e15 e14 3utr i13 K36me3 / H i4 ** ** ** ** ** tss IIIb K36me2 / H IIIb BCOR (% input)

Transcription

1 a Epithelial cells (2) Mesenchymal cells () Alternatively spliced region i13 e14 e15 / CycA e14 e13-e K27me3 / H3 b 2.5 c v5 K36me3 / H3 d e v5 K9me3 / H3 HP1α (% input) f j g h i k v5 HMGA1 i2 e3 e5 K36me2 / H3 BCOR (% input) l # # # # # # # # # # # # # # # m # # # # # # v5 # # v5# # # p15# BMI1 (% input) n o v5 p15 RING1B (% input) p q # # # # # # # # v5# # # p15# r. qrt-pcr SUZ12 KDM2b Cbx8 HP1g s. co-ip 2 WB: WB: CBX8 WB: SUZ12 Input 2% Input % IP: no Ab IP: CBX8 IP: SUZ12 2 no Ab MSC no Ab Supplementary Figure 1 A chromatin signature characteristic of splicing pattern. (a) Schematic representation of locus and the oligonucleotide pairs used in ChIP (see Supplementary Table 1 for details), together with general expression levels of exons and the alternatively spliced exons and in epithelial 2 cells (in red) and mesenchymal cells (in black). qrt-pcr values (mean SEM, n=6) are normalized to Cyclophilin A. (b-q) Enrichment levels of H3K27me3, H3K9me3, H3K36me3, H3K36me2, HP1, BCOR, BMI1 and RING1B along and control

2 alternatively spliced or HMGA1 loci in 2 (red) and (black) cells. ChIP values (mean SEM of n=2-5) are either normalized to H3 or depicted as the percentage of input. No antibody ChIP is used as negative control in 2 (yellow) and (light grey) cells. The transcription start site of p15 is used as a positive control in BCOR, BMI1 and RING1B ChIPs. p<5 and p<1 in two tailed Student s t-test relative to values. (r) mrna expression levels of, SUZ12,, KDM2b, CBX8 and HP1 in 2 and cells. qrt-pcr values (mean SEM, n=3) are normalized to Cyclophilin A. (s) Western blot (WB) of (upper panel) and CBX8 or SUZ12 (lower panels) as positive IP controls, after immunoprecipitation (IP) with CBX8 (lane 4) or SUZ12 (lane 5) in 2 cells. No antibody IP is used as a negative control (lane 3) and serial dilutions of the total input are used as a reference (lane 1 and 2).

3 + PHF1 / a b c d ±19 ± PHF1 1 +PHF1 / ±4 ± PHF1 e13-e14/cyc PHF1 2 v6- / e. +PHF1 f e1-e2/hmga PHF1 HMGA1 g -e8/ TPM2 +PHF1 TPM2 h e8-/pkm PHF1 PKM2 ESRP1 / CycA i ESRP1 j ESRP2 k TIA1 l RBFOX EZH +PHF ESRP2 / CycA EZH +PHF TIA1 / CycA 3 1 +PHF1 RBFOX / CycA CTR EZH +PHF PTB / CycA m PHF1 PTB hnrnpa/ CycA n hnrnp A1 EZH +PHF hnrnph/ CycA o hnrnp H EZH +PHF /CycA p q r PHF1 1 E+P / CycA EZH +PHF PHF1 / CycA EZH +PHF - s - / pcdna3 t u / v - / pcdna3 / pcdna3 v6- / pcdna3 Supplementary Figure 2 overexpression promotes exon inclusion in mesenchymal cells. (a-r) Inclusion levels of exons, constitutive e14 (a-d), alternatively spliced control -v6 and HMGA1-e2 (e-f), PTBdependent spliced PKM2- and TPM2- (g-h), splicing regulators ESRP1, ESRP2, TIA1, RBFOX2, hnrnpa1, hnrnph and PTB (i-o) and, and PHF1 (p-r) after combined overexpression of the H3K27 methyltransferase and its co-factor PHF1 for 72h in 2 (red) and (black) cells. qrt-pcr values (mean SEM, n=9) are normalized to Cyclophilin A or total

4 mrna of the corresponding gene. (s-v) Inclusion levels of exons, and control -v6 in after overexpression for 72h of alone. qrt-pcr values (mean SEM, n=6) are normalized to total mrna of the corresponding gene and depicted as the x-fold change of transfection with empty vector (). p<1 and p<1 in two tailed Student s t-test relative to transfection. /: splicing ratio of exons and.

