supporting information - proceedings of the national ... · pdf filesupporting information ......

8
Supporting Information Pelisch et al. 10.1073/pnas.1004653107 SI Materials and Methods Plasmids. The expression vectors used were: pcDNA3.1-HA- SUMO1 and pcDNA3.1-HA-SUMO3 (1, 2), pcDNA3.1-FLAG- HA-SENP1 (3, 4), pcDNA3-HA-PIAS1 and pCDNA3-FLAG- PIAS1 (5), GFP-SUMO and GFP-SUMO(GA) (6), Myc-Topo I and GST-Topors (7), HA-Sam68 (8). Immunouorescence. Fixed and permeabilized cells were blocked with 3% BSA in PBS. Afterward, cells were incubated with the primary antibody for 1 h in blocking buffer. After washing with PBS, cells were incubated with Alexa 637-conjugated secondary antibody for another hour. Cells were extensively washed with PBS and mounted. Immunoprecipitation. For coimmunoprecipitation experiments, 30 μL of anti-T7-agarose beads (Novagen) or 1 μg of anti-HA (Covance) plus 30 μL of Protein A/G Plus agarose (Santa Cruz Biotechnology) were used. After incubation for 1 h at 4 °C, beads were pelleted, and washed three times with lysis buffer and once with PBS. Immunoprecipitates were resuspended in 2× Laemmli sample buffer. SUMO Transfer Reactions. Recombinant E1 (150 ng), E2 (300 ng), and SUMO1 (small ubiquitin-related modier-1) (200 ng) were incubated for 30 min at 30 °C in sumoylation assay buffer (9) supplemented with 0.5 mM ATP. Reactions were then diluted 3- fold in the same buffer but lacking ATP and containing EDTA (10 mM nal concentration) to prevent further loading of the E2 by inhibiting the Mg 2+ -dependent activation by the E1. The SUMO-loaded E2 was then incubated with GST-p53 (200 ng) either with or without 200 ng GST-SF2/ASF for the indicated time points. Reactions were stopped by addition of an equal volume of 2× Laemmli sample buffer. Western Blot and Antibodies. The antibodies used were: anti-SF2/ ASF (mAb 103 hybridome culture supernatant, provided by Adrián Krainer, Cold Spring Harbor Laboratory, NY), anti-HA (Co- vance), anti-T7 (Bethyl Laboratories and Novagen), anti-T7 coupled to agarose beads (Novagen), anti-FLAG (Sigma), anti- Myc, anti-β-actin, anti-p53, anti-GFP, and anti-Sam68 (Santa Cruz Biotechnology), anti-SUMO1 (Zymed/Invitrogen), anti- SUMO2/3 (IMGENEX), anti-Ubc9 (BD Biosciences), anti-Topo I (LAE Biotech), anti-GST (Abcam), anti-GAPDH (Abcam), and anti-Nop58 (Human Protein Atlas). SI ACKNOWLEDGMENTS. The authors thank M. de la Mata and M. Blaustein for helpful discussions and suggestions; V. Buggiano and A. Turjanski for technical help; A. Kornblihtt forcritical reading of the manuscript and support; R. Hay (Dundee, UK), D. Wotton (Charlottesville, NC), S. Weger (Berlin, Germany), J. Palvimo (Kuopio, Finland), C. Sette (Rome, Italy), J. Manley (New York, NY), F. Fuller-Pace (Dundee, UK), M. Dasso (Bethesda, MD), A. Mayeda (Aichi, Japan), R. Fisher (New York, NY), H. Ulrich (Herts, UK), A. Pichler (Vienna, Austria), F. Melchior (Heidelberg, Germany), P. OHare (London, UK), E. Wahle (Halle, Germany), F. Hirose (Hyogo, Japan), T.R. Reddy (Detroit, MI), C. Abate- Shen (New York, NY), H. Ariga (Sapporo, Japan), B. Chabot (Sherbrooke, Quebec, Canada), M. Carmo-Fonseca (Lisbon, Portugal), D. Fujita (Calgary, Canada), G. Biamonti (Pavia, Italy) and J. Cáceres (Edinburgh, UK) for providing plasmids, A. Krainer (New York, NY) for antibodies and C. Lima (New York, NY) for protocols. F.P., J.G., I.E.S., and M.J.M. are postdoctoral fellows from Consejo Nacional de Investigaciones Cientícas y Tecnicas of Argentina. J.D. and G.R. are doctoral fellows from Consejo Na- cional de Investigaciones Cientícas y Tecnicas of Argentina. E.P. is a doctoral fellow of the University of Buenos Aires. B.J.W. is supported by an EU Marie-Curie International Incoming Fellow- ship. A.I.L. is a Wellcome Trust Principal Research Fellow; E.A. and A.S. are career investigators of Consejo Nacional de Inves- tigaciones Cientícas y Tecnicas of Argentina. A.I.L. and A.S. are members of the European Alternative Splicing Network. 1. Desterro JM, et al. (1998) SUMO-1 modication of IkappaBalpha inhibits NF-kappaB activation. Mol Cell 2:233239. 2. Tatham MH, et al. (2001) Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. J Biol Chem 276:3536835374. 3. Bailey D, O'Hare P (2002) Herpes simplex virus 1 ICP0 co-localizes with a SUMO-specic protease. J Gen Virol 83:29512964. 4. Bailey D, O'Hare P (2004) Characterization of the localization and proteolytic activity of the SUMO-specic protease, SENP1. J Biol Chem 279:692703. 5. Lee H, et al. (2006) PIAS1 confers DNA-binding specicity on the Msx1 homeoprotein. Genes Dev 20:784794. 6. Poukka H, et al. (2000) Covalent modication of the androgen receptor by small ubiquitin-like modier 1 (SUMO-1). Proc Natl Acad Sci USA 97:1414514150. 7. Hammer E, et al. (2007) The E3 ligase Topors induces the accumulation of polysumoylated forms of DNA topoisomerase I in vitro and in vivo. FEBS Lett 581: 54185424. 8. Babic I, et al. (2006) SUMO modication of Sam68 enhances its ability to repress cyclin D1 expression and inhibits its ability to induce apoptosis. Oncogene 25: 49554964. 9. Pichler A, Knipscheer P, Saitoh H, Sixma TK, Melchior F (2004) The RanBP2 SUMO E3 ligase is neither HECT- nor RING-type. Nat Struct Mol Biol 11:984991. Pelisch et al. www.pnas.org/cgi/content/short/1004653107 1 of 8

