SUPPLEMENTARY FIGURE LEGENDS

Supplementary Fig. 1. Controls from primary RNAi viability screen. (A) Cell number relative to siNTC was determined six days after transfection of the indicated siRNA (or TOX transfection control). Data is from the primary RNAi viability screen. (B) The data from (A) was converted to a Z-score as described in the Materials and Methods section. (C) Relative cell number (Z-score) was determined six days after transfection of CTNNB1 (β-catenin; left) or

KRAS (right) siRNA in the indicated cell lines. The cell lines were grouped based on mutation of APC or CTNNB1 (top) or KRAS (bottom). P-values were calculated between each group of cell lines; Student’s t-test.

Supplementary Fig. 2. PRPF6 is overexpressed and amplified in primary human colon cancer. (A) Immunoblot analysis of normal colon, primary colon tumors, and metastatic colon tumors. (B) Shown is the relative PRPF6 expression in primary colon tumors with the indicated

PRPF6 copy number (data is from The Cancer Genome Atlas). (C) Quantitative PCR (mean ±

S.D.) for PRPF6 copy number in normal colon or primary colon tumors. (D) IHC of PRPF6 in normal colon and colon tumors. (E) Shown is the mean ± S.D. of the H-score

(quantification of PRPF6 protein from IHC samples) for colon tumors with PRPF6 gain (>2.9 copies by qPCR) or disomic for PRPF6 (<2.9 copies); data from (C). P-value is a Student’s t- test. (F) Copy number of PRPF6 for each cell line was determined by quantitative PCR (mean ±

S.D.). Mean ± S.D. copy number of each group of cell lines is shown on the right. P-value is a

Student’s t-test.

1

Supplementary Fig. 3. RNAi of tri-snRNP in colon cancer cell lines. (A)

Immunoblot of select cell lines three days after siRNA transfection. (B) PRPF6 levels from (A) were quantified relative to ACTIN, and the mean ± S.D. protein level relative to siNTC for each group of cell lines is shown. P-value is a Student’s t-test. (C) Relative cell number (compared to shNTC; mean ± S.D.) is shown for eight doxycycline inducible PRPF6 shRNAs seven days after addition of doxycycline (1 µg/ml) to induce shRNA expression. Immunoblot for PRPF6 was three days after doxycycline addition. (D) Relative cell number (compared to siNTC; mean ±

S.D.) is shown for two independent siRNAs six days after siRNA transfection in human primary colon cells. Immunoblot was three days after siRNA transfection. (E) Immunoblot of select cell lines for the indicated tri-snRNP proteins three days after siRNA transfection. A representative

ACTIN is shown that is similar to the expression levels for each of the four cell lines (data not shown). (F) Relative cell number (compared to siNTC; mean ± S.D.) is shown for two independent siRNAs six days after siRNA transfection in the indicated cell lines. P-value was calculated between each group of cell lines; Student’s t-test.

Supplementary Fig. 4. Tri-snRNP complex has reduced expression and is mislocalized upon PRPF6 inhibition. (A) Immunoblot of the indicated tri-snRNP complex proteins three days after siRNA transfection (left). Protein levels were quantified relative to ACTIN, and the mean ± S.D. protein level of all three PRPF6 siRNA lanes relative to siNTC for each cell line is shown (right). (B) Immunofluorescence of PRPF31 and PRPF3 protein four days after siRNA transfection in SW620 and COLO 678 cells. The punctuate spots visible upon PRPF6 inhibition are Cajal bodies, where the tri-snRNP is formed.