5 / a b c d ±7 ±9 ±6 ± / 2 1 e13-e14/cyc v6- / e. HMGA1 TPM2 PKM2 f g h e1-e2 / HMGA e8 / TPM2 # # # # e8- / PKM i ESRP1/ CycA ESRP1 j ESRP2 k TIA1 l RBFOX2 ESRP2 / CycA TIA1 / CycA RBFOX /CycA CTR PTB / CycA m PTB hnrnpa / CycA n hnrnp A1 o hnrnp H p MRG15 q hnrnph/cyca 3 1 MRG15 / CycA 9 3 / CycA. Supplementary Figure 3 overexpression promotes exon inclusion in mesenchymal cells. Inclusion levels of exons, constitutive e14 (a-d), alternatively spliced control -v6 and HMGA1-e2 (e-f), PTBdependent spliced PKM2- and TPM2- (g-h), splicing regulators ESRP1, ESRP2, TIA1, RBFOX2, hnrnpa1, hnrnph and PTB (i-o) and MRG15 and (p-q) after overexpression of for 72h in 2 (red) and (black) cells. qrt-pcr values (mean SEM, n=6-9) are normalized to Cyclophilin A or total mrna of the corresponding gene. p<1 and p<1 in two tailed Student s t-test relative to. /: splicing ratio of spliced exons and.

6 EZH1 + sirnas a b c / - / # # sineg# # - / # # sineg# # / # # sineg# # v6- / d. sineg e1-e2/hmga e HMGA sineg ESRP1/ CycA f ESRP1 g PTB h TIA1 i RBFOX PTB / CycA si TIA1 / CycA si RBFOX / CycA sineg sineg j / CycA sineg sineg EZH1 / CycA k EZH1 si 2 sirna l m n / - / ## # si# - / ## # si# / ## # si# v6- / o. si e1-e2 / HMGA p HMGA1 si ESRP1 / CycA q ESRP1 r PTB s TIA1 t RBFOX2 u PTB / CycA. si TIA1 / CycA si RBFOX / CycA sineg sineg / CycA si Supplementary Figure 4 or downregulation reduces exon inclusion in epithelial cells. Expression levels of exons,, control -v6 and HMGA1-e2, splicing regulators ESRP1, PTB, TIA1 and RBFOX2 and EZH1 and or after downregulation for 72h of EZH1 and (a-k) or (l-u) in 2 (red) and (black) cells. qrt-pcr values (mean SEM, n=6) are normalized to total mrna of the corresponding gene and depicted as the x-fold change of transfection with a negative control sirna. p<5 and p<1 in two tailed Student s t-test relative to negative sirna. /: splicing ratio of exons and.

7 ChIP - +PHF1 K27me3 / H3 / H3 a c.1 K27me3 / H3 / H3 b d.1 HMGA1 i2 e3 e5 i2 e3 e5 K36me3 / H3 e K36me3 / H3 f i2 e3 e5 K36me2 / H3 MRG15 / H3 g i K36me2 / H3 MRG15 / H3 h j HMGA1 i2 e3 e5 i2 e3 e5 Supplementary Figure 5 and induce changes in chromatin. Enrichment levels of H3K27me3,, H3K36me3, H3K36me2 and MRG15 along and control HMGA1 loci after combined expression of +PHF1 (a-f) or (g-j) for 72h in s. Note that the amplitude of reduction in H3K36me2 and K36me3 levels are comparable to the fold increase in H3K27me3 levels. ChIP values (mean SEM, n=3-4) are normalized to control H3. p<5 and p<1 in two tailed Student s t-test relative to.