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Page 1: Supporting Information - Proceedings of the National ... · PDF fileSupporting Information ... Splicing reporter I : Fibronectin EDI exon Pre-mRN A mRN A + EDI - EDI ... minigene shown

Supporting InformationPelisch et al. 10.1073/pnas.1004653107SI Materials and MethodsPlasmids. The expression vectors used were: pcDNA3.1-HA-SUMO1 and pcDNA3.1-HA-SUMO3 (1, 2), pcDNA3.1-FLAG-HA-SENP1 (3, 4), pcDNA3-HA-PIAS1 and pCDNA3-FLAG-PIAS1 (5), GFP-SUMO and GFP-SUMO(GA) (6), Myc-Topo Iand GST-Topors (7), HA-Sam68 (8).

Immunofluorescence. Fixed and permeabilized cells were blockedwith 3% BSA in PBS. Afterward, cells were incubated with theprimary antibody for 1 h in blocking buffer. After washing withPBS, cells were incubated with Alexa 637-conjugated secondaryantibody for another hour. Cells were extensively washed withPBS and mounted.

Immunoprecipitation. For coimmunoprecipitation experiments,30 μL of anti-T7-agarose beads (Novagen) or 1 μg of anti-HA(Covance) plus 30 μL of Protein A/G Plus agarose (Santa CruzBiotechnology) were used. After incubation for 1 h at 4 °C, beadswere pelleted, and washed three times with lysis buffer and oncewith PBS. Immunoprecipitates were resuspended in 2× Laemmlisample buffer.