2

Supplementary Fig. 5. CRISPR mediated knockout of PRPF6 and tri-snRNP leads to growth defects in both PRPF6-high and PRPF6-low cell lines. (A-C) Relative cell number

(compared to sgNTC; mean ± S.D.) is shown for two independent sgRNAs seven days after sgRNA transduction in the indicated CAS9 expressing PRPF6-low (COLO 678, HT55, HCA-7) and high (SW48, RKO, SW620) cell lines. P-values were calculated between each group of cell lines, Student’s t-test. Immunoblot analysis of the indicated tri-snRNP complex proteins four days after sgRNA transduction is shown for a representative PRPF6-high and low cell line

(right). (D, E) Schematic cartoon depicts the PRPF6 and BRR2 genomic . “E1” and “E2” represent 1 and 2. Vertical red arrowheads mark the location of sgRNAs co-transduced to generate the indicated deletion. Black horizontal arrowed line shows the position of PCR primers used for the indicated genotype determination. DNA gel shows representative cell line clones with wildtype (WT) or heterozygous (HET) PRPF6 (D) and BRR2 (E) alterations. Allelic frequencies are shown below from all the tested clones (HOM, homozygous). Immunoblot analysis shows protein levels for each target for representative wildtype and heterozygous clones.

Supplementary Fig. 6. Consistent changes upon PRPF6 inhibition in KM-

12 and SW620 cells. (A) Relative log2 PRPF6 expression is shown from the microarray data for each cell line (mean ± S.D.). (B) The similarity of the effect of each siRNA on the global gene expression in each cell line was determined with Pearson correlations between all between the indicated pairs of siRNAs. The average Pearson correlation ± S.D. is shown for each pair-wise siRNA comparison. Positive correlation indicates the siRNA have similar effects on the global gene expression changes in the given cell line. (C) Genes that were either

3 downregulated or upregulated upon PRPF6 knockdown relative to siNTC were selected based on the indicated criteria. The relative log2 expression level of the genes that were significant in both

KM-12 and SW620 cells are shown. The expression data was normalized to the median expression of the respective siNTC samples. (D, E) For each cell line, genes that had due to downregulated exon expression (D) or upregulated exon expression (E) upon

PRPF6 knockdown relative to siNTC were selected based on the indicated criteria. The overlap of the genes in each cell line is shown. P-values of overlap between the indicated pairs of cell lines were calculated with a hypergeometric distribution.

Supplementary Fig. 7. In situ analysis of ZAK isoform mRNA expression in colonic tissues.

Photomicrographs show representative expression of the indicated ZAK isoforms in normal colon and colon cancer samples. The level of expression corresponds to the intensity of the chromogenic red signal. DapB (top row) is a non-human specific probe (derived from a bacterial gene) that is used as a negative control for background. Scale bar denotes 100 µm.

Supplementary Fig. 8. Ectopic ZAK-LF expression induces oncogenic effects in PRPF6-low colonic cells but is not sufficient to compensate for PRPF6 knockdown. (A) Bar graph shows cell growth in SW948 and SW1463 cell lines after lentiviral infection with the indicated expression constructs (KD, kinase dead). Cells were plated at equal number at day 0 and raw cell titer glo values x 106 (cell number) for each cell line is shown at the indicated time points from triplicate measurements (mean ± S.D.). (B) Focus formation assay in SW948 cells. Cells were plated at low dilution and focus forming units were measured one week after plating. Bar graph depicts mean ± S.D. from triplicate measurements. *, p<0.05, Student’s t-test. Representative

4 images are shown on the right. (C) ZAK-LF rescue of PRPF6 knockdown effects. SW620- shPRPF6Dox cells were first transduced with the indicated lentiviral ZAK constructs and selected for stable expression. Bar graph shows relative cell number seven days after doxycycline induced

PRPF6 knockdown. Mean ± S.D. from five replicates is shown. (D) Immunoblot analysis shows the level of ZAK isoform expression for the SW948 and SW620-shPRPF6Dox cell lines used in the prior experiments.