8 rk4me3 / H3 mk4me3 / H3 Ser5 Pol / Pol II ChIP a b c.1 9.2±3 15.3±4 i6 i1 13.8±3 1±3 i6 i1 5.2±.1 5.1± H1 i6 2 no Ab MSC no Ab d. Conservation of as and RACE clones in 2 and MCF7 e. Mapping of as by Northern blot locus as GAPDH sense() MCF7 2 # as(e7) as(e7) MCF7 2 # as(-) as(-) MCF7 2 intron 8 as(-) sense() as(-) MCF7 2 # as() as() MCF7 2 5 RACE 5 RACE MCF7 2 mrna/ f. qrt-pcr g. Strand-specific RT MCF7 2 PRC3 CRL x-fold No primer RT RACE as (5RACE) e4-e5 (e4-e5) MCF7 2 h. Strand-specific and qrt-pcr Spliced HMGA1 (relative to TBP) nucl cytopl Nuclear RNA Cytopl. RNA Unspliced CycA (relative to TBP) nucl cytopl Nuclear RNA Cytopl. x-fold No primer RT RNA cyca as (5RACE) (e4-e5) nuclear cytoplasm nuclear cytoplasm Epithelial-to-mesenchymal transition sirna anti-ptb in Control sirna PTB sirna i CDH1 / TBP j k 3 Fibronectin/TBP E-cadherin/ TBP E-cadherin Fibronectin / / FBN / TBP l x-fold No primer RT ssrt UTR/no primer e4-5/no primer as CTL (5RACE) (e4-5) T T6 m n e e15-e16 PTB PTBP1 PTB Supplementary Figure 6 Expression of a nuclear lncrna antisense to when exon is included. (a-c) Enrichment levels of H3K4me3, using two different antibodies, and the transcription initiation complex RNA polymerase II phosphorylated in serine 5 along locus in 2 (red) and (black) cells. No antibody control is shown for 2 (yellow) and (grey) cells. ChIP values (mean SE, n=3) are normalized to H3 and total RNA Pol II, respectively. The transcription start site of H1 is used as a positive control in ser5 Pol II ChIP. p<5 and p<1 in two tailed Student s t-test relative to s. (d)

9 UCSC browser mapping of locus (blue), as transcription start site (5 RACE, orange) and end (3 RACE, orange) and the full sequence of as (red). A human EST (CV ) antisense to that shares 99.4% identity with as and three mouse ESTs (BI , BG , BG ) with 89%, 91.4% and 89% homology, respectively, are shown as an evidence of evolutionary conservation of the lncrna. Conservation values of alternative splicing region and as with 46 vertebrate species are depicted in green. (e) Representative Northern blots (n=4) in MCF7, 2 and cells detecting different regions of as. The lncrna was detected with probes complementary to the region identified by 5 RACE, antisense and antisense intron. Probes complementary to antisense exon, antisense and sense intron were used to verify that the detected band is not an artifact nor corresponds to sense RNA. As a loading control, we used a probe complementary to sense GAPDH (lower panel). The GAPDH panel corresponding to as(-) and 5 RACE loading control comes from the same gel. A band of 1 kbp was detected in MCF7 and 2 cells, but not s, and it spans the 5 RACE region, antisense and stops along intron, before reaching antisense exon. A scheme of loci and the position of the probes used (colored arrows) is represented on top of the Northern blots for better comprehension. (f) Inclusion levels of exon and in the cell lines used for Northern blot. MCF7 and 2 include, CRL and include and is not expressed in PRC3. qrt-pcr values (mean SEM, n=3) are normalized to total mrna levels. (g) Strand-specific RT-PCR for detecting as in MCF7 (dark red), 2 (red) and (black) cells. qrt-pcr values (mean SEM, n=6) are depicted as the x-fold change relative to an RT with no primer. The as is detected by amplification of the 5 RACE- region. As a negative control, we amplified a region upstream to the cdna retrotranscribed by the strand-specific primer ( e4-e5). (h) Nuclear and cytoplasmatic RNA was extracted and retrotranscribed for detection of as by strand-specific qrt-pcr. qrt-pcr values (mean SEM, n=5) are normalized to TBP or depicted as the x-fold change of an RT with no primer. Spliced HMGA1 (exons e4-e5) cdna and unspliced Cyclophilin A (intron i1) pre-cdna are used to detect cytoplasmatic and nuclear RNA, respectively. (i-l) Expression levels of the EMT-markers E-cadherin and Fibronectin, exons and, and as upon induction for 6 days (T6) of the epithelial-to-mesenchymal transition in MCF1a-Snail-ER cells. qrt-pcr values (mean SEM, n=4) are normalized to TBP or total. Strand-specific RT is normalized to no primer RT. p<1 in two tailed Student s t-test relative to T. (m-n) Inclusion levels of exons, and constitutive e15 after downregulation of PTB in cells (grey) for 72h. The expression levels of PTB are shown as control. qrt-pcr values (mean SEM, n=5) are normalized to or Cyclophilin A and depicted as percentage of inclusion or the x-fold change of transfection with negative sirna, respectively. p<5 and p<1 in two tailed Student s t-test relative to negative control sirna.