SUMO Transfer Reactions. Recombinant E1 (150 ng), E2 (300 ng),and SUMO1 (small ubiquitin-related modifier-1) (200 ng) wereincubated for 30 min at 30 °C in sumoylation assay buffer (9)supplemented with 0.5 mM ATP. Reactions were then diluted 3-fold in the same buffer but lacking ATP and containing EDTA(10 mM final concentration) to prevent further loading of the E2by inhibiting the Mg2+-dependent activation by the E1. TheSUMO-loaded E2 was then incubated with GST-p53 (200 ng)either with or without 200 ng GST-SF2/ASF for the indicatedtime points. Reactions were stopped by addition of an equalvolume of 2× Laemmli sample buffer.

Western Blot and Antibodies. The antibodies used were: anti-SF2/ASF(mAb103hybridomeculturesupernatant, providedbyAdrián

Krainer, Cold Spring Harbor Laboratory, NY), anti-HA (Co-vance), anti-T7 (Bethyl Laboratories and Novagen), anti-T7coupled to agarose beads (Novagen), anti-FLAG (Sigma), anti-Myc, anti-β-actin, anti-p53, anti-GFP, and anti-Sam68 (SantaCruz Biotechnology), anti-SUMO1 (Zymed/Invitrogen), anti-SUMO2/3 (IMGENEX), anti-Ubc9 (BD Biosciences), anti-TopoI (LAEBiotech), anti-GST (Abcam), anti-GAPDH (Abcam), andanti-Nop58 (Human Protein Atlas).

SI ACKNOWLEDGMENTS. The authors thank M. de la Mata andM. Blaustein for helpful discussions and suggestions; V. BuggianoandA.Turjanski fortechnicalhelp;A.Kornblihtt forcriticalreadingof the manuscript and support; R. Hay (Dundee, UK), D.Wotton(Charlottesville, NC), S. Weger (Berlin, Germany), J. Palvimo(Kuopio, Finland), C. Sette (Rome, Italy), J. Manley (New York,NY), F. Fuller-Pace (Dundee, UK), M. Dasso (Bethesda, MD),A. Mayeda (Aichi, Japan), R. Fisher (New York, NY), H. Ulrich(Herts,UK),A.Pichler(Vienna,Austria),F.Melchior(Heidelberg,Germany), P. O’Hare (London, UK), E.Wahle (Halle, Germany),F. Hirose (Hyogo, Japan), T.R. Reddy (Detroit, MI), C. Abate-Shen (New York, NY), H. Ariga (Sapporo, Japan), B. Chabot(Sherbrooke, Quebec, Canada), M. Carmo-Fonseca (Lisbon,Portugal), D. Fujita (Calgary, Canada), G. Biamonti (Pavia, Italy)and J. Cáceres (Edinburgh,UK) for providing plasmids,A.Krainer(New York, NY) for antibodies and C. Lima (New York, NY) forprotocols. F.P., J.G., I.E.S., and M.J.M. are postdoctoral fellowsfromConsejoNacional de InvestigacionesCientíficas y Tecnicas ofArgentina. J.D. and G.R. are doctoral fellows from Consejo Na-cional de Investigaciones Científicas y Tecnicas of Argentina. E.P.is a doctoral fellow of the University of Buenos Aires. B.J.W. issupported by an EU Marie-Curie International Incoming Fellow-ship. A.I.L. is a Wellcome Trust Principal Research Fellow; E.A.and A.S. are career investigators of Consejo Nacional de Inves-tigaciones Científicas y Tecnicas of Argentina. A.I.L. and A.S. aremembers of the European Alternative Splicing Network.

1. Desterro JM, et al. (1998) SUMO-1 modification of IkappaBalpha inhibits NF-kappaBactivation. Mol Cell 2:233–239.

2. Tatham MH, et al. (2001) Polymeric chains of SUMO-2 and SUMO-3 are conjugated toprotein substrates by SAE1/SAE2 and Ubc9. J Biol Chem 276:35368–35374.

3. Bailey D, O'Hare P (2002) Herpes simplex virus 1 ICP0 co-localizes with a SUMO-specificprotease. J Gen Virol 83:2951–2964.

