Supplementary Figure 1

A B C

AR in 22Rv1 cells cryptic exon 3 cryptic exon 3 exon 3 22Rv1AR-V7 cryptic 1 2 3 4 5 6 7 8 -GFP200kDa 1.2 exon 3 exon 8 150kDa 5’ 3’ 22Rv1 AR-V7 AR-V7-GFP 100kDa -GFP 1 AR-FL tandem duplication exon 8 0.8 Duplication site 75kDa AR-V7 0.6 50kDa H3 0.4 37kDa 25kDa 0.2 0 AR-V7-GFP22Rv1LNCaP95 WTno template

ACTB Relative copy number Duplication site exon 8 22 Rv1AR-V7-GFP WT 22 Rv1AR-V7-GFP WT

D control gRNA E AR-V7 gRNA 100 14 12 WT 80 AR-V7-GFP 10 60 8 40 6 Count 4 20 2

Relative expression 0 0 AR-V7 AR-FL 100 101 102 AR-V7-GFP Supplementary Figure 2

A

control gRNA control gRNA 1.4 control gRNA control gRNA control gRNA 1.4 control gRNA 1.4 1.4 control gRNA 1.4 ACIN1 gRNA CELF1 gRNA 1.2 CLSN1A gRNA 1.2 CPSF1 gRNA 1.2 EFTUD2 gRNA GEMIN5 1.2 1 CHERP gRNA GEMIN6 gRNA 1.2 1 1 1 1 0.8 0.8 0.8 1.4 0.8 0.6 0.8 0.6 1.4 0.6 0.6 1.2 0.6 0.4 1.2 0.4 0.4 1 0.4 0.2 0.4 0.2 1 0.2 0.2 0.8 0.2 0 0.8 0 0.6 0 Relative expression 0 0 Relative expression Relative expression Relative expression 0.6 0.4 Relative expression 0.4 CELF1 0.2 ACIN1 0.2 CPSF1 non-target CLSN1A 0 GEMIN6 non-target 0 non-target non-target Relative expression non-target Relative expression

EFTUD2 CHERP non-target non-target

control gRNA 1.4 control gRNA 1.4 control gRNA control gRNA 1.2 control gRNA control gRNA control gRNA PABPC1 gRNA PDCD7 gRNA 1.2 PRPF40A gRNA 1.4 1.4 1 1 PRPF3 gRNA PRPF4 gRNA RBM15 gRNA 1.2 SFSWAP gRNA 1.2 0.8 0.8 1 1.4 1 0.6 0.6 1.4 0.8 0.8 1.4 1.2 1.2 0.4 0.4 1.2 0.6 0.6 0.2 0.2 1 1 0.4 1 0.8 0.4 0 0 0.8 0.2 0.2

Relative expression 0.8 Relative expression 0.6 0.6 0.6 0 0 Relative expression 0.4 0.4 Relative expression Count 0.4 PABPC1 PDCD7 0.2 0.2 non-target non-target 0.2 0 0 RBM15 Relative expression 0 Relative expression non-target SFSWAP

Relative expression non-target

PRPF4 PRPF3 non-target non-targetPRPF40A non-target

3

control gRNA control gRNA control gRNA 1.4 control gRNA 1.4 control gRNA 1.4 control gRNA 1.4 SNRNP48 gRNA 1.2 SRRM2 gRNA 1.2 SRSF6 gRNA 1.2 TRA2B gRNA U2AF2 gRNA UPF3B gRNA 1.2 1 1 1 1 0.8 0.8 0.8 1.4 1.4 0.8 0.6 0.6 0.6 1.2 1.2 0.6 0.4 0.4 0.4 1 1 0.4 0.2 0.2 0.2 0.8 0.8 0.2 0 0 0.6 0.6 Relative expression

Relative expression 0 0 Relative expression Relative expression 0.4 0.4 0.2 0.2 SRRM2 SRSF6 0 0 non-targetSNRNP48 non-target Relative expression UPF3B non-target Relative expression non-target