10 a e b c sineg si si PTB sineg si si f 2.5 ESRP1 sineg si sineg si si sineg si si g E14 / CycA ESRP2 5.E-3 4.E-3 3.E-3 2.E-3 1.E-3.E+ si sineg si si h RBFOX sineg si si i d v6 sineg si si TIA sineg si si j Control sirna anti-as oligo hnrnp H sineg si si k hnrnp A1 1 2 si l 1 m n sineg si si 2.5 sineg si si E14 / CycA E-3 E-3 E-3 5.E-4 E+ sineg si si o 3 1 v6 sineg si si Control sirna sirna / p q r s t ±4 ±2 ±9 ± as as as as / as as as e13-e14 / CycA as as as as as lncrna/cyca u.1 as pebb as asfgfr as ESRP1/CycA v ESRP1 w ESRP2 x TIA1 y RBFOX2 z PTB as as as ESRP2/CycA as as as TIA1 / CycA as as as RBFOX/CycA as as as PTB / CycA as as as 2 - / CycA a FGFR1- b FGFR1- c FGFR1 d FGFR3- e FGFR3- f FGFR as as as -e11/cyca as as as e17-e19/cyca as as as - / CycA as - / CycA as as as as as e13-e14/cyca as as as Supplementary Figure 7 as lncrna specifically affects splicing. (a-k) Expression levels of exons,, control -v6, constitutive -e14 and splicing regulators ESRP1, ESRP2, TIA1, RBFOX2, hnrnpa1, hnrnph and PTB after downregulation of as with LNA-modified antisense DNA oligos against antisense exon and the 5 UTR of as in 2 cells for 72h (si). qrt-pcr values (mean SEM, n=8) are normalized to Cyclophilin A, total or as percentage of inclusion. (l-o) Inclusion levels of exons,, control

11 -v6 and constitutive -e14 after treatment of 2 cells with an sirna against antisense exon for 72h (si). qrt-pcr values (mean SEM, n=3) are normalized to Cyclophilin, total or as percentage of inclusion. p<5 and p<1 in two tailed Student s t-test relative to negative sirna. (p-f ) Expression levels of exons, and constitutive e14 (p-s), control -v6 (t), as (u), splicing regulators ESRP1, ESRP2, TIA1, RBFOX2, hnrnpa1, hnrnph, PTB (v-z) and homologs FGFR1 exons, and total (a -c ) and FGFR3 exons, and total (d -f ) after overexpression of as for 72h in 2 (red) and (black) cells. Despite the sequence complementarity with exons and, as does not affect the tissue-specific splicing of FGFR1 and FGFR3. qrt-pcr values (mean SEM, n=3-9) are normalized to Cyclophilin A or total or as percentage of inclusion. p<1 and p<1 in two tailed Student s t-test relative to transfection. /: splicing ratio of exons and.

12 ChIP and UV-RIP - SUZ12 (% input) a.1 i13 e15 b.1 RING1B (% input) c.1 d # # # #.1# # p15 (% input) e.1 i13 e15 f.1 KDM2b (% input) g h MRG15 (% input) i.1 i13 e15 j.1 % input k utr SUZ SUZ12 PTENpg SUZ12 5utr RING RING PTENpg RING as no Ab as no Ab UV-RIP: α-suz12 α-ring1b l. RNAse A digestion - 2 m. qrt-pcr - Control sirna sirna RNAse A / RNAse A + (x- fold change) as (5UTR) () () PTENpg1 / CycA sineg + as +asr as RACE- / CycA sineg + as as +asr as Supplementary Figure 8 as lncrna regulates splicing via recruitment of PRC2 and to the locus. (a-f) Levels of SUZ12,, MRG15, RING1B and KDM2b along and control loci after ectopic expression of as in (grey). ChIP values (mean SEM, n=3-6) are depicted as the percentage of input. No antibody ChIP is used as negative control in (brown) and as (light brown) expressing cells. The transcription start site of p15 is used as a positive control in RING1B ChIPs. p<5 and p<1 in two tailed Student s t-test relative to (black). (k) UV crosslinked RNA