4. Bailey D, O'Hare P (2004) Characterization of the localization and proteolytic activity ofthe SUMO-specific protease, SENP1. J Biol Chem 279:692–703.

5. Lee H, et al. (2006) PIAS1 confers DNA-binding specificity on the Msx1 homeoprotein.Genes Dev 20:784–794.

6. Poukka H, et al. (2000) Covalent modification of the androgen receptor by smallubiquitin-like modifier 1 (SUMO-1). Proc Natl Acad Sci USA 97:14145–14150.

7. Hammer E, et al. (2007) The E3 ligase Topors induces the accumulation ofpolysumoylated forms of DNA topoisomerase I in vitro and in vivo. FEBS Lett 581:5418–5424.

8. Babic I, et al. (2006) SUMO modification of Sam68 enhances its ability to represscyclin D1 expression and inhibits its ability to induce apoptosis. Oncogene 25:4955–4964.

9. Pichler A, Knipscheer P, Saitoh H, Sixma TK, Melchior F (2004) The RanBP2 SUMO E3ligase is neither HECT- nor RING-type. Nat Struct Mol Biol 11:984–991.

Pelisch et al. www.pnas.org/cgi/content/short/1004653107 1 of 8

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PIAS1/SENP1

-- - +

250

150

100

FLAG-SENP1:T7-SF2/ASF:

FLAG-PIAS1:HA-SUMO1:

-+ - +

-++ - +

--

++ +

++ - +

+++ - +

+ -

++ +

75

WB: HA

WB: FLAG

50

WB: -actin

SUMO1 conjugates

-actin

SF2/ASF 37

WB: T7

1 2 3 4 5 6 7

B

WB: -actin

WB: SF2/ASF

SF2/ASF

-actin

SUMO1 conjugates

+ - +

- + +

:siRNA Ctl :siRNA SF2/ASF :HA-SUMO1

A

WB: HA

WB: SF2/ASF

Fig. S1. (A) HEK 293T cells were transfected with HA-SUMO1, FLAG-PIAS1 (protein inhibitor of activated STAT-1) and FLAG-SENP1 (SUMO-specific proteases-1)(500 ng each, 2 μg total amount of DNA) and 100 ng (+) or 500 ng (++) of T7-SF2/ASF, as indicated. Cells were lysed 48 h post transfection in Laemmli samplebuffer and proteins were separated by SDS/PAGE and subject to Western blot as indicated at the bottom of each panel. (B) Cells were transfected with a controlsiRNA (siRNA Ctl) or with an SF2/ASF-specific siRNA (25 nM). After 24 h, cells were retransfected with HA-SUMO1. Cells were lysed 48 h posttransfection inLaemmli sample buffer, proteins were separated by SDS/PAGE, and subject to Western blot, as indicated at the bottom of each panel.

Pelisch et al. www.pnas.org/cgi/content/short/1004653107 2 of 8

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COS7

182 115

82

64 WB: HA

T7-SF2/ASF:

T7-SF2/ASF ΔRRM2:

SENP1:

HA-SUMO3:

-

-

-

+

-

-

+

+

+

-

-

+

++

-

-

+

++

-

+

+

-

+

-

+

-

++

-

+

-

++

+

+

SUMO3-conjugates

182 115

82

64 WB: HA

T7-SF2/ASF:

T7-SF2/ASF ΔRRM2:

SENP1:

HA-SUMO1:

-

-

-

+

-

-

+

+

+

-

-

+

++

-

-

+

++

-

+

+

-

+

-

+

-

++

-

+

-

++

+

+

SUMO1-conjugates

WB: HA

T7-SF2/ASF:

T7-SF2/ASF ΔRRM2:

HA-SUMO3:

