Supplemental information

Figure S1. Genome-wide alternative splicing events affected by inducible expression SRSF3 in mouse MEF3T3 cells. To examine the effects of SRSF3 (SRp20) on genome-wide RNA splicing in mouse cells, we utilized Exonhit SpliceArray to compare genome-wide changes of RNA splicing and transcription in MEF3T3 mouse fibroblasts with or without ectopically inducible expression of SRp20.

(A–C) Clustered heat maps for the top events regulated by SRSF3 identified by ExonHit splice arrays.

SRSF3-targeted in three experimental repeats consist of 65 evidenced splicing events (A), 74 novel splicing events (B) and 11 genes with expression changes (C), with a threshold of ≥2-fold changes

(p<0.01) in log2 as determined by a B/E method and FDR cutoff of 0.1 for expression change and

0.2 for splicing alternation. Individual gene names and event ID are indicated on the right. Event ID specifies individual splicing events being detailed in Supplementary Table S1-S3. MEF3T3 cells with an empty vector transfection were included as vector controls. Color key scales in log2 values are indicated at the bottom of each panel. The full list of B/E ratio analysis result is available at NCBI GEO

(http://www.ncbi.nlm.nih.gov/geo/) (accession number GSE60147).

Figure S2. Isoform profiling of mouse Ilf3 and human ILF3. (A) Diagrams of RNA isoforms of mouse

Ilf3. Mouse Ilf3 consists of 22 exons and 21 introns, which produces 6 spliced isoforms of mRNAs by alternative splicing as illustrated. (B) Diagrams of splice isoforms of human ILF3. Human ILF3 gene consists of 21 exons (boxes) and 20 introns (lines between boxes), which produce 8 splice isoforms of mRNAs by alternative splicing as depicted. Isoform 7 and 8 are two novel isoforms identified in this study. ILF3 Isoform-3, previously reported as TCP80 was not detectable in our study. Diagrams do not reflect exon/intron scale. (C) Profiling of human ILF3 isoforms in U2OS cells. U2OS cells with

(lanes 1-5) or without (lanes 6-8) transfection by Myc/Flag-tagged ILF3 isoform-1 (pCMV6-ILF3 isoform-1), isoform-7 (pJR16), isoform-8 (pJR15), isoform-7 and -8, or an empty vector were blotted by

1 an anti-ILF-3 antibody (Ab) or by anti-Myc antibody. All ectopically expressed ILF3 protein isoforms have an additional 31 amino acid (aa) residues as a C-terminal Myc/FLAG tag and were immunoblotted with anti-ILF3 or anti-Myc antibody. U2OS cells transfected with 40 nM of si-SRSF3 (a SRSF3-specific siRNA) or si-NS (a non-targeting siRNA) or without (none) siRNA were served for endogenous ILF3 detection by Western blotting. ILF3 isoform profile (1, 2/5, 7 and 8) is indicated on the left. hnRNP K served as a sample loading control. *, endogenous isoform 7 without a tag in lane 7. Exp, exposure. (D-E)

Knockdown of SRSF1 expression in U2OS cells does not affect alternative splicing of ILF3 RNA. Total cell protein extract and total RNA from U2OS cells transfected with 40 nM of si-SRSF1 or si-NS twice at a 48 h interval were examined, respectively, by Western blotting for SRSF1 knockdown efficiency (D) and by RT-PCR for production of ILF3 isoforms with the primer pairs used in Figure 2A (E)

Figure S3. Roles of RBM5 and RBM39 in alternative RNA splicing of ILF3. (A-B) RBM5 and

RBM39 are not downstream targets of SRSF3. SRSF3 in HEK293T cells were knocked down twice in a

48 h interval with 40 nM of a non-specific si-NS or a SRSF3-specific si-SRSF3. Total protein extract and total cell RNA were prepared at 24 h after the last time of siRNA treatment. The knockdown efficiency of

SRSF3 in HEK293 was examined by Western blot analysis (A). The knockdown of SRSF3 on RBM5 and RBM39 RNA expression was examined by RT-qPCR (B). (C-D) RBM5 and RBM39 in alternative

ILF3 RNA splicing and ILF3 isoform-2 production. Total RNA prepared from HEK293T cells with or without twice knockdown of RBM5 or RBM39 expression by the indicated siRNA (C) as described above was examined by RT-PCR for alternative RNA splicing of ILF3 with the indicated primer pairs (a, b, c, d and e, see Figure 2A). The detected ILF3 isoforms were indicated on the gel right (D). Triplicates in each experiment were repeated twice.

