Sandra Vorlová, Gina Rocco, Clare V. LeFave, Francine M. Jodelka, Ken Hess, Michelle L. Hastings, Erik Henke, and Luca Cartegni

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1 Molecular Cell, Volume 43 Supplemental Information Induction of Antagonistic Soluble Decoy Receptor Tyrosine Kinases by Intronic PolyA Activation Sandra Vorlová, Gina Rocco, Clare V. LeFave, Francine M. Jodelka, Ken Hess, Michelle L. Hastings, Erik Henke, and Luca Cartegni Supplemental Experimental Procedures Oligonucleotide sequences # Name Sequence Gene 1 IGF.Ex6.F 5'-CTT GCA GCA ACT GTG GGA CTG G-3' IGF1R 2 IGF.In7R.R 5'-CTC TCC CAT TCG TAG CCC TGT C-3' IGF1R 3 IGF.In13R.R 5'-TCC TGG ACC ACC CTG CTT TCA G-3' IGF1R 4 IGF.Ex15FLR.R 5'-CAG AGG CAT ACA GCA CTC CAT TCC-3' IGF1R 5 INS.Ex6.F 5'-AAC CTA AGG CAG CTC TGG GAC TG-3' INSR 6 INS.Ex10.F 5'-CTG GTC TCC ACC ATT CGA GTC TG-3' INSR 7 INS.In7R.R 5'-GCC TCC TCA GCG TCT CTC TAA CTC-3' INSR 8 INS.In9R.R 5'-CAT CAG ACA CAC GTG TGC AAA CC-3' INSR 9 INS.In13R.R 5'-CCC ATC TTC CTG CGA AGT CAG G-3' INSR 10 INS.In14R.R 5'-CCC ACC ATG CTC AGT GCT AAG C-3' INSR 11 INS.Ex16.FLR.R 5'-TCA CTG GCA CTG AGA TAC TCA GGG-3' INSR 12 FG1.Ex6.F 5'-ATA ATG GAC TCT GTG GTG CCC TC-3' FGFR1 13 FG1.In8R.R 5'-GAG CTG AGA GGA TTC AGC CCT C-3' FGFR1 14 FG1.In8.2R.R 5'-CGC CTG CAA AAC TAG GGA AGC TC-3' FGFR1 15 FG1.Ex10FLR.R 5'-GTT CAT GGA TGC ACT GGA GTC AGC-3' FGFR1 16 FG2.Ex2.F 5'-GGC CCT CCT TCA GTT TAG TTG AGG-3' FGFR2 17 FG2.In4R.R 5'-AGA ACT TCC CTC CAT GCT CCT CTC-3' FGFR2 18 FG2.In5R.R 5'-GAA TCG CTT GAA CCT GGG AGA TGG-3' FGFR2 19 FG2.In8R.R 5'-GTG AGT GTG GGA TCT CAC TGT GTG-3' FGFR2 20 FG2.Ex10FLR.R 5'-TGT TGG AGT TCA TGG AGG AGC TGG-3' FGFR2 21 FG3.Ex5.F 5'-AGC GGA TGG ACA AGA AGC TGC TG-3' FGFR3 22 FG3.In7R.R 5'-ACC CCT AGA CCC AAA TCC TCA CGC-3' FGFR3 23 FG3.In8R.R 5'-CCT TGG AGC TGG AGC TCT TGT GC-3' FGFR3 24 FG3.Ex10FLR.R 5'-GTG GTG TGT TGG AGC TCA TGG ACG-3' FGFR3 25 FL3.Ex3.F 5'-ATG TAT CTG CTT CCA TCA CAC TGC-3' FLT3 26 FL3.In5R.R 5'-TAC AGG CGT GAG CCA CTT CAC-3' FLT3 27 FL3.In12R.R 5'-TCT GGG GAT TCT TGC CTG ACT CAA G-3' FLT3 28 FL3.Ex14FLR.R 5'-TCA CCT GTA CCA TCT GTA GCT GGC-3' FLT3 29 FL3.In4CR.R 5'-AGA GGA GCT GGT CAA GCT AAC G-3' FLT3 30 KIT.Ex1.F 5'-CTC TGC GTT CTG CTC CTA CTG C-3' KIT 31 KIT.Ex6.F 5'-CAA ACC TGA ACA CCA GCA GTG G-3' KIT 32 KIT.In2R.R 5'-ATT CTT GAA AGC ACA GCA CTG CAG-3' KIT 33 KIT.In2.2R.R 5'-TTT AGG CCT TCT AGA CCC AGC CAG-3' KIT 34 KIT.In9R.R 5'-TGG TAG ACA GAG CCT AAA CAT CCC-3' KIT 35 KIT.Ex11FLR.R 5'-CTC AGC CTG TTT CTG GGA AAC TCC-3' KIT 36 PDA.Ex8.F 5'-GCT AAG GAA GAA GAC AGT GGC C-3' PDGFRA 37 PDA.In10R.R 5'-CAG CTG ATG AGT TGT CCT GAC TG-3' PDGFRA 38 PDA.Ex11FLR.R 5'-CAA TCA CCA ACA GCA CCA GGA C-3' PDGFRA 39 PDB.Ex6.F 5'-GAA GAC TCG GGG ACC TAC ACC-3' PDGFRB 40 PDB.In8R.R 5'-GGG TCA GTG GCC TAG AAT CCA TC-3' PDGFRB 41 PDB.In10R.R 5'-TAA CTC CTT TGG CCT GCT GTG G-3' PDGFRB 42 PDB.Ex12FLR.R 5'-CCG TCA GAG CTC ACA GAC TCA ATC-3' PDGFRB 43 PDB.In7CR.R 5'-GCC TAG GTT TGT GGC TGA AAG C-3' PDGFRB 44 VE1.Ex5.F 5'-GGC TTC TGA CCT GTG AAG CAA CAG-3' VEGFR1 45 VE1.In7R.R 5'-GGG AAC CAT GGC CAA GCT GTA TTC-3' VEGFR1 46 VE1.In9R.R 5'-CTA ATG ACT TTC TCC TGG GCC ACA C-3' VEGFR1 47 VE1.In13R.R 5'-TTT GGA GAT CCG AGA GAA AAC AGC C-3' VEGFR1 48 VE1.In14R.R 5'-GTG ACG GGA CTG TTA AGG GAA AGG-3' VEGFR1 1