5

Supplementary Fig. 1

A 1.2

1

0.8

0.6

0.4

0.2 SK-CO-1 Cell number (relative to siNTC) C2BBe1 0 HT55 SW1417 B COLO 741 TOX SW48 4 siNTC siPLK1 siKRAS siCTNNB1 SW1463 LoVo 2 LS1034 RKO SW837 0 DLD-1 HCT-15 -2 HCA-7

-4 Relative cell number (Z-score) -6

-8

C WT Mutant WT KRAS Mutant KRAS APC/CTNNB1 APC/CTNNB1 2 2

1 1

0 0

-1 -1

-2 -2

siKRAS (Z-score) -3 -3 siCTNNB1 (Z-score) Relative cell number after Relative ell number after -4 -4 RKO LoVo HT55 SW48

DLD-1 -5 RKO HCA-7 LoVo HT55 SW837 LS1034 SW48 -5 HCT-15 DLD-1 C2BBe1 HCA-7 SW1417 SW1463 SW837 SK-CO-1 LS1034 HCT-15 C2BBe1 SW1463 SW1417 COLO 741 SK-CO-1 COLO 741

p-value WT vs. Mutant = 0.05 p-value WT vs. Mutant = 0.0002 Supplementary Fig. 2

A Normal Colon Primary Colon Tumor Metastatic Colon Tumor Liver Lung Ovary PRPF6

ACTIN

B 8 C 8

7

6 7 5

4

6 3

PRPF6 copy number 2

PRPF6 expression in colon tumors 1 5 0

Relative Normal Colon tumor (n = 127) colon 2 3 4 5 6 7 8 PRPF6 DNA copy number

D Colon tumor E Normal colon (no expression) 300 Colon tumors (n = 119)

p = 0.001

200

100 H-score (PRPF6 protein level)

0

PRPF6 gain PRPF6 disomic Colon tumor Colon tumor PRPF6 gain = (weak expression) (moderate expression) >2.9 copies by qPCR F p = 0.05 9 8 8 7 6 6 5 4 4

PRPF6 copy # 3 2 2 PRPF6 copy number 1 0 RKO HT55 SW48 CL-40 PRPF6-low PRPF6-high DLD-1 KM-12 HCA-7 SW837 SW620 SW948 HCT-15 LS1034 C2BBe1 SW1463 SW1417 SK-CO-1 COLO 678 Supplementary Fig. 3

A B COLO 678 HCA-7 HT55 CL-40

1.2 siPRPF6 -3 siPRPF6 -4 siNTC 1 siNTC siPRPF6 -3 siPRPF6 -4 siNTC siPRPF6 -3 siPRPF6 -4 siNTC siPRPF6 -3 siPRPF6 -4 siNTC siPRPF6 -3 siPRPF6 -4 PRPF6 Low PRPF6 0.8 cell lines ACTIN 0.6 SW620 LS1034 RKO KM-12 SW48 0.4

p = 0.3 0.2 siNTC siPRPF6 -3 siPRPF6 -4 siNTC siPRPF6 -3 siPRPF6 -4 siNTC siPRPF6 -3 siPRPF6 -4 siNTC siPRPF6 -3 siPRPF6 -4 siNTC siPRPF6 -3 siPRPF6 -4

High PRPF6 PRPF6 relative protein expression Average 0 cell lines Low High PRPF6 PRPF6 ACTIN C shNTC shNTC + dox shPRPF6 -1 shPRPF6 -1 + dox shPRPF6 -2 shPRPF6 -2 + dox shPRPF6 -3 shPRPF6 -3 + dox shPRPF6 -4 shPRPF6 -4 + dox shPRPF6 -5 shPRPF6 -5 + dox shPRPF6 -6 shPRPF6 -6 + dox shPRPF6 -7 shPRPF6 -7 + dox shPRPF6 -8 shPRPF6 -8 + dox

PRPF6 KM-12 cells (High PRPF6 expression) ACTIN

1.6 1.4 1.2 1 shNTC 0.8 0.6

Cell number 0.4

(relative to shNTC) 0.2 0 D F Low PRPF6 High PRPF6 1.4 expression expression 1.4 1.2 siNTC 1.2 1 siNTC 1 0.8 siPRPF6 -3

siPRPF6 -4 siNTC siPRPF6 -3 siPRPF6 -4 0.8 siPLK1 0.6 p = 0.54 between PRPF6 0.6 high and low PRPF6