TRA2B U2AF2 non-target non-target

AR-V7-GFP

B C D 30 100 80 20 1.4 1.4 1.4 1.4 1.4 1.2 1.2 1.2 1.2 1.2 60 1 1 1 1 1 0.8 0.8 0.8 0.8 0.8

negative cells 40 10 0.6 0.6 0.6 0.6 0.6 P Control 0.4 0.4 0.4 0.4 0.4 20 0.2 0.2 0.2 0.2 0.2 Control 0 0 0 0 0 %GF 0 Relative expression Relative expression Relative expression Relative expression Relative expression 0

SF3A1SF3A3 SF3B1 SF3B2 U2AF1 U2AF2 non-target non-target non-target non-target non-target %PI / Annexin V positive cells SF3A1SF3A3SF3B1SF3B2U2AF1U2AF2 gRNA SF3A1 gRNA SF3A3 gRNA SF3B1 gRNA SF3B2 gRNA U2AF1 gRNA gRNA

E p=0.0001 p<0.0001 9 p<0.0001 p=0.0003 7000 G SF3B2 expression H downstream_gene_variant 8 ● intron_variant 15 5000 intron_variant&non_coding_ transcript_variant 7 missense_variant 3000 10 non_coding_transcript_exon_variant &non_coding_transcript_variant U2AF2 expression 6 ● ● ● ● splice_region_variant ● &synonymous_variant 5 ●● upstream_gene_variant ● ● Benign CRPC ● ● ●● ● ● ●● ●● ● 16000 ● ●● ● ●●●●● ● ●● ●●●●●● ●● ● ● ● ●●● ●●●● ● ●● ●●● ●● ●● Localized PC ●●●●●●●● ●●●●● ●● p=0.00077 AR-V7 positive ●● ●●●●●●●●●● ●●●● ●●●●● ● ●● ● ●●●●●●●●●●●● ● ● ●● ● ●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●● ●● AR-V7 negative ●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●● AR V7 expression (RPKM) 0 ●●●●●●●●●●●● ● 12000 AR-V7 high positive

●

● 0 20 40 60 ● ● ● 8000 ● ● F ● ● ● ● ● AR Last exon expression (RPKM) ● RPKM 1000 4000

40.0 SF3B2 expression AR-V7 negative AR expression 11 1000 41.4 p=0.95 15 ● 10 1000 47.1 9 AR-V7 positive 1000 61.2 8 10 7

● Gleason score 6 SF3B2 1 kb ● ● 5 RPKM ● 1000 53.3 5 ●● ● ● ● 15 AR-V7 negative ● ● p=0.40 1000 ● ●● ● 31.6 ●● ●● ● ● ● ●● ● ● ● ● ● ● 10 ● ● ● ●● ● ●● ● ● ●● ● ●●● ●● ● ●●●●●●● ● ● ● ●● ●●●●●●●●● ● ● ●● ●●● ●● ●● 1000 ●●●●●●●●●●●●●●●● ● ● ●●●●● ● ●●● ●●● ●●●●●●●● ●● ● ●● ●● ●●●●●●●●●●●●●●●●●●●●● ● ●● ● ● ● ●●● ●●● ● ● ●● ●●●●●● ● ●●●●● ● ● ● ● ● ● ● ● ● ● ●●●● ●●● ●●● ●●● ● ●●●● ● ●●●●● ● ● ● ●● ●●●●●●● ● ● ●●● ● ● ● ● ● ● ● ● ●● ●● ●● ● ● ●●●●●●●●●●●●●●●● ●● ● ● ● ●● ● ●●●● ●● ● ●● ●●●●●● ● ●● ● ● ●●● ● ● 18.3 ● ●●●●●●●●●●●●●●●●●●●●●●● ●● ● ● AR V7 expression (RPKM) 0 ●●●●●●●●●●●●●●●●●●●●● ●● ●

● AR-V7 positive 5 ● 1000 ● ● ● ● 57.0 ● ● ● ● ● ● ● ● 0 ● 20 40 60 0 U2AF2 1 kb AR Last exon expression (RPKM) AR-V7 expression

mutation normal Supplementary Figure 3

Bladder cancer (TCGA) AML (TCGA) 1.0 1.0 SF3B2 High (n=202) SF3B2 High (n=79) 0.8 SF3B2 Low (n=203) 0.8 SF3B2 Low (n=80)

