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.