Cell number 0.4 Cell number 0.4 (relative to siNTC) ACTIN

0.2 (relative to siNTC) 0.2 0 Human primary Human primary colon cells 0 colon cells RKO HT55 SW48 CL-40 KM-12 HCA-7 SW948 SW620 HCT-15 COLO 678 E siNTC siSF3B1 -1 siSF3B1 -4 siNTC siSNRNP70 -1 siSNRNP70 -4 Cell line siNTC siPRPF31 -1 siPRPF31 -3 siNTC siBRR2 -2 siBRR2 -4 siNTC siPRPF8 -1 siPRPF8 -2 siNTC siPRPF3 -1 siPRPF3 -3 COLO 678 Low PRPF6 HT55 BRR2 SF3B1 PRPF8 PRPF3

SW620 PRPF31 High PRPF6 SNRNP70 KM-12

Representative ACTIN: Supplementary Fig. 4

A HCT-15 SW48 SW620

PRPF31 1.5 BRR2 PRPF8

siNTC siPRPF6 -3 siPRPF6 -4 siPRPF6 -33 siNTC siPRPF6 -3 siPRPF6 -4 siPRPF6 -33 siNTC siPRPF6 -3 siPRPF6 -4 siPRPF6 -33 SNU114 PRPF6 1 siNTC PRPF31

BRR2 0.5 PRPF8 level after siPRPF6

SNU114 relative protein Average 0 ACTIN HCT-15 SW48 SW620

B siNTC siPRPF6 -3 siPRPF6 -4

PRPF31 protein

SW620 cells (High PRPF6 expression)

PRPF3 protein

siNTC siPRPF6 -3 siPRPF6 -4

PRPF31 protein

COLO 678 cells (Low PRPF6 expression)

PRPF3 protein Supplementary Fig. 5

A sg-NTC 1 0.9 0.8 0.7 sg.PRPF6-1 0.6 sg.PRPF6-2 sg.NTCsg.PRPF6-1sg.PRPF6-2 0.5 sg.NTCsg.PRPF6-1sg.PRPF6-2 PRPF6 0.4 p = 0.36 between high and low PRPF6 0.3 ACTIN 0.2 HT-55 RKO 0.1

Relative cell number to sg-NTC 0

RKO HT55 SW48 HCA-7 SW620 COLO 678 B sg-NTC 1 0.9 0.8 0.7 sg.PRPF8-1 0.6 sg.PRPF8-2 sg.NTCsg.PRPF8-1sg.PRPF8-2 0.5 sg.NTCsg.PRPF8-1sg.PRPF8-2 PRPF8 0.4 p = 0.18 between high and low PRPF6 0.3 ACTIN 0.2 HT-55 RKO 0.1

Relative cell number to sg-NTC 0

RKO HT55 SW48 HCA-7 SW620 COLO 678 C sg-NTC 1 0.9 0.8 0.7 sg.BRR2-1 0.6 sg.BRR2-2 0.5 sg.NTCsg.BRR2-1sg.BRR2-2sg.NTCsg.BRR2-1sg.BRR2-2 0.4 p = 0.89 between BRR2 high and low PRPF6 0.3 ACTIN 0.2 HT-55 RKO 0.1

Relative cell number to sg-NTC 0

RKO HT55 SW48 HCA-7 SW620 COLO 678 D E

WT PRPF6 E1 E2 499 bp WT BRR2 E1 E2 891 bp

ΔPRPF6 E1 E2 372 bp ΔBRR2 E1 E2 273 bp

WT HET WT HET WT HET WT HET Clone: 1 1 2 3 Clone: 1 2 1 2 3 Clone: 1 1 2 3 Clone: 1 2 1 2 3 900 bp WT 500 bp PRPF6 WT BRR2 400 bp 700 bp ΔE1/E2 500 bp 300 bp ACTIN ACTIN ΔE1/E2 300 bp