0.6 p=0.0237 0.6 p=0.0316

0.4 0.4 Overall survival 0.2 Overall survival 0.2

0.0 0.0 0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 Survival time (months) Survival time (months)

Lung adenocarcinoma (TCGA) Head and Neck squamous cell carcinoma (TCGA) 1.0 1.0 SF3B2 High (n=252) SF3B2 High (n=259) 0.8 0.8 SF3B2 Low (n=252) SF3B2 Low (n=258) 0.6 p=0.0109 0.6 p=0.0491

0.4 0.4 Overall survival 0.2 Overall survival 0.2

0.0 0.0 0 50 100 150 200 250 0 50 100 150 200 Survival time (months) Survival time (months)

Breast cancer (microarray) 1.0 SF3B2 High (n=952) 0.8 SF3B2 Low (n=951) 0.6 p=0.0441 0.4

Overall survival 0.2

0.0 0 50 100 150 200 250 300 350 Survival time (months) Supplementary Figure 4

A Control gRNA transfected 22Rv1 RNAseq SF3B2 depleted 22Rv1 RNAseq 1e+04 R = 0.95 1e+03 R = 0.99 1e+01 1e+00 1e-02 replicate 1 replicate 1 1e-03 1e-05

1e-05 1e-02 1e+01 1e+04 1e-03 1e+00 1e+03 replicate 2 replicate 2 B GFP overexpressed 22Rv1 RNAseq SF3B2 overexpressed 22Rv1 RNAseq 1e+04 R = 0.91 1e+05 R = 0.99

1e+02 1e+01 1e-01 replicate 1 replicate 1 1e-02 1e-04

1e-03 1e-01 1e+01 1e+03 1e-05 1e-02 1e+01 1e+04 replicate 2 replicate 2

C ACTB 0-1000

Control 0-1000

0-1000 SF3B2 RNAseq dep 0-1000

D PAR-CLIP of SF3B2 E 1e+03 R = 0.97 1e+03 R = 0.99 Overlap of SF3B2 Binding sites (all) Replicate1 Replicate2 1e+01 1e+01 999 1e-01 1e-01 4185 1909 replicate 2 replicate 3 21109 1e-03 1e-03 3229 2873

1e-03 1e-01 1e+01 1e+03 1e-03 1e-01 1e+01 1e+03 7376 replicate 1 replicate 2 Replicate3 1e+03 R = 0.96 Overlap of SF3B2 Binding sites (T to C conversion) 1e+01 Replicate1 Replicate2

1e-01 257

replicate 3 507 313

1e-03 2579

226 274 1e-03 1e-01 1e+01 1e+03 replicate 1 663 Replicate3 Supplementary Figure 5

A B 1.00 6714 Significant 7500 Not significant 0.75 ● Significant ● Not Significant 5000 3535 0.50

2500 Number of LSVs 2233 0.25 1231 0 x ry e 0.00 bina compl complexity 0.00 0.25 0.50 0.75 1.00 C D 1.00 0-100 IR Control ES 0-100 A5SS 0-100 0.75 A3SS

RNAseq SF3B2 dep 0-100

0-500 SF3B2 0.50 PAR CLIP 0-500 MATR3 SNHG4 MATR3 0.25 0-100 Cumulative distributuin Control 0-100

0.00 0-100

RNAseq SF3B2 0-100 0.0 0.5 1.0 dep 0-500 depletion / Control SF3B2 PAR CLIP 0-500 ATP5O Supplementary Figure 6

A

GFP-TAPSF3B2-TAPSF3B2-TAP/AR-V7 KO Acceptor splice site stop codon SF3B2-TAP CE3 aactt(112 bp)agaaaaattcc(35 bp)ccctgaagaaaggctgacttgcc AR-FL 1 0.7 1 aactt------gcc AR-V7 178 bp deletion 1 2.5 0.1 ACTB 1 1 1 22Rv1 B

Acceptor splice site stop codon CE3 GFP GFP-SF3B2GFP-SF3B2/AR-V7 KO GFP-SF3B2 catactagaaaaattccgggttggcaattgcaagcatctcaaaatgaccagaccctga cat------ctcaaaatgaccagaccctga AR-FL 1 1.1 0.3 34 bp deletion AR-V7 1 2.9 0.005 ACTB 1 1 1 LNCaP95