13 immunoprecipitation of as (5 UTR) to SUZ12 or RING1B in cells ectopically expressing as (grey) or control (black). Exon of pre-mrna and PTENpg1 asrna are used as negative controls. RIP values (mean SEM, n=2) are depicted as the percentage of input. (l) RNA expression levels of as 5 UTR, exons and pre-mrna and, as a positive control, the region of PTENpg1 RNA known to interact with a divergent antisense RNA after treatment with or without RNAse A for detection of double stranded RNA in 2 cells. qrt-pcr values (mean SEM, n=2) are depicted as the fold change in RNA levels in untreated (RNAse A -) relative to treated (RNAse A +) nuclear RNAs. Contrary to PTENpg1, neither pre-mrna nor as lncrna are protected upon RNAse A digestion, suggesting that there are no RNA:RNA hybrids. (m) Expression levels of and as after combined ovexpression of as and downregulation of for 72h in cells. qrt-pcr values (mean SEM, n=6) are normalized to Cyclophilin A and depicted as the x-fold change to transfection with a negative control sirna and.

14 Assay PrimerName FOR REV Product(bp) Ta( o C) RT<PCR h%(e1)e2) GACAAGTTTTGGTGGCACG CACGTGGAATACACCTGCAA 15 h.v6%(v6)) ATCAAGCAGGAAGAAGGATGGATA TGAGAATTACTCTGCTGCGTTGTC 139 hhmga1%(e4)e5) CCCAGCGAAGTGCCAACACCTAAG GCCCTCCTCTTCCTCCTTCTCCAGT 15 hhmga1.e2%(e1)e2) GACTCCGAGCCGGGGCTATTTCTG CGGTGCTGGGCGCTGAGGAC 111 h.e14%(e13)e14) ATGACATTAACCGTGTTCCTGAG ATCTCTGGCGAGTCCAAAGTCT 188 h.e15%(e15)e16) GCTCCAGAAGCCCTGTTTGATAGA GGGCGAGCCCCCTAAAGTG 99 h.%()) AAGTGCTGGCTCTGTTCAATGT GTAGTCTGGGGAAGCTGTAATCTC 161 h.%()) TGAGGACGCTGGGGAATATACG TAGTCTGGGGAAGCTGTAATCTCCT 123 htpm2.e6%(e5)e6) CTGGAGCGCTCGGAGGAGAG TGAGGCCATCAGGGACTTGAGG 15 htpm2.%()e8) GGAGGCCCAGGCGGACAA GCCACAGACCTCTCGGCAAACTC 1 hpkm2.e1%(e1)e11) TGCCGTGGAGGCCTCCTTCAAGT GGGGCACGTGGGCGGTATCTG 12 hpkm2.%(e8)) GGAGAAACAGCCAAAGGGGACTAT GAGGCTCGCACAAGTTCTTCAAAC 119 hcyclophilin%a GTCAACCCCACCGTGTTCTT CTGCTGTCTTTGGGACCTTGT 97 hcyca%(i1))unspliced CCCCACCCCACCTATGAGTGTAGT ACCCCTCCATTCTCATCAAGACCT 149 hfgfr1%()) TGACCCGCAGCCGCACAT GGGTCAGCACCTCCGCATCC 134 hfgfr1%()e11) ACGTGCTTGGCGGGTAACTCTA CTCTTCTTGGTACCACTCTTCATCTTG hfgfr1(e17)e19) TCGCACGGGACATTCACCACAT