WT HET HOM WT HET HOM Frequency: 67% 33% 0% (n = 18) Frequency: 74% 26% 0% (n = 23) Supplementary Fig. 6

A B

COLO 678 cells KM-12 cells SW620 cells Compare siPRPF6-3 to -4 0.7 Compare siNTC to siPRPF6-3 Compare siNTC to siPRPF6-4 0.6

0.5

siNTC siPRPF6-3 siPRPF6-4 siNTC siPRPF6-3 siPRPF6-4 siNTC siPRPF6-3 siPRPF6-4 0.5 0 0.4 -0.5 -1 0.3 -1.5 PRPF6 expression

2 0.2 -2 -2.5 0.1

-3 0 Relative log Average Pearson correlation of all genes Average COLO 678 KM-12 SW620

C D Gene expression downregulated upon PRPF6 knockdown (p<0.05; >1.3-fold over siNTC; 2 siRNAs each cell line) p = 10-59

KM-12 SW620 cells 315 377 572 cells

COLO678 siNTC COLO678 siNTC COLO678 siNTC COLO678 siPRPF6-3 COLO678 siPRPF6-3 COLO678 siPRPF6-3 COLO678 siPRPF6-4 COLO678 siPRPF6-4 COLO678 siPRPF6-4 KM12 siNTC KM12 siNTC KM12 siNTC KM12 siPRPF6-3 KM12 siPRPF6-3 KM12 siPRPF6-3 KM12 siPRPF6-4 KM12 siPRPF6-4 KM12 siPRPF6-4 SW620 siNTC SW620 siNTC SW620 siNTC SW620 siPRPF6-3 SW620 siPRPF6-3 SW620 siPRPF6-3 SW620 siPRPF6-4 SW620 siPRPF6-4 SW620 siPRPF6-4 9 3 1 10 COLO 678 cells E Gene expression upregulated upon PRPF6 knockdown (p<0.05; >1.3-fold over siNTC; 2 siRNAs each cell line)

p = 10-44

KM-12 SW620 cells 278 203 517 cells 12 Gene expression upregulated or downregulated upon PRPF6 knockdown 10 3 (p<0.05; >1.3-fold over siNTC; 2 siRNAs each cell line) 14 fold change COLO 678 cells -2 2 Supplementary Fig. 7

Normal colon Colon tumor

DapB

ZAK-SF

ZAK-LF

In situ hybridization Supplementary Fig. 8

A B 3 Vector 4 SW948 50 * ZAK-LF 3.5 2.5 ZAK-LF (KD) 40 ZAK-SF 3 ZAK-SF (KD) 30 2.0 2.5 20 1.5 2 10 1.5 1.0 0 Focus formation (per 6 well dish) Relative cell number 1 Vector 0.5 0.5 ZAK-LF WT ZAK-LK KD ZAK-SF WT ZAK-SF KD Vector ZAK-LF-WT ZAK-SF-WT 0 0 Day 0 Day 3 Day 6 Day 0 Day 3 Day 6 SW1463 SW948

C D 1.2 SW620-shPRPF6Dox

1 VectorZAK-LF-WTZAK-LF-KDVectorZAK-LF-WTZAK-LF-KD ZAK-LF 0.8 ACTIN SW948 SW620- 0.6 shPRPF6Dox

0.4 Relative cell number

0.2 VectorZAK-SF-WTZAK-SF-KDVectorZAK-SF-WTZAK-SF-KD ZAK-SF 0 DOX: - + - + - + - + - + ACTIN Vector ZAK-LF WT ZAK-LF KD ZAK-SF WT ZAK-SF KD SW948 SW620- shPRPF6Dox