Figure S4. Expression of ILF3 isoform-1 and -2 in WI-38 fibroblast cells is regulated by SRSF3 and is important for cell growth. (A) Ectopic expression of SRSF3 in WI-38 cells reduces the expression of suppressive ILF3 isoform-5. Total RNA extracted from WI-38 cells 48 h after the second transfection

2 with a SRSF3 expression vector was examined by RT-PCR with an ILF3-specific primer pair. RT, reverse transcription. (B) Ectopic expression of ILF3 isoform-1 and -2 in WI-38 cells promotes cell proliferation. A total of 1 x 105 WI-38 cells in each well of a 12-well plate were transfected twice in a 48- h interval by reverse transfection with 1 μg FLAG-ILF3 isoform-1 in combination with 1 μg FLAG- tagged ILF3 isoform-2 or an empty control vector pFLAG-CMV-5.1 (2 μg). The cells were counted on day 4 from the first transfection and cell numbers from each group were statistically analyzed with a student t-test. The ectopic expression of ILF3 isoform-1 and -2 was examined by Western blot with an anti-FLAG antibody and is shown above the bar graph.

Figure S5. Effects of human ILF3 isoform-1, -2, -5 and -7 at ectopic expression on the growth of

NIH/3T3 cells. (A) Effects of transient expression of ILF3 isoforms on cell growth. NIH 3T3 cells at 5 x

105/well were seeded into a 6-well plate and reversely transfected immediately with 2 μg of FLAG-ILF3 isoform-1 (pCMV6-ILF3), -2 (pJR9), -5 (pJR18), or -7 (pJR16), or a control vector (pSB21). The cells at

24 h were passed and reversely transfected again with the same amount of each plasmid. The cell counts were obtained at 96 h after the first transfection. (B) Effects of stable expression of ILF3 isoforms on cell growth. NIH 3T3 cells stably transfected with FLAG-ILF3 isoform-1, -2, -5, -7 or pSB21 plasmid at 2.5 x

105 per well were seeded into a 6- well plate and passed once on Day 3. The total cell counts were obtained on Day 5. Below each bar graph are individual FLAG-tagged ILF3 isoforms blotted by an anti-

FLAG antibody. (C) Effects of ectopic transient expression of ILF3 isoform-1 and -2 on U2OS cell growth. The cell counts were obtained at 96 h after twice transfection of individual plasmids.

Figure. S6. Stable expression of human ILF3 isoform-2 in MEF3T3 cells promotes cell cycle progression. MEF3T3 cells stably transfected by pJR19 encoding FLAG-ILF3 isoform-2 or an empty vector. The cells at 2 x 105 cells per well were seeded into a 6-well plate in DMEM medium containing

10% calf serum for 24 h, synchronized by starvation for 48 h in DMEM medium containing 0.1% calf

3 serum, and collected at the indicated duration of serum stimulation in DMEM medium containing 10% calf serum for cell cycle flow cytometry.

Figure S7. SRSF3 regulates RNA splicing of human ILF3 minigene. (A) Diagram of an ILF3 minigene from its exon 16 to exon 19 under CMV IE promoter control (arrow), with its splicing directions and the primers (short lines) used for detection of alternative 3’ ss selection in the exon 18. (B)

Examination of ILF3 minigene splicing in U2OS cells. U2OS cells were cotransfected by 2 μg of pJR8 and 40 nM of si-SRSF3 or NS siRNA. After 48 h of transfection, total cell RNA was extracted for RT-

PCR analysis by oJR51 (pJR plasmid-specific primer) and oJR26 in the ILF3 exon 18 to examine ILF3 isoform-2 and -5 splicing in the minigene expression.

Table S1. Genes with evidence splicing events (p<0.01, fold change >2). spliceEventId: EHT (ExonHit company) nomenclature used in array design; p-value (SR vs Vec):

Statistical analysis of SRSF3 overexpression (SR) vs control (Vec) group; Fold-Change (SR vs. Vec): The fold change of the F1 vs F2 ratio between SRSF3 overexpression (SR) vs control (Vec) group;

F1 to F2 Ratio - SR 1.CEL: The ratio of expression level between isoform F1 and F2; entrezId:

Gene Number; formId: Splice event ID (above) appended with F1 or F2;

Accession: NCBI/Genbank accession # used for event design; net bp delta: Size (bp) of splice event;

Gene name: Entrez Gene name; spliceEventName: Type of splice event, see Table S1.

Table S2. Genes with predicted novel splicing events.