-

-

+

+

-

+

-

+

+

SUMO3

conjugates

SCp2

182

115

82

A

B

WB: HA

:T7-SF2/ASF

:T7-SF2/ASF ΔRRM2

:HA-SUMO1

-

-

+

+

-

+

-

+

+

SUMO1

conjugates

182

115

82

Fig. S2. (A) COS7 cells were transfected with HA-SUMO (1 or 3) and FLAG-SENP1 as indicated (500 ng each), either with or without T7-SF2/ASF or T7-SF2/ASFΔRRM2 [250 (+) or 500 (++) ng]. Total amount of DNA was brought up to 2 μg. After 48 h, cells were lysed in 2× Laemmli sample buffer and subject to Westernblot with an anti-HA antibody. (B) Functionally normal mouse mammary epithelial cells (SCp2) were transfected with HA-SUMO (1 or 3, 500 ng), either with orwithout T7-SF2/ASF or T7-SF2/ASF ΔRRM2 (RNA-recognition motif-2) (500 ng). Total amount of DNA was brought up to 2 μg. After 48 h, cells were lysed inLaemmli sample buffer and subject to Western blot as in A.

SF2/ASF

Wild type

ΔΔRRM1

ΔRRM2

ΔRS

RRM2

Fig. S3. The scheme represents the modular structure of SF2/ASF consisting of two RRMs and one C-terminal domain rich in arginine and serine dipeptides (RSdomain), and the deletion mutants used in this study.

Pelisch et al. www.pnas.org/cgi/content/short/1004653107 3 of 8

Page 4: Supporting Information - Proceedings of the National ... · PDF fileSupporting Information ... Splicing reporter I : Fibronectin EDI exon Pre-mRN A mRN A + EDI - EDI ... minigene shown

••

A C:ltCANRis

:FSA/2FSANRis

FSA/2FS-7T :1MRR

2MRRFSA/2FS-7T

:1OMUS-AH

+

-

-

-

+

-

+

-

-

+

-

+

+

-

+

-

+

-

+

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AH:BW

1OMUS

setagujnoc

FSA/2FS:BW

:BW nitca-

FSA/2FS

nitca-7T:BW

FSA/2FS 1MRR2MRRFSA/2FS

TGCTTAGTGTGGTGGAGGGCTGTA CAACAAGGGACGCCCCGGCAATGGGTGCATCTACGCCGTTAG AGCCTACAGACCTCCATT

A TGCAGGAGTTACAGGAACC CTATCGCGGCATAAACATCTTGCGCTAAGAACTCCAGCTACAGCGC CTTCCCGCCAGGGGGCGCGCCCAGGAGCTTGAGTTGCTTCCG GCGCAGAAGGCGCAGAGCTAGTATCGGCAGCGCTGGTATGTG GTCTGCCATGGGTAGCATCGGCGAAGCTCCTTTGAGGTGGGC CGGAGCCGGACAAGGTGCAGGCGGTGGAGGTGGGGGCGGCGG TATCGCTGGAGCCCCTCGAAGTCTGGCGGACCTACCCCCCGG TCTCTGTTGGTGAGACAAGGTTGAAGGTGAACCTCCGTCAGG CACTAGGAAATTTAGGACTGTATGTAGTGGACGAAGTGCGTA AGCCATTTGTAGTCGTATTTTGAGGTGCTGTGGTCACGGTAG GTATAGAAGAAAGGCATGTAGGTCAAAAGCTTGACGTATCCA TCTAGATTTGAATCACAACTACATCCGTCAAAGAGGGAGTAC GGGTAGTTGAAATTGGGCAGAAGGTATTGAACCTGAAGACCC TGCCGAAGCTCTAGCTCTAGACGATGCCGAAGACGATGCTGA CGCTGAGGACGACAACGAAGACGAAGAGGAACCCCTCATTGA TCTTATCGCACCACTAGG

GCTCTAGACGATACTGCCCC AATACATGCTCTC

7T

7T

stcurtsnoctnatsiser-ANRisB

Δ

Δ

b

b

Fig. S4. (A) SF2/ASF cDNA showing (in red) the sequence targeted by the siRNA used to knock-down SF2/ASF expression. (B) SF2/ASF deletion constructsrefractory to SF2/ASF siRNA because of the lack of the RRM1 domain and therefore the sequence targeted by the siRNA. (C) Cells were transfected witha control siRNA (siRNA Ctl) or with an SF2/ASF-specific siRNA (40 nM). After 24 h, cells were retransfected with HA-SUMO1 (500 ng) and either one of the siRNA-resistant consturcst shown in B (300 ng). After 48 h from the second transfection, cells were lysed in Laemmli buffer and subject to Western blot analysis withthe antibodies indicated at the bottom of each panel.