2 49 VE1.In15R.R 5'-TTG CCA GAC AGA ACC AGA CAG TCC-3' VEGFR1 50 VE1.Ex17FLR.R 5'-CAC TTG CTG GCA TCA TAA GGG AGC-3' VEGFR1 51 VE1.In6CR.R 5'-ACT AAC ACT GCC ACA GGA GTC AGG-3' VEGFR1 52 VE2.Ex11.F 5'-CTG TCT CAG TGA CAA ACC CAT ACC C-3' VEGFR2 53 VE2.In13R.R 5'-TTA AGG CAT TCC AAC TGC CTC TGC-3' VEGFR2 54 VE2.In15R.R 5'-TAA TGG CTT CCA GCT CCA TCC ATG-3' VEGFR2 55 VE2.Ex17FLR.R 5'-CCA TGA CGA TGG ACA AGT AGC CTG-3' VEGFR2 56 VE2.Ex1.F 5'-GCA GAG CAA GGT GCT GCT GGC CGT-3' VEGFR2 57 VE2.EX3.R 5'-GAA CAT AGA CAT AAA TGA CCG AGG C-3' VEGFR2 58 Met.Ex9.F 5'-GAA AAT TGA CTT AGC CAA CCG-3' MET 59 Met.In12R.R 5'-TGG CTT CCT TAT TTA CAT CAT GAG-3' MET 60 Met.Ex13FLR2.R 5'-TTC TCT TTT TCA GCC ACA GG-3' MET 61 NT1.Ex1.2.F 5'-AGC CTG CTG GCT TGG CTG ATA C-3' NTRK1 62 NT1.In3R.R 5'-GGA AGC CCC AGG AGA ATG GTG AG-3' NTRK1 63 NT1.In3.2R.R 5'-TTG GGC CCT CCT CGC AAT CAT TC-3' NTRK1 64 NT1.In9R.R 5'-GGT CCC AAA GTC TGC ACA AGC TTC-3' NTRK1 65 NT1.Ex11FLR.R 5'-ATT GTG GGT TCT CGA TGA TGT GGC-3' NTRK1 66 NT2.Ex6.F 5'-CTG CAA ATC TGG CCG CAC CTA AC-3' NTRK2 67 NT2.In7R.R 5'-AAT TTA AGC AGC ACC CAG AGT GCC-3' NTRK2 68 NT2.In9R.R 5'-AAA TGT CAA GGG AAA ACG GCC ATC-3' NTRK2 69 NT2.Ex11FLR.R 5'-GAC GCA ATC ACC ACC ACA GCA TAG-3' NTRK2 70 NT2.In8CR.R 5'-CGC TGA CCT ATG GAT CCA TGA GC-3' NTRK2 71 EGFR.Ex6.F 5'-GAC CAA AAT CAT CTG TGC CC-3' EGFR 72 EGFR.In8R.R 5'-AGA ACA AGC CTC GTC CGC AC-3' EGFR 73 EGFR.In10R.R 5'-TCT CTC TCT AAA ACA CTG ATT TCC-3' EGFR 74 EGFR.In11R.R 5'-GCC TGC TCA TGT GAG ATA GC-3' EGFR 75 EGFR.In16R.R 5'-AGG ACA GTC AGA AAT GCA GGA AAG C-3' EGFR 76 EGFR.Ex18FLR.R 5'-CTT CTC CAC TGG GTG TAA GAG GC-3' EGFR 77 EGFR.In7CR.R 5'-CCA AAG ACT CTC CAA GAT GG-3' EGFR 78 EGFR.In9CR.R 5'-ATA CAA GCA ACT GAA CCT GTG AC-3' EGFR 79 HE2.Ex6.F 5'-ACT GAC TGC TGC CAT GAG CAG TG-3' ERBB2 80 HE2.In7R.R 5'-AAA GCA AGA TGC AGA CAG TGC TCC-3' ERBB2 81 HE2.In8R.R 5'-CTA GGT CCC AAG AGG GTC TGA GG-3' ERBB2 82 HE2.In8.2R.R 5'-CAG AGG GAC AGG AAC TGC AGC TG-3' ERBB2 83 HE2.In12R.R 5'-TGC ACA AGT CCA AGA ACG CTG C-3' ERBB2 84 HE2.In15R.R 5'-TCT GCA GAA AAG ACC GTT GGA CTC-3' ERBB2 85 HE2.In16R.R 5'-GGG CAA TGA AGG GTA CAT CCT GG-3' ERBB2 86 HE2.Ex18FLR.R 5'-CAA GCA CCT TCA CCT TCC TCA GC-3' ERBB2 87 HE2.In9CR.R 5'-CCT GAC ACT GTC TCA GGT CAT CG-3' ERBB2 88 ERB3.Ex12.F 5'-TTG AAC TGG ACC AAG GTG CTT CG-3' ERBB3 89 ERB3.In14R.R 5'-GGC CTC TCT GTG CTA TCC TTA GCC-3' ERBB3 90 ERB3.In16R.R 5'-CAG AAG CCT GCA TTC TTG AGT CCC-3' ERBB3 91 ERB3.Ex18FLR.R 5'-TTC CAA AGA CAC CCG AGC CAA GC-3' ERBB3 92 ERB4.Ex10.F 5'-CCC AGA GAA ACT GAA CGT CTT TCG G-3' ERBB4 93 ERB4.In16R.R 5'-CGA TGG TGT TAC TAA CTG GGA CTC TTG-3' ERBB4 94 ERB4.Ex18FLR.R 5'-GTG CCA CTG GGA GTT AAT GGT TCC-3' ERBB4 95 ERB4.In11CR.R 5'-TGT AAT ACC TCA CAC CAT CAT CGG AGG-3' ERBB4 96 MER.Ex5.F 5'-AAG GGA GTG CAG ATC AAC ATC-3' MER 97 MER.In9R.R 5'-CAT CAC ATC CTA TCA GCC CTT AC-3' MER 98 MER.Ex11FLR2.R 5'-GTA AGT TCA ATG GCT CGC CGA CAG-3' MER 99 MER.In6CR.R 5'-CTG TAC AGA GAT GGA GAA ATG C-3' MER 100 TYR.Ex1.F 5'-TTC TCT GCT GCT CCC GGA GTC-3' TYRO3 101 TYR.In3.R 5'-ACG CCC ATA TGG CTC TGC CTC-3' TYRO3 102 TYR.In9.R 5'-CCT CTG CCC CTC TCC ACC TAC-3' TYRO3 103 TYR.Ex10FLR.R 5'-AGG ATG TGC GGC TGT GAG GAG-3' TYRO3 104 TI1.Ex1.F 5'-CTC CCC ATC CTC TTC TTG GCT TC-3' TI1 105 TI1.Ex9.F 5'-CCA GAG AAG ACC ACA GCT GAG TTC G-3' TI1 106 TI1.Ex6.F 5'-CTA TGG CTG CTC TTG TGG ATC TGG-3' TI1 107 TI1.In3R.R 5'-GCC CAT GGC AAC CAT CAT CCT AC-3' TI1 108 TI1.In10R.R 5'-ATT ACC CAA CAC CTC TGT CCT GCC-3' TI1 109 TI1.In15R.R 5'-TGT GGG ACA AAG GAG AGC ATC AGG-3' TI1 110 TI1.Ex17FLR.R 5'-AAA GCT GGG TCA GTC TCT AGG ACC-3' TI1 111 RYK.Ex3.F 5'-TTT TGG CAA TGG ATA TGC CCC AGG-3' RYK 112 RYK.In6R.R 5'-ACC ACT GTG CAT CGA TGT GTA TGG-3' RYK 113 RYK.In5R.R 5'-TCC ACC AGT CTA CAA AGC ACC AGC-3' RYK 114 RYK.Ex7FLR.R 5'-TGA GAC AGC CCT TGG GAA CTA CTG-3' RYK 115 DD2.Ex11.F 5'-GCT GCA ATG TAC AAC AAC TCT G-3' DDR2 116 DD2.Ex10.F 5'-CTC TGA AGC CAG TGA GTG GGA AC-3' DDR2 117 DD2.In11R.R 5'-CTG GTG TAA AGC CTC CCA CAC AG-3' DDR2 118 DD2.In13R.R 5'-GCA GCG GCA GAC CAA ATC TAG-3' DDR2 119 DD2.Ex14FLR.R 5'-CTT GGA GGT TCA CTA TGT CAG C-3' DDR2 120 DD2.In12CR.R 5'-ATC ATG ACC ATT CTG CAC CTC C-3' DDR2 121 RO2.Ex3.F 5'-AGA CAC TGG CTA CTA CCA GTG CG-3' ROR2 122 RO2.Ex5.F 5'-TGA TTA CCA CGA GGA TGG GTT CTG C-3' ROR2 123 RO2.In4R.R 5'-AAT TCG GCA AAG ACA TGA GCT GGC-3' ROR2 2

