p53 is activated in response to disruption of the pre-mrna splicing machinery.

Transcription

1 p53 is activated in response to disruption of the pre-mrna splicing machinery. Nerea Allende-Vega, Saurabh Dayal, Usha Agarwala, Alison Sparks, Christophe Bourdon and Mark K. Saville. Jean- Supplementary Information Figure Legends S1. UBL5 mrna levels corresponding to the experiments shown in Figures 1a, 2a and 5a respectively. Values are the mean +/- range of values of two experiments. S2. The approaches used have an impact on the splicing machinery. The snrnps and some splicing factors are organized in subnuclear speckles. These can de detected by immunofluorescent staining for splicing factors including SC35. Upon splicing inhibition, the number of speckles is reduced but the remaining speckles increase in size and intensity. This is used as a marker for disruption of the splicing machinery. A375 cells were transfected with the indicated for 24 h or treated with 100 M TG003 for 4 h. After splicing inhibition nuclear speckles increase in size and become less abundant. Immunofluorescence was carried out using anti-sc35 antibody, to visualize the nuclear speckles and cells were co-stained for p53 using the antibody CM1. S3. Spliceosomal protein knockdown or treatment with TG003 selectively alters RNA splice form expression. A375 cells were transfected with or treated with TG003 as indicated. RBM5, Clk1, FAS and GAPDH mrna expression was analysed by semiquantitative RT-PCR using the indicated primers. The exon composition of the major products is shown. A no template control (NTC) PCR was also performed for each of the primer pairs. Spliceosomal protein knockdown results in an increase in the expression of a form of RBM5 lacking exon 16. With the exception of SF3b1, knockdown of spliceosomal proteins did not alter the other splicing events examined. Suppression of SF3b1 caused an increase in the levels of a splice form of FAS mrna lacking exon 6 and a corresponding decrease in exon 5, 6 and 7 containing mrna. TG003 treatment resulted in a decrease in Clk1 mrna lacking exon 4 and an increase in mrna containing exons 2 to 5. TG003 did not alter the other splicing events analysed. S4. SF3b1 knockdown results in the accumulation of alternative protein isoforms of Mdm2. A375 cells were transfected with or treated with TG003 as indicated. Mdm2 expression was analysed using three antibodies that recognize epitopes in different regions of Mdm2: 4B2 (amino acids 19-50, encoded within exons 3 to 4), 2A9 (amino acids , encoded within exons 8 to 9) and 2A10 (one epitope, amino acids encoded within exon 10 and a second epitope, amino acids encoded within exon 12). SF3b1 knockdown caused a redistribution of Mdm2 isoforms from full length to lower molecular weight species that could be detected with 2A9 and 2A10. For the other approaches employed to target the spliceosome minor protein species increased in parallel with the full length protein. This is most likely due to increased transcriptional activity of p53. S5. Primers used for PCR are listed along with the catalogue numbers of the ON-TARGETplus (Dharmacon) used for spliceosomal protein knockdown. The that were used in experiments where only a single complementary to the targets was employed are underlined. S6. Primary antibodies used for Western blotting.

2 S1 - Prpf8(1) Prpf8(2) UBL5(2) - control Prpf8(1) Prpf8(2) UBL5(2) 120 Fig1 % UBL5 mrna UBL5(2) UBL5(3) Fig2 % UBL5 mrna p53(1) p53(2) HCT166 p53 +/+ HCT166 p53 -/ Fig5 % UBL5 mrna

3 S2 Merge CM1 SC35 U1-70 USP39 Prpf31 SF3b1 Prpf8 UBL5 TG M

4 S3 Long exposure KB RBM5 (Primers: E15 and E17) 0.2 Exons 15, 16 and 17 Exons 15 and Exons 15, 16 and 17 Exons 15 and Clk1 (Primers: E2 and E5) Exons 2, 3, 4 and 5 Exons 2, 3 and FAS (Primers: E5 and E7) 0.2 Exons 5, 6 and Exons 5 and 7 GAPDH NTC TG003 (100 M) DMSO Prpf8 TG003 (50 M) UBL5 SF3b1 USP39 Prpf31 U Short exposure 0.2

5 S4 U1-70 Mr B long exposure short exposure 97 2A long exposure short exposure 97 2A long exposure short exposure actin USP39 Prpf31 SF3b1 UBL5 Prpf8 DMSO TG003 (50 M) TG003 (100 M)

6 S5 Primers and Probes for RT-qPCR TBP: Primers: 5 -CACGAACCACGGCACTGATT-3, 5 -TTTTCTTGCTGCCAGTCTGGAC-3 Probe: 6-FAM-TGTGCACAGGAGCCAAGAGTGAAGA-TAMRA UBL5: Primers: 5 -GGATGATCGAGGTTGTTTGCA-3, 5 -CTTCTTAAGGTCCCCGATGGT-3 Probe: 6-FAM-CCGCGTTAAATGCAACACGGATGA-TAMRA Primers for semiquantitative RT-PCR Mdm2 forward E3: 5 -GTACCTACTGATGGTGCTGTAAC-3 Mdm2 reverse E12: 5 -AGTTAGCACAATCATTTGAATTG-3 MdmX forward E2: 5 -ATCATTTTCCACCTCTGCTC-3 MdmX reverse E6: 5 -TCCATACTGTGATCCTGTGC-3 MdmX forward E6: 5 -TCGCACAGGATCACAGTATGG-3 MdmX reverse E11: 5 -ACAGTACCTCTTGCTTGGAGA-3 RBM5 forward E15: 5 -CGGCTGTAGTGTCCCAGAGT-3 RBM5 reverse E17: 5 -TTGCGAGTTGGGGTCATAAT-3 Clk1 forward E2: 5 -AAGGATTGGGATTATGGAAA-3 Clk1 reverse E5: 5 -CTTTATGATCGATGCACTCC-3 FAS forward E5: 5 -TGTGAACATGGAATCATCAAGG-3 FAS reverse E7: 5 -GCATGTTTTCTGTACTTCCTTTCTCT-3 GAPDH forward: 5 -GAAGCTTGTCATCAATGGAA-3 GAPDH reverse: 5 -CCTCTTCAAGGGGTCTACAT-3 Prpf8 (1): Prpf8 (2): Prpf8 (3): UBL5 (1): UBL5 (2): UBL5 (3): U1-70K (1): U1-70K (2): U1-70K (3): U1-70K (4): SF3b1 (1): SF3b1 (2): SF3b1 (3): Prpf31 (1): Prpf31 (2): Prpf31 (3): Prpf31 (4): USP39 (1): USP39 (2): USP39 (3): MdmX (1): MdmX (2): MdmX (3): MdmX (4):

7 S6 Primary antibodies used for western blotting 4B2 for Mdm2 unless otherwise indicated, DO1 for p53, BL1258 for MdmX (Bethyl Laboratories), Ab-1 for p21 (Calbiochem), F-6 for Prpf8 (Santa Cruz), H-280 for U1-70K (Santa Cruz), anti- Sap155 for SF3b1 (MBL), ab11175 for H2A.X (Abcam), JBW301 for anti-phospho-histone H2A.X (Upstate), H5 for the phosphoserine 2 form of RNA polymerase II (Covance), N-20 for total RNA polymerase II (Santa Cruz) and Ab-1 for -actin (Calbiochem). Anti-USP39 and anti-prpf31 antibodies were a gift from Dr.Luhrmann.