CAGGAGCACCCCGAAAGACCAC 1 hfgfr3%()) TGACGCACAGCCCCACATCCAG CGCACGTCGGCCTCCACACTC 129 hfgfr3%()) GGCGGGCGCTAACACCAC CACACTGCCCGCCTCGTCA hfgfr3%(e13)e14) TCGGGAAACACAAAAACATCATCAACC GCCGCGCCCGCAGAAACT hesrp1 AATATGCACAGAATGCGTTGAGGAAG ATGAGAGGGGCCGAGGAGAATC 124 hesrp2 TGTATAAAGCGACAGGGGAGGAGT AGCCGCAGGATCACTTGGTCT hrbfox2 ACAGCACGTGTAATGACCAATAAGAAG TAGGGTAAGGGAAGCCAGGAATG 217 hnrnp%a1 ACGTTCGTCAGCTTGCTCCTTTCT TAGCTGCATCCACCTCCTCCACA 292 hnrnp%h CAGCAGCTGAGTGGGGGTTAC TGCTGTTCACTGCTCCTTGGTTAC 135 hptb%(ptbp1) ACGTCACGGAGGGGGAAGTCATCT GTTGGCAGCCTCCTCCGTGTTCAT 119 htia1 TAGCCAGATTGGACCTTGTAAAA ATCTTCCGTCCATTCATAGCAG 132 hezh1 CGCGGAAAGGTCTATGACAAATAC TTGGCATAACAGTTGGGATTCAC 134 h CCCGCTGAGGATGTGGATACT ACAAGGCTGCCGTGGATGAT 15 hphf1 GCTTCTCCTGCTCCCACCTCTG CTCCCTAGCACTGTCCACCTTTTTG 123 hsuz12 CTTTTAGTCGCAACGGACCAGTTA TTTGCTGTTCTACTTCCCCATCTTC 126 hcbx8 GGCCGAAGCCCTCCTGAAG GCGAGCAAGCGAGCATCCA 129 h GCATAACCAACCGTTCCCACCTAA CATCCCCATTCCCAGACTCCAAC 96 hkdm2b GACCCAGCGCTCCCACCTCAC CCTCGCCCTCCTCGTCCTTCTC 17 hhp1g TCGACGTGTAGTGAATGGGAAAGT CGCTTCAATCAATTCTGGACAATC 115 hago1 AGCGCTCTGCCGTTTTTC ACCCGCACAGTAGCACAGTATC 148 hago2 GCCAGAGAAGTGCCCGAGGAGAGT ATCGGAAGGGGCATGGCTGTGTAT 138 hpcsk6 (e3-e4) GACCCGCAGGCCCTTTACTTCA CACGCTGCCTGGACATTCATTTC 11 hpcsk6.ase (e17-18) AATCCACCCCAGGCTCTGCTAATA TCTTGACACTCCCCAGGCTGAAGT 132 hdicer1.rt TCCTCGCATTTTGGGACTAACTG GTCCACAATCCACCACAATCTCAC 179 PTENpg1 asrna GGCTGCACAGTTAGAAAAGACGAA CGGCCTGAAGCCCCTCTC Assay PrimerName FOR REV Product(bp) Ta( o C) Strand<specificRT GSP_as GTTAACACCACGGACAAAGAG as%(5race)) TAAAAGGGGCCATTTCTGATAACA ACTCTGCATGGTTGACAGTTCTG 124 CTL:%%(e4)e5) CCGGAGATGATGAGGATGACAC TGGGCAGCGAAACTTGACAG 152 Assay PrimerName FOR REV Product(bp) Ta( o C) 5'RACE R_5.2% CACCACGGACAAAGAGATTGAGGTT R_5.1% CCTCCACAATCATTCCTGTGTCGT 5'%RACE%Outer%Primer% GCTGATGGCGATGAATGAACACTG 5'%RACE%Inner%Primer CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG 3'RACE polya F_3.1 GCAGAACTGTCAACCATGCAGA F_3.2 AGAATTACCCGCCAAGCACGTA F_3.3 AAGGCTAGACGACACAGGAATG F_3.4 ACAAAGCCCACAACCGAGAG polyt)adapter% CCAGTGAGCAGAGTGACG RT)polyT CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT!