3 124 RO2.In7R.R 5'-CAG GAC AGA ACG CTC ATC ACA AGG-3' ROR2 125 RO2.Ex9FLR.R 5'-GCC GAA CAG GTG ACC TTT GTA GAC-3' ROR2 126 RO2.In6CR.R 5'-ACC GAC ACC CCC ATA CAC ATC AAG-3' ROR2 127 SMNexon6Xho 5'-CGATCTCGAGATAATTCCCCCACCACCTCCC-3' SMN 128 SMNexon8NotI 5'-ATATGCGGCCGCCACATACGCCTCACATACA-3' SMN 129 hve12.f, 5 -GCTTCCAATAAAGTTGGGACTGTGG-3 VEGFR1/Flt-1; 3-oligo PCR 130 hve13b.r 5 -TCCGAGAGAAAACAGCCTTTT-3 VEGFR1/Flt-1; 3-oligo PCR 131 hve14.r 5 -ACACCATTAGCATGACAGTCTAAAG-3 VEGFR1/Flt-1; 3-oligo PCR 132 hkd11.f 5 -GAATGGAGAAGTGTGGAGGACTTCC-3 VEGFR2/KDR; 3-oligo PCR 133 hkd15.r 5 -CTCACTCTGCGGATAGTGAGGTTCC-3 VEGFR2/KDR; 3-oligo PCR 134 hkd13b.r 5 -ACATTTAAGGCATTCCAACTGCCTC-3 VEGFR2/KDR; 3-oligo PCR 135 Met.Ex9.F 5'-GAAAATTGACTTAGCCAACCG-3 MET; 3-oligo PCR 136 Met.In12R.R 5'-TGGCTTCCTTATTTACATCATGAG-3 MET; 3-oligo PCR 137 Met.Ex13FLR2.R 5'-TTCTCTTTTTCAGCCACAGG-3 MET; 3-oligo PCR 138 EGFR.Ex6.F 5'-GAC CAA AAT CAT CTG TGC CC-3' EGFR; 3-oligo PCR 139 EGFR.In10R.R 5'-TCT CTC TCT AAA ACA CTG ATT TCC-3' EGFR; 3-oligoPCR 140 EGFR.Ex18FLR.R 5'-CTT CTC CAC TGG GTG TAA GAG GC-3' EGFR; 3-oligo PCR sirna sequences U1-70K sirna 5 -GCUCCGGAGAAUGGGUAUUUGAUGG-3, 3 -ACCGAGGCCUCUUACCCAUAAACUACC-5 sic sequence was the Dicector DS scrambled negative control duplex 5 -CUUCCUCUCUUUCUCUCCCUUGGA-3. Morpholino sequences MoKD1 5 -ACACGCTCTAGACACACAAAAA-GAA-3 MoKD2 5 -GATCCAGAATTGTCTCCCTACCTAG-3 MoKD3 5 -ACAC-TTTAGATTTATTCTTTCTTCA-3 MoKD4 5 -CTAGAATGAATCCTTACCTGCA-AGT-3 MoFL2 5 -TTTTTGTTGCAGTGCTCACCTCTGA-3 MoME2 5 -GAGGAATGCAGGAATCCCACCTCT -3 MoEG2 5 -GTGATAATTCAGCTCAAACCTGTGA-3 MoC 5 -GATCCATCCCTCTGTTAAG-ACCTAG-3 3