C SF3B2 OE / SF3B2 OE ARv7 KO

log10 FPKM + 1

2

1 AR-target genes Supplementary Figure 7

A B 1.00 SF3B2 intron-bound genes (n = 4466) p < 2.0E-16 SF3B2 exon-bound genes (n = 1066) p = 1.4E-05 SF3B2 intergenic regions-bound genes (n = 1889) p = 4.2E-06 SF3B2 intron and exon-bound genes (n = 1328) p = 1.0E-04 Control genes (n = 4259) 0.75 ● Significant 1.00 ● Not Significant

0.50 0.75

0.50 0.25

SF3B2 overexpression Ε[Ψ] 0.25

0.00 Cumulative distributuin 0.00

0.00 0.25 0.50 0.75 1.00 0.0 2.5 5.0 GFP overexpression Ε[Ψ] −5.0 −2.5 Log2 fold change of gene expression (SF3B2 overexpression / GFP overexpression)

C 0-3000 GFP-OE 0-3000

0-3000 D NOTCH1 RNAseq SF3B2-OE 0-3000 0-100 GFP-OE KLK4 0-100 0-500 0-100 GFP-OE SF3B2-OE 0-100 0-500 0-100 Control 0-500 RNAseq gRNA 0-100 RNAseq SF3B2-OE 0-500 SF3B2 0-100 dep 0-100 UBE2C 0-100 0-1000 SF3B2 0-100 GFP-OE PAR CLIP 0-1000

0-1000 RNAseq SF3B2-OE 0-1000

PSMA E AR target genes (n = 98) p = 0.0013 Control genes (n = 12910) 1.00

0.75

0.50

0.25 Cumulative distributuin 0.00

0.0 2.5 5.0 −5.0 −2.5 Log2 fold change of gene expression (SF3B2 overexpression / GFP overexpression) Supplementary Figure 8

A C LNCaP95

1.2 p = 0.0005

Cytoplasm Nuclear AR-V7 exogenous SF3B2 1 SF3B2 endogenous SF3B2 0.8 0.6 ACTB 0.4

H3 0.2 0 Relative expression of DMSO PLA-B

B D DMSO PLA-B 4nM PLA-B 8nM 1.0K 1.0K 1.0K 120 800 800 800 600 600 600 100 400 400 400

200 1.05% 200 3.24% 200 20.5% 80 0 0 0 100 101 102 103 100 101 102 103 100 101 102 103 60

FSC PLA-B 12nM PLA-B 16nM PLA-B 20nM 1.0K 1.0K 1.0K 40 800 800 800 600 600 600 20 400 400 400 % AR-V7 depleted population 200 57.8% 200 83.4% 200 94.3% 0 0 0.4 0.8 1.2 1.6 2 nM 0 0 0 100 101 102 103 100 101 102 103 100 101 102 103 Spliceostatin A concentration AR-V7-GFP F E 30 PNT2 30 22RV1 1.2 PNT2 1.2 22RV1 25 25 1 1 20 20 0.8 0.8 15 15 0.6 0.6 10 10 %Dead cells 0.4 0.4 %Dead cells 5 5 0.2 0 0 0.2 4 8 12 16 20 4 8 12 16 20 0 0 PLA-B (nM) PLA-B (nM) Relative cell proliferation Relative cell proliferation 0 4 8 12 16 20 0 4 8 12 16 20 DMSO DMSO PLA-B (nM) PLA-B (nM) non-treatment non-treatment G H I SF3B2-overexpressed LNCa95 PLA-B * P < 0.05 ** ** P < 0.01 25 200 DMSO 200 PLA-B * GFP DMSO PLA-B SF3B2 20 ** 150 * 150 * 15 ** * ** eight(g)

100 100 W 10

5 50 50 0 Relative tumor volume (%)

Relative tumor volume (%) 0 2 4 6 8 10 0 0 Days 0 7 14 21 28 0 7 14 21 28 days after initiation of treatment days after initiation of treatment