15 Assay PrimerName FOR REV Product(bp) Ta( o C) ChIP H1 GGCGAGCTCTCCCGACACC ATCTGCTCCTGAGCCCCTGACTAC 11 p15ink4b CATGATTCTCGGGATTTTTCTCTA CAGGCAGTACTTGGGCTTCAC 122. TTCCCTGCTGAGGCTATTAGTCTG CCCACTTGTTTCCCTACTCTCC 115. AGTCACCCTCCGCTTTCCCTCCA CTTCGCCACTTGTCCCGCACAT 111.v5 TCACCTGAACAGGAATGGATACAA AGGGCCTCTGCTACAATCTCACTA 128. TGGAATGGTGCTATGTGGCTTAC AGAAATGAAGGGAAAATCAGGTTG 12. TCCTTTGCCCCAGCTTGCCTAA TGGGAAGAATCTTGCTGCCTGATG 121 HMGA1. CCCCGCCCCCTGAGTGACAC CCGCGCCAGCGCCAGAAATA HMGA1.i2 GGAGCAGGATAAATCACCAACCA GCATCAAGAGACCCTCCAGACAG 18 HMGA1.e3 GGGCAGACCCCTCCATCC CATCTCGCTTCAGCCACTTTCTAC 1 HMGA1.e5 AACCCTCTAGAAAACCACCACAAC ACTCAACACCCTCAACTCAAAAGA 122 HMGA1.3UTR CCAGGATTCCCCCAGCCAAACT CACCCCTCCTGCCTTCCTGTAACC 126. GGCAACCCTCCCCGCAGTATCAAG GCTGGCCAACGGCTCGCTGAG 139.i6 TATCTTAAAACGCACCCACACTAC CAAGGTCACAAACTATGCTCCTAT 162. TGTTATTTCAAAGGTGTCAGCCA GAGAGGGAAGAAAGGAGGAGTG 131. GTGTTGATTGTTACTCTGATGTTGTT TATTGTCTAAATCACCTCTGAATGG 159.e AGCCCTTTAATGCCGCTGTTTAGA TAACGGCCAACCAGGAAGGTCTTAG TGATCCTTATGAGTTGCTGTTCTTG TTGCCTTTAGTAGCGTCCAGTAGTA 93.e GAATATACGTGCTTGGCGGGTAAT TAAAAGGGGCCATTTCTGATAACA 166. GGAGCGGCACCTCTGAATGTCA CAAGCCCAAGATGGCAGGAGCAT CCTGGGTCCTGGTGGTCAAAT TAAGCAGGCCATAGAGTTAGCACAC FGFR1.i1 GGAATGAAAAGCCCCAGAAA GCCTCATAAATGCCTAGAAACAAC 134.i13 TGTGGCTGGACAACTGGAGGTA AAAGAAAACGCAGTGTGGCTCTAA 175.e14 TTCCTTTTTGTTCTGGCGGTGTT CTCTGGCGAGTCCAAAGTCTGCTA 148.e15 AGGGGCGGCTTCCAGTCAAGT CAAGCCCAGGAAAAAGCCAGAGAA 112.i15 TCCCAGAGATTGAGCCTCCTGAAC AATCACATGCCCCAGATGAGTTGC 128.3UTR CCGTGCGTACTGGCTGTG CTGCATTTGTGCTCTGTAAGTGTG 121! Supplementary Table 1. List of oligonucleotide pair sequences used in real-time qpcr. Product: product length in bp. Ta: recommended annealing temperature. The combination of spliced exons detected by qrt-pcr are specified in brackets.!!!

16 as (875 nt) gggatgggctaatttatacattggctcaatgactcactaaaaatatgagatcccttcaggttttaagggtgaaatttcccttgatcataaatgtgagtgtgggatctc actgtgtgtgaatttccaaggcagttttcttatccctgagggatcatttttaacattttttatatctttatgcaaggataaaaggggccatttctgataacagaagctgtgt taattttatagcagtcaaccaagaaaagggaaaaaaacccagagagaaagaacagtatatacctggcagaactgtcaaccatgcagagtgaaaggatat cccaatagaattacccgccaagcacgtatattccccagcgtcctcaaaagttacattccgaatatagagaacctcaatctctttgtccgtggtgttaacaccggc ggcctagaaaacaagggaagcaaaagaaaaggctagacgacacaggaatgattgtggagggggctgtggaaccacaaggcgtcgcaccgggggcttc agggggtgctggccactgggagattccgactgcagcccatccacaaagcccacaaccgagagacacggagcaacactgaccagctcacctccacaggc ccgtcaccacaccggcttcacctcctacacatgcacgcaatcaaacgcatggaaaaaatcaacccacagaggtccgttctcttggcctgtgctttggtttgcattt aggggtggttttccaaaagtatggtgcccagaaggccaagctggttctggtcctgttggctgcagactcccaccaagaggaaagggtagttctatctaaattgct gtgttttaagacaacacactgcacataagcccagatggacacctc Supplementary Table 2. as full sequence according to 5 /3 RACE and validated by Northern blot and qrt-pcr. 5 RACE detected a single start point 282 bp upstream exon in the antisense direction.!