4 Figure S1. Related to Figure 2 (A) RT-PCR validation and DNA sequencing of selected amplified soluble RTK isoforms. PCR validation was performed using forward primers located one or more exons upstream of the predicted intronic polya site. Reverse primers were located within the intronic hit (soluble RTK) or within an exon downstream of the TM domain (full-length RTK). Amplified products for all 31 validated sdrtks were extracted from the agarose gel and sequenced to validate correct exon/intron boundary and intron retention leading to an alternative 3 UTR. Eleven of them, corresponding to the examples in Figure 2, are shown here with the sequence pictogram spanning the exon/intron boundary next to the RT-PCR gels showing their expression. (B) Evidence of polyadenylation for the sdrtk variants selected for further studies. 3 RACE experiments were performed to directly show intronic polyadenylation for EGFR. Sequencing of RACE products revealed the correct exon/intron junction and a polya tail 14 nucleotides downstream of the predicted polya signal within intron 10. Analysis of EST GenBank entries retrieved from the EST screen revealed polya tails within 5 or 30 nucleotides (MET Intron 12) or 6 nucleotides (VEGFR-1/Flt-1) downstream of the respective predicted polya signals. Direct evidence for and mapping of polya signals in VEGFR2/KDR intron 13 is reported in Supplementary figure s3g-j and Figure 4C). 4

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6 Figure S2. Interference with U1 snrnp Functions Induces Intronic Polyadenylated Variants, Related to Figure 3 (A) Inhibition of U1 snrnp function by U1 decoy RNAs. U1 snrnp promotes splicing by recognizing sequences at the 5 splice site by direct base pairing. Roca and colleagues (Roca and Krainer, 2009) demonstrated that a perfectly matched RNA decoy (D1-wt) strongly inhibits U1 snrnp functions, whereas a single nucleotide mismatch in the key +2 position of the intron renders the decoy ineffective in blocking U1 activity (D1-2C). Modified from (Roca and Krainer, 2009), =pseudouridine. (B) Quantitation of IPA activation of MET and VEGFR1 by U1 snrnp inhibition. As shown in Figure 3B, 293T cells where mock-transfected or transfected with a plasmid overexpressing either D1-wt (a short RNA complementary to the 5 end of U1 snrnp that mimics a 5 ss) or the D1-2C mutant (with a mutation in the U1-binding region, see Fig. s2a). Activation of intronic PAS in EGFR1, Met, VEGFR1/FLT-1 and VEGFR2/KDR was assessed by 3-oligo PCR, with two exonic (F and R-fl) and one intronic (R-s) primer. Here we show quantitation and statistical analysis of the induction of the soluble isoforms of MET and VEGFR1 but functional knock-down of U1 snrnp. The RT-PCR bands were quantified using ImageJ and the amount corresponding to the soluble variant is expressed as % value. Error bars represent Standard Deviations from at least 3 experiments. P-values were calculated using t-test (unpaired, two-tailed). Induction of the soluble variants of MET and FLT-1 following D1-wt treatment is significant in comparison to both mock-treated cells and cells overexpressing the D1-2C mutant oligo. (C) Knock-down of U1-snRNP protein U1-70K activates intronic polyadenylation. Immunoblot of whole cell lysates from HEK-293T cells transfected with either a scrambled control (C) sirna or a sirna targeted to U1-70K. Blots were probed with primary antibodies to indicated proteins. (B) Threeoligo RT-PCR analysis of RNA from control or U1-70K knock-down HEK-293T cells using primers specific for indicated transcripts as described in Figure 2A. Quantification of the isoform corresponding to the soluble decoy variant is shown underneath the gel (n 3). (D) Three oligo PCR using different ratios of soluble and full-length VEGFR-2/KDR cdnas reflects accurate readout for endogenous template ratios. Soluble and full-length VEGFR-2/KDR cdnas were cloned in a vector backbone and used for control experiments. Different ratios of soluble and full-length cdna were used as templates in a standard PCR experiment using a common forward primer upstream and two reverse primers, one specific for the soluble, the other one specific for the full-length isoform. Ratios determined by agarose gel electrophoresis using ethidium-bromide confirmed appropriate readouts for both VEGFR-2/KDR isoforms. (E) Target sites for morpholino oligonucleotides in EGFR, MET, VEGFR-1/Flt-1 and VEGFR-2/KDR to induce alternative intronic polyadenylation. The scheme depicts the exonic (upper case) and intronic (lower case) sequences of the target genes around the exon/intron boundary aligned to the corresponding morpholino ASO sequences (in red). (F-I) Morpholino induced usage of alternative polya sites in EGFR, MET and Flt-1 leads to decreased expression of full-length isoforms and increased expression of soluble isoforms at RNA and protein levels. (F) The indicated morpholinos (MoEG2, MoME2 or MoFL2 for the respective targets, and or MoC as control) we used at 4μM in 200 l of total media volume/well to treat for 24 h HeLa (EGFR), MDA231 (MET) or HUVEC (Flt-1), seeded in 48-well plates at 8x10 3 cells/well. Q-PCR analysis showed decreased levels of full-length EGFR, MET and Flt-1 respectively and a significant increase of the soluble isoforms normalized to MoC treated samples. Values are the mean of three independent measurements ± SD and were quantified by the ddc(t) method. (G) HeLa were seeded in 48-well plates at 8x10 3 cells/well, treated with 4μM MoEG2 or control morpholino in 200 l of total media volume/well and analyzed at different time points post-treatment. 8 g of cell lysates were analyzed by Western Blot with anti-egfr antibody for full-length EGFR or tubulin for loading control (left panels). Treatment with MoEG2 leads to a rapid downregulation of full-length EGFR. 50 l of conditioned media for each time point was analyzed for soluble EGFR by solid-phase ELISA assay (right 6