Splice Event Id: EHT (ExonHit company) nomenclature used in array design; p-value (SR vs Vec):

Statistical analysis of SRSF3 overexpression (SR) vs control (Vec) group; Fold-Change (SR vs Vec): The fold change of the ratio of exon inclusion vs exon exclusion between SR vs Vec group; SR 1.CEL: The ratio of expression levels between exon inclusion and exon exclusion in SR 1 sample; entrezId: Entrez

Gene Number; start: Position on chromosome of event start; Chromosome end: Position on

4 chromosome of event end; Orientation: Orientation of probe set on chromosome; Accessions/descriptions at: NCBI/Genbank accession number used for predicted event design. Predicted net bp delta: Predicted size (bp) change caused by alternative splicing in a transcript.

Table S3. Genes with expression level changes.

Table S4. DNA/RNA oligonucleotides used in this study.

5

Figure S1

A B C

Evidence splicing events Novel splicing events Gene expression changes Splice Splice event ID Gene name event ID Gene name Gene ID Gene name Figure S2

Protein Mous e Ilf3 isoform name A size (aa) 1 2 3 4 5 17 18 19 20 21 22 912 1L (Ilf3, NF110, NFAR-2

1 2 3 4 5 17 18 19 20 21 22 898 1S

1 2 3 4 5 17 18 19 716 2L (DRBP76, NF90, NFAR-1

1 2 3 4 5 17 18 19 703 2S

1 2 3 4 5 17 18 19 20 21 22 790 3L

1 2 3 4 5 17 18 19 20 21 22 776 3S

B Human ILF3 Protein size (aa) isoform name 13 14 15 16 17 18 19 20 21 894 1 (ILF3, NF110, NFAR-2, TCP110)

13 14 15 16 17 18 702 2 (DRBP76, NF90, NFAR-1, NFAR90, MPP4)

13 14 15 16 17 18 706 6 (NF90b)

13 14 15 16 17 18 690 5 (DRBP76δ)

13 14 15 16 17 18 694 4 (DRBP76α)

13 14 15 16 17 18 19 20 21 773 7

13 14 15 16 17 18 19 20 21 730 8

13 14 15 16 17 18 19 20 21 777 3 (TCP80)

C D siRNA NS SRSF1 ILF3 isoform SRSF1 SRSF3 NS Ab - -

1 7 8 7+8 vec None si si GAPDH 1 lane 1 2 7 short exp. 8 2/5 E 1 ILF3 7 siRNA NS SRSF1 * long exp. isoform 8 RT + - + - 2/5 (a+d) 16 17 19 1 1 7 17 18 2 8 (b+c) 2/5 17 5

16 17 20 21 7 hnRNP K (a+e) ILF3 primers ILF3 16 17 21 8 lane 1 2 3 4 5 6 7 8 GAPDH lane 1 2 3 4 Figure S3

A B

1.6 P>0.05 1.5 P>0.05 1.4 NS SRSF3 1.2 - - si si 1 1 SRSF3 0.8 RBM39 RNA RNA RBM39 level level 0.6 0.5 GAPDH 0.4 0.2 Relative RBM5 RNA RNA RBM5 Relative lane Relative 1 2 0 0 Si-NS Si-SRSF3 Si-NS Si-SRSF3

C D RBM5 NS - RBM39 - RBM5 RBM39 - si si ILF3

1.2 si isoform 1 RT + - + - + - 0.8 (a+d) 16 17 19 1 17 18 2 RBM RNA RNA RBM 0.6

level (b+c) 0.4 17 5 ILF3 0.2 Relative Relative 16 17 20 21 7 0 (a+e) 16 17 21 8 GAPDH lane 1 2 3 4 5 6 Figure S4

A WI-38 SRSF3

- 17 18 19 T7 Vector

RT + - + - 17 19 isoform 1 17 18 isoform 2 ILF3 17 isoform 5 GAPDH

B ILF3 isoform 1+2 Vec. Isoform 1 Anti-FLAG Non-Specific Isoform 2

1st expt. 2nd expt. Figure S5

A NIH3T3 (transient) B NIH3T3 (Stable ILF3)

p=0.037 p=0.043 p=0.0087 p=0.0059 100 ) 5 ) 120 80 5 10 10 60 80

40 40

Cell number (x number Cell 20 Cell number (x numberCell 0 0 ILF3 isoform 1 7 5 2 Vec. ILF3 isoform 1 7 5 2 Vec. 98kD Non-specific 98 kD FLAG-ILF3 FLAG-ILF3

C U2OS 80 ) 5 60 10

40

20 Cell number (x numberCell

0 ILF3 isoform 1 2 vector Figure S6

ILF3 Isoform 2 Vector

0 h

7 h

10 h Figure S7

A

16 17 18 19

B

siRNA

SRSF3 NS RT + - + - Isoform-2 ILF3 Isoform-5 GAPDH