A

SF2/ASF SRp30c

SRp40

B

SF2/ASF

SRp30c

SRp40

SRp20

----+

+---+

-+--+

--+-+

---++

T7-SF2/ASF:T7-SRp20:T7-SRp40:

T7-SRp30c:HA-SUMO1:

250

150

100

75

50 WB: HA

WB: T7

WB:

SUMO1 conjugates

SF2/ASFSRp30c SRp20

•-actin

SRp40

50

37

25

1 2 3 4 5

C

β

-actin β

Fig. S5. (A) Scheme of SF2/ASF and other members of the Ser/Arg-rich (SR) family of proteins used to compare their sumoylation stimulatory activity. (B)Alignment of the RRM2 domains of SF2/ASF, SRp30c, and SRp40 according to ClustalW2 (www.ebi.ac.uk/Tools/clustalw2/index.html) and BOXSHADE 3.21(www.ch.embnet.org/software/BOX_form.html) software. Identical amino acids are shown in black and similar ones in gray. (C) HEK 293T cells were transfectedwith 500 ng HA-SUMO1 and 100 ng of T7-SF2/ASF, T7-SRp20, T7-SRp40, or T7-SRp30c. Cells were lysed 48 h posttransfection in Laemmli sample buffer; proteinswere separated by SDS/PAGE and subject to Western blot as indicated at the bottom of each panel.

Pelisch et al. www.pnas.org/cgi/content/short/1004653107 4 of 8

Page 5: Supporting Information - Proceedings of the National ... · PDF fileSupporting Information ... Splicing reporter I : Fibronectin EDI exon Pre-mRN A mRN A + EDI - EDI ... minigene shown

A

D

RT-PCR: FN EDI

T7-SF2/ASF:

T7-SF2/ASF ΔΔRRM1:

T7-SF2/ASF ΔRRM2:

T7-SF2/ASF ΔRS:

T7-SF2/ASF RRM2:

-

-

-

-

-

+

-

-

-

-

-

+

-

-

-

-

-

+

-

-

-

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-

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-

-

-

+

1 2 3 4 5 6

Splicing reporter I: Fibronectin EDI exon

Pre-mRNA

mRNA

+ EDI - EDI

+ EDI

- EDI

G

(-) SF2/ASF ΔRRM1 ΔRRM2 ΔRS RRM2

**

*

T7-SF2/ASF:

T7-SF2/ASF RRM2:

-

-

+

-

-

+

+ ex. 9

- ex. 9

RT-PCR: CFTR

B

E

H

*

Splicing reporter II: CFTR exon 9

Pre-mRNA

mRNA

+ exon 9 - exon 9

T7-SF2/ASF:

T7-SF2/ASF RRM2:

-

-

+

-

-

+

13S

12S

9S

RT-PCR: E1a

C

F

Splicing reporter III: adenovirus E1a (alternative 5´ splice sites)

(-) SF2/ASF RRM2

I

**

*

*

(-) SF2/ASF RRM2

Pro

po

rtio

n o

f s

plicin

g i

so

form

Fig. S6. Three SF2/ASF alternative splicing targets (“splicing reporter minigenes”) were used to analyze the effect of over-expressing wild type SF2/ASF or itsdeletion mutants: fibronectin EDI exon (A), CFTR exon 9 (B), and adenovirus E1a (C) (1–3). (D–I) Cells were transfected with 500 ng of each splicing reporterminigene shown in A to C, and 50 ng of the indicated SF2/ASF construct. Forty-eight hours later, RNA was extracted and subject to RT-PCR as described (4).Radioactive samples were run on native 6% polyacrilamide gels, which were subsequently dried and exposed to X-ray films (Agfa).