7 panel, Quantikine, Human EGFR Immunoassay, R&D systems). Increased levels of soluble EGFR were detectable for both, MoEG2 and MoC treated media after 24 h. After 48 h, higher levels of soluble EGFR were measured for MoEG2 versus MoC treated samples. (H) MDA231 cells were treated for 48 h with MoME2 and control morpholino. 8 g of cell lysates were analyzed by Western Blot with anti-met antibodies for full-length MET (with tubulin as loading control). Upper panel shows effective downregulation for full-length MET after 24 and 48 h, whereas MoC control treatment has no effect on MET expression. No suitable antibodies were available for soluble MET detection/quantification. (I) HUVEC cells were seeded in 48-well plates and treated for 48 h with increasing concentrations of MoFL2 and MoC morpholino (0.25 to 4μM). 100 μl of conditioned media was collected and subjected to a human soluble VEGFR-1/Flt-1 solid-phase ELISA assay (Quantikine, Human svegf-r1/flt-1 Immunoassay, R&D Systems). An increase in soluble VEGFR-1 of MoFL2 treated cells compared to MoC treated cells was evident at 1μM compound concentration, and increased further to 1.8 ng/ml of soluble VEGFR-1 at 4 μm MoFL2 versus 0.9 ng/ml for MoC-treated samples. 7

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9 Figure S3 Additional Characterization of VEGFR2/KDR Intron 13 polya Site Modulation, Related to Figure 4 (A-D) Induction of VEGFR2/KDR intron 13 polyadenylation in HUVEC by MoKD2 is time-dependent and leads to skdr accumulation. (A) The MoKD2 and MoC morpholinos were used at 42μM in 200 l of total media volume/well to treat HUVEC seeded in 48-well plates at 8x10 3 cells/well, and the effect was analyzed at different time points post-treatment by three-oligo RT-PCR. Top band corresponds to full-length product, middle band to soluble product and the lower band corresponds to partial skipping of exon 13, which is occasionally observed. Under these conditions, the switch in RNA processing is already evident after only 3 hours of treatment, and is virtually complete by 6-12 hours. The control compound MoC or a mock treatment (NT) have no effect (lower panels). (B-C) Whole cell lysates of MoKD2-treated (B) or MoC-treated (C) HUVEC were collected at different time points. 8 μg of respective lysates were analyzed by Western Blot with anti-kdr antibodies for full-length KDR (upper panel) or tubulin for loading control (lower panel). 20 ul of conditioned media for each time point was analyzed for soluble KDR by IB using N-terminal directed KDR-antibody (middle panel). Like for the switch at the RNA level, treatment with MoKD2 leads to a rapid downregulation of FL VEGFR2/KDR, consistent with the short half-life of the VEGFR2 receptor protein (Calera et al; Exp Cell Res Oct 15;300(1): Meissner et al, Cancer Res Mar 1;69(5): ). It takes over 24 hours, however, for the soluble isoform to be detectable in the media by Western blot. (D) Quantification of soluble KDR accumulation in conditioned media over time. 50 l of conditioned media was collected at indicated time points for 2μM MoKD2, 2μM MoC and untreated HUVEC and subjected to a human svegfr2 solid-phase ELISA assay (Quantikine, Human Soluble VEGFR-2 Immunoassay, R&D Systems). The presence of the soluble protein was detectable after 12 hours and kept increasing up to 72 hours, when it reached a concentration of ~3ng/ml in the media. Of note, the level of soluble KDR did not seem to have reached plateau under these conditions. (E) Targeting of 5 splice site versus 3 splice site to activate intronic polya sites. Solid-phase Elisa analysis was performed to quantify soluble KDR in conditioned media. 50 l of medium supernatant for HUVEC treated for 48h with 2-8 M of MoKD1, MoKD2 or MoC were collected and subjected to the ELISA assay as above (Figure s3d). The results confirm and extend the data presented in (Figure 4B). Blocking U1 binding to the 5 ss is much more effective (~4ng/ml skdr) than targeting the downstream site (~1 ng/ml skdr). Both MoKD1 and MoKD2 treatment can induce the intron 13 soluble variant at the RNA level, but whereas the switch is complete with MoKD2, it is much less pronounced with MoKD1 (Figure 4B). Immunoblot analysis confirms the efficacy of MoKD2 treatment at the protein level, with complete disappearance of the full-length protein from the lysate, and appearance of the soluble one in the media, without any need for concentration procedures (Figure 4B). On the other hand the MoKD1 treatment shows a reduction in the full-length protein but under these condition the induced soluble protein is not detectable by Western blot, and can only be observed using the more sensitive ELISA assay. (F) Induction of exon skipping by splice site blocking vivo-morpholino oligos MoKD1 and MoKD2. To compare the intrinsic effectiveness of compounds MoKD1 and MoKD2, they were tested for their ability to induce exon skipping in addition to activate intronic polyadenylation. In principle, blocking the 5 ss by MoKD2 could induce skipping of exon 13 (indicated by orange lines) and blocking the 3 ss by MoKD1 can induce skipping of exon 14 (indicated by brown lines), as exon-skipping is the most common effect of blocking a splice site. By using exonic primers upstream and downstream of the potentially skipped exons (forward primer in exon 11, reverse primer in exon 15 as indicated in scheme) we could use a PCR strategy to only measure the relative abundance of full-length vs. exon-skipping, independently of polya activation. The PCR result is non-quantitative in terms of absolute amount of templates and semi-quantitative in terms of relative amount of templates. In other words, in both treatments there is a net loss of template spanning exon 11-exon15 because of the usage of the polya sites in intron 13, which is much more pronounced in the MoKD2 treatment (Figure 4B). Since these products do not reach exon 15, they cannot be detected by 9

