTGCGCGC

TMEM201 TGCTCAGGCCCAGGCTTTGCCAGG

HIVEP3 CTGATGGTGAAGCCTCCTGACCCTC

CSK GGACTGAACCTGGAAGATCATGGACC

PHF12 ACACCCACCCAAGACTCCTGCAATGC

HAPLN3 CCTGCCGCATTCCCTCACTGGCTG

RBFOX2 CCCAGTTCATGAGGCCTGGCTATTGC

Mutagenesis mUE-C2

GAAACAGTCAAACTTATTTTGCTGGCCGGCCTCGCCCTCCTTCCCAGTTTTTTGCTTCTGTCTCC

GGAGACAGAAGCAAAAAACTGGGAAGGAGGGCGAGGCCGGCCAGCAAAATAAGTTTGACTGTTTC

mDE-C2

CTTGTTTCTCTCTCTGCTTCTGCTATTCTGCTGCTGCATTATATCCTGTTGATACATCTGCACACCTCAC

GTGAGGTGTGCAGATGTATCAACAGGATATAATGCAGCAGCAGAATAGCAGAAGCAGAGAGAGAAACAAG

mUE

CGTGTAGTTGAAACAGTCAAACCAACACACCCACACAACACAACAACACAACCCAGTTTTTTGCTTCTGTCTCC

GGAGACAGAAGCAAAAAACTGGGTTGTGTTGTTGTGTTGTGTGGGTGTGTTGGTTTGACTGTTTCAACTACACG

mDE

CCAATATTAAACCATTTTCCTAATACCACACACACACACCACCACACCAACACCACATAGTCATTATATGTTGGTG

CACCAACATATAATGACTATGTGGTGTTGGTGTGGTGGTGTGTGTGTGTGGTATTAGGAAAATGGTTTAATATTGG

Hex-Mut GCTTCTGTCTCCAATAATAAACCATTTTCCTAATACTTG

CAAGTATTAGGAAAATGGTTTATTATTGGAGACAGAAGC

UV crosslinking CELF2 3’UTR Intron

GCGGGATCCGATTTGCATTAGTTTTTCTCCTGCAC

GCGTCTAGATAGGCAAACGATGAACTAACGGGC

PAS2 oligonucleotide

GCGGGATCCGTCAAACTTATTTTTGTAATGTATGTTATTGTG

GCGTCTAGAGCAGATGTATCACCAACATATAATGAC

* Primer sequence is a part of Renilla Luciferase gene.