10 this PCR, and therefore the only conclusions that can be drawn here are about the relative effect of the treatments on exon skipping. HUVEC were treated for 48 h with 2 μm of the compounds and the effect on exon-skipping was assessed by PCR using the primers described above. Top bands correspond to the full-length products, lower bands to exon 14 (for MoKD1, missing 147 nt from exon 14) or exon 13 (for MoKD2, missing 342 nt from exon 13) skipped products. Both treatments have comparable effects on exon skipping, suggesting that their ability to target and inhibit the splice sites is similar. As a consequence, the difference in induction of IPA (Figure 4B) could be ascribed to a non-splicing function associated to the 5 ss, such as the proposed U1 snrnp inhibition of IPA. (G-J) Mapping of soluble KDR polya site usage (G) Scheme shows primers used to amplify VEGFR2/KDR regions between its exons 11 and 14 corresponding to the putative soluble KDR (reverse primer within intron 13) and full-length KDR (reverse primer in exon 14). Forward primer used for RT-PCR for both isoforms is located in exon 12. Described primers were used to amplify endogenous soluble (left lane) and full-length (right lane) KDR from HUVEC RNA. RT-PCR products for skdr were sequenced to confirm intron 13 retention. The sequencing chromatogram shows the boundary between VEGFR2/KDR exon 13 and intron 13. (H) Scheme shows the portion of the VEGFR2/KDR gene exon intron structure spanning exon 12 to 14 and indicates the primer positions for a primer walking strategy to map the 3 end of skdr mrna. Reverse primers R1 to R6 are located upstream of the proximal putative polya signal, R7 to R9 are in between proximal and distal putative polya signal and R10 to R11 are located downstream of the distal polya signal. PolyA signals are depicted in green. For each PCR a common forward primer located in exon 12 was used. (I) MDA231 RNA was used as RT-PCR template to map the 3 end of skdr by primer walking. Numbers represent number of reverse primers as described in (s3h), green bars indicate position of polya signals in VEGFR2/KDR intron 13. The RT-PCR signal drops when using reverse primers located downstream of the proximal sites, suggesting that the amount of template is reduced following polyadenylation there. (J) A KDR minigene was used as control template in a PCR using the same primer sets as in (C) to confirm efficient amplification. This rules out that the differences in the PCR results are due to inefficient primer binding or disadvantaged amplification of the longer products, and supports that they are derived from variations in template abundance (i.e. usage of the polya site). 10

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12 Table S1. RTK Genes and Validation of Predicted Truncated RTK Isoforms, Related to Figure 1 A total of 39 Ensembl ( RTK gene entries were analyzed. For all, the first 180 bps of each intron upstream and around the exon that encodes for the TM domain were selected and used as probes in a BLASTN analysis ( against Expressed Sequence Tag (EST) GenBank database ( Positive hits with alignment scores >80 were retrieved and were aligned to their respective genomic RTK sequences. Alignments that didn t reach the upstream exon-intron boundary were discarded. Hits that spanned the upstream exonintron boundary but ended within the first upstream exon were considered weak since neither upstream processing nor unprocessed RNA could be excluded. Hits for which the alignment continued into the upstream intron were not considered further, since they most likely reflect unprocessed RNA or genomic contaminations. Similarly, hits that continued into the downstream exon were also discarded as intron retention rather than polyadenylation products. Alignments to the exon-intron junctions and their continuation into upstream exons, but not introns, indicated that the intronic sequence probed was part of processed mrna, and therefore these alignment clusters were considered strong positive hits. Weak and strong positive hits were further confirmed by RT-PCR analysis of selected cdnas (Universal Human Reference RNA, Stratagene, HUVEC and MDA231 RNA, isolated as described). The table shows RTK families and respective genes that revealed positive hits in the EST screen. Genes with no hits at all in the EST database (using intronic probes as described above) are indicated with an asterisk. For each gene we annotated the exon encoding the transmembrane domain (TM), the predicted retained intron and the strength (weak or strong) of each prediction. If not already predicted, the last intron upstream of the TM domain was included in the analysis to assess for a full-length extracellular domain (prediction= no ). Binding sites for forward primers were chosen at least two exons upstream of the predicted intron to ensure amplification of processed mrna. A prediction was considered validated when PCR amplification led to the predicted product size (indicated) and subsequent DNA sequencing confirmed the expected isoform. Overall, we analyzed 56 possible candidates for intronic polyadenylation (20 strong, 22 weak, 14 no predictions) and validated 15/20 (75%) 12/22 (54%), and 4/14 (28%) of the candidates, respectively. 12

13 Supplementary Table S1 13

14 Table S2. Characteristics of Validated Soluble sdrtks, Related to Figure 2 Validated hits are grouped by Gene Family (11) and Gene (19) and ordered by the intron containing the polya site. The size of the intron and the position of the first candidate polya signal is indicated. Several introns contain multiple canonical polya sites (only the first one is indicated). Two introns (in the FGFR3 gene) do not contain a canonical polya signal. This is not uncommon, as ~17.5% of polya sites do not use any of the 12 canonical hexamers (Kamasawa et al.; In Silico Biol (3-4)347-61). For each validated hit where expression of intronic sequences within mrnas were detected, we annotated the number of the retained intron; the size of the intron; the position of the first putative polya signal; the kind of evidence of polyadenylation available; the size of the putative secreted decoy receptor generated by induction of alternative intronic polyadenylation; the amino acid sequence of the newly generated unique C- terminal domain; a literature reference, when the polyadenylated variant had been described before. More specifically, direct and indirect evidence that supports that the validated variants are generated by IPA includes the direct sequencing of all amplified products corresponding to the IPA variants, the identification of canonical PAS in the intron involved (29/31), the differential expression between 5 and 3 regions of the introns in 21 cases, the previous description and publication of 7 of the variants as polya products, and the identification of polya tails in the correct position directly on the EST sequences. In addition, The four variants for which we did follow-up work are efficiently translated as truncated version following activation of the IPA site (MET, EGFR, VGFR1 and VGFR2, Fig 3 and 4), which implies efficient polyadenylation. Furthermore, in the case of EGFR we mapped the polya sites by 3 RACE (Figure s1b), and in the case of VEGFR2 by primer walking (s3h-j). Notes: a) no canonical polya signal identified within the intron; b) EST sequence contains polya; c) the polya variant has been published; d) there is evidence of polyadenylation from this work (3 RACE, oligo walking, polya signal blockade with morpholino compounds; e) there is evidence of differential expression between the upstream and downstream regions of the intron. 14

15 Supplementary Table S2 15

16 Table S3. Characteristics of sdrtks Selected for Further Studies, Related to Figure 3 Four of the validated sdrtk variants (EGFR, MET, VEGFR1/FLT1 and VEGFR2/KDR) were selected for further analysis in order to assess more specifically the role of U1 in controlling IPA. These are all very prominent members of the RTK family, with well-characterized biology and important pathological roles. When more than one sdrtk variant was expressed for one of these genes (EGFR, and VEGFR1), we decided to focus initially on the better characterized ones (e.g.: EGFR intron 10) and for which either a pathological role has been shown (like in the case of sflt-1 in pre-eclampsia). Expression of all these 4 sdrtks has been associated to important biological properties and has been proposed as a possible therapeutic avenue to address pathologies characterized by defective signaling from their respective fulllength genes (see also Table s4). 16

17 Table S4. Examples of Biologically Active Recombinant sdrtk, Related to Figure 7 Ectopic expression of recombinant truncated version of RTKs that might possess dominant-negative properties have been explored for their potential therapeutic application either because they bind the ligand and sequester it (Figure 7-1) or because they engage the endogenous receptor(s) in non-productive heterotypic interactions (Fig 7-3). Typically these are experimentally developed variants that do not necessarily correspond to natural isoforms. The table lists some published examples of Receptor Tyrosine Kinases where expression by genetic vectors or direct administration of recombinant portions of the extracellular domain show biological activity in vitro or in vivo as soluble decoy receptors. 17

18 Supplemental References Adams, T.E., Koziolek, E.J., Hoyne, P.H., Bentley, J.D., Lu, L., Lovrecz, G., Ward, C.W., Lee, F.T., Scott, A.M., Nash, A.D., et al. (2009). A truncated soluble epidermal growth factor receptor-fc fusion ligand trap displays antitumour activity in vivo. Growth Factors 27, Aigner, A., Juhl, H., Malerczyk, C., Tkybusch, A., Benz, C.C., and Czubayko, F. (2001). Expression of a truncated 100 kda HER2 splice variant acts as an endogenous inhibitor of tumour cell proliferation. Oncogene 20, Albuquerque, R.J., Hayashi, T., Cho, W.G., Kleinman, M.E., Dridi, S., Takeda, A., Baffi, J.Z., Yamada, K., Kaneko, H., Green, M.G., et al. (2009). Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth. Nat Med 15, Bhargava, P., and Robinson, M.O. (2011). Development of second-generation VEGFR tyrosine kinase inhibitors: current status. Curr Oncol Rep 13, Brantley, D.M., Cheng, N., Thompson, E.J., Lin, Q., Brekken, R.A., Thorpe, P.E., Muraoka, R.S., Cerretti, D.P., Pozzi, A., Jackson, D., et al. (2002). Soluble Eph A receptors inhibit tumor angiogenesis and progression in vivo. Oncogene 21, Calera, M.R., Venkatakrishnan, A., and Kazlauskas, A. (2004). VE-cadherin increases the half-life of VEGF receptor 2. Exp Cell Res 300, Canadas, I., Rojo, F., Arumi-Uria, M., Rovira, A., Albanell, J., and Arriola, E. (2010). C-MET as a new therapeutic target for the development of novel anticancer drugs. Clin Transl Oncol 12, Cecchi, F., Rabe, D.C., and Bottaro, D.P. (2010). Targeting the HGF/Met signalling pathway in cancer. Eur J Cancer 46, Ciardiello, F., and Tortora, G. (2008). EGFR antagonists in cancer treatment. N Engl J Med 358, D'Ambrosio, C., Ferber, A., Resnicoff, M., and Baserga, R. (1996). A soluble insulin-like growth factor I receptor that induces apoptosis of tumor cells in vivo and inhibits tumorigenesis. Cancer Res 56, Duan, D.S., Pazin, M.J., Fretto, L.J., and Williams, L.T. (1991). A functional soluble extracellular region of the platelet-derived growth factor (PDGF) beta-receptor antagonizes PDGF-stimulated responses. J Biol Chem 266, Ellis, L.M., and Hicklin, D.J. (2008). VEGF-targeted therapy: mechanisms of anti-tumour activity. Nat Rev Cancer 8, Fischer, C., Mazzone, M., Jonckx, B., and Carmeliet, P. (2008). FLT1 and its ligands VEGFB and PlGF: drug targets for anti-angiogenic therapy? Nat Rev Cancer 8, Flynn, P.C., and Kaufman, W.R. (2011). Female ixodid ticks grow endocuticle during the rapid phase of engorgement. Exp Appl Acarol 53, Goldman, C.K., Kendall, R.L., Cabrera, G., Soroceanu, L., Heike, Y., Gillespie, G.Y., Siegal, G.P., Mao, X., Bett, A.J., Huckle, W.R., et al. (1998). Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate. Proc Natl Acad Sci U S A 95, Johnson, D.E., Lu, J., Chen, H., Werner, S., and Williams, L.T. (1991). The human fibroblast growth factor receptor genes: a common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain. Mol Cell Biol 11, Kamasawa, M., and Horiuchi, J. (2008). Identification and characterization of polyadenylation signal (PAS) variants in human genomic sequences based on modified EST clustering. In Silico Biol 8, Kawamura, N., Kawamura, K., Manabe, M., and Tanaka, T. (2010). Inhibition of brain-derived neurotrophic factor/tyrosine kinase B signaling suppresses choriocarcinoma cell growth. Endocrinology 151,

19 Kendall, R.L., and Thomas, K.A. (1993). Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci U S A 90, Kerbel, R.S. (2008). Tumor angiogenesis. N Engl J Med 358, Kuo, C.J., Farnebo, F., Yu, E.Y., Christofferson, R., Swearingen, R.A., Carter, R., von Recum, H.A., Yuan, J., Kamihara, J., Flynn, E., et al. (2001). Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer. Proc Natl Acad Sci U S A 98, Lee, H., Akita, R.W., Sliwkowski, M.X., and Maihle, N.J. (2001). A naturally occurring secreted human ErbB3 receptor isoform inhibits heregulin-stimulated activation of ErbB2, ErbB3, and ErbB4. Cancer Res 61, Malik, N.M., Jin, P., Raatz, Y., Sumariwalla, P.F., Kiriakidis, S., Shepard, M., Feldmann, M., and Paleolog, E.M. (2010). Regulation of the angiopoietin-tie ligand-receptor system with a novel splice variant of Tie1 reduces the severity of murine arthritis. Rheumatology (Oxford) 49, Meissner, M., Reichenbach, G., Stein, M., Hrgovic, I., Kaufmann, R., and Gille, J. (2009). Down-regulation of vascular endothelial growth factor receptor 2 is a major molecular determinant of proteasome inhibitormediated antiangiogenic action in endothelial cells. Cancer Res 69, Michieli, P., Mazzone, M., Basilico, C., Cavassa, S., Sottile, A., Naldini, L., and Comoglio, P.M. (2004). Targeting the tumor and its microenvironment by a dual-function decoy Met receptor. Cancer Cell 6, Mitsudomi, T., and Yatabe, Y. (2010). Epidermal growth factor receptor in relation to tumor development: EGFR gene and cancer. FEBS J 277, Ogawa, T., Takayama, K., Takakura, N., Kitano, S., and Ueno, H. (2002). Anti-tumor angiogenesis therapy using soluble receptors: enhanced inhibition of tumor growth when soluble fibroblast growth factor receptor-1 is used with soluble vascular endothelial growth factor receptor. Cancer Gene Ther 9, Reiter, J.L., and Maihle, N.J. (1996). A 1.8 kb alternative transcript from the human epidermal growth factor receptor gene encodes a truncated form of the receptor. Nucleic Acids Res 24, Sather, S., Kenyon, K.D., Lefkowitz, J.B., Liang, X., Varnum, B.C., Henson, P.M., and Graham, D.K. (2007). A soluble form of the Mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation. Blood 109, Scott, G.K., Robles, R., Park, J.W., Montgomery, P.A., Daniel, J., Holmes, W.E., Lee, J., Keller, G.A., Li, W.L., Fendly, B.M., and et al. (1993). A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells. Mol Cell Biol 13, Sela, S., Itin, A., Natanson-Yaron, S., Greenfield, C., Goldman-Wohl, D., Yagel, S., and Keshet, E. (2008). A novel human-specific soluble vascular endothelial growth factor receptor 1: cell-type-specific splicing and implications to vascular endothelial growth factor homeostasis and preeclampsia. Circ Res 102, Shelton, D.L., Sutherland, J., Gripp, J., Camerato, T., Armanini, M.P., Phillips, H.S., Carroll, K., Spencer, S.D., and Levinson, A.D. (1995). Human trks: molecular cloning, tissue distribution, and expression of extracellular domain immunoadhesins. J Neurosci 15, Shibuya, M. (2006). Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1): a dual regulator for angiogenesis. Angiogenesis 9, ; discussion 231. Trusolino, L., Bertotti, A., and Comoglio, P.M. (2010). MET signalling: principles and functions in development, organ regeneration and cancer. Nat Rev Mol Cell Biol 11, Vivanco, I., and Mellinghoff, I.K. (2010). Epidermal growth factor receptor inhibitors in oncology. Curr Opin Oncol 22,