1. Caputi M, et al. (1994) A novel bipartite splicing enhancer modulates the differential processing of the human fibronectin EDA exon. Nucleic Acids Res 22:1018–1022.2. Niksic M, Romano M, Buratti E, Pagani F, Baralle FE (1999) Functional analysis of cis-acting elements regulating the alternative splicing of human CFTR exon 9. Hum Mol Genet 8:

2339–2349.3. Cáceres JF, Stamm S, Helfman DM, Krainer AR (1994) Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors. Science 265:1706–1709.4. Cramer P, et al. (1999) Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer. Mol Cell 4:251–258.

Pelisch et al. www.pnas.org/cgi/content/short/1004653107 5 of 8

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A

B

SF2/ASF

Input GST-Ub c9

DNase/RNase: - +

20

37

WB: T7

GS T

37

GS

T

GS

T-U

bc

9

Inp

ut

SF2/ASF

WB: SF2/ASF

C

50

100

75

Mr (KDa)

37

25

15 0

T7

-SF

2/A

S F

DIn

pu

t

GS

T

WB: T7

37

GS

T-U

bc

9

SF2/ASF

Fig. S7. (A) Pull-down assay was performed essentially as in Fig. 2A. Before addition of the recombinant GST fusion protein, the lysate was divided in twoaliquots: one of them was mock treated and the other was treated with DNase and RNase. (B) T7-SF2/ASF was purified from transfected HEK 293T lysates asindicated inMaterials and Methods. An aliquot of the purified protein (∼2 μg) was analyzed by SDS/PAGE and Coomassie staining. (C) Purified recombinant T7-SF2/ASF (500 ng) was incubated with 2 μg GST or GST-Ubc9 and pulled down with glutathione Sepharose beads. SF2/ASF binding was analyzed by Westernblotting with an anti-T7 antibody. (D) GST pull-down assay was performed as in A but from nontransfected HEK 293T cell lysates. Samples were analyzed byWestern blotting with an anti-SF2/ASF antibody.

Pelisch et al. www.pnas.org/cgi/content/short/1004653107 6 of 8

Page 7: Supporting Information - Proceedings of the National ... · PDF fileSupporting Information ... Splicing reporter I : Fibronectin EDI exon Pre-mRN A mRN A + EDI - EDI ... minigene shown

-

+

+

+

75

50

37

WB: SUMO1

Sp100-SUMO1

RanBP2 FG:

GST-SF2/ASF:

GST-Sp100:

E2:

E1/SUMO1:

-

-

+

-

+

-

-

+

+

+

-

+

+

+

-

+

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-

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1 2 3 4 5 6

WB: Myc (input)

IP: T7 / WB: Myc

IP: T7 / WB: T7

Myc-Topo I:

T7-SF2/ASF:

+

-

+

+

Topo I

SF2/ASF

Topo I

100

37

100

T7-SF2/ASF:HA-p53:

WB: HA (input)

IP: T7 / WB: HA

-+

IP: T7 / WB: T7

50

50

37

p53

p53

SF2/ASF

+-

++

A

F

C D

E

1 2 3 4

p53

p53-SUMO1100

75

WB: p53

GST-SF2/ASF:

GST-p53:

E2:

E1/SUMO1:

-

+

-

+

-

+

+

+

+

+

+

+

+

+

150

100

T7-SF2/ASF: siRNA Ctl:

siRNA SF2/ASF: His-SUMO1:

-+-+

--++

++-+

WB: Nop58

Nop58

75

-+-+

--++

++-+

Ni2+ pull-downCell lysates

His-SUMO1-Nop58

T7-SF2/ASF:siRNA Ctl:

siRNA SF2/ASF:His-SUMO1:

-+-+

--++

++-+

Cell lysates

WB: -actin / SF2/ASF

SF2/ASF

-actin 50

37

WB: Topo I

G

B

100

75

150

50

GST-Topo I:

GST-SF2/ASF:

E1/E2/SUMO1:

Topo I

Topo I-SUMO1

+

-

+

+

+

+

+

Fig. S8. (A) SF2/ASF stimulates SUMO1 conjugation to Topo I in vitro. GST-Topoisomerase I (“Topo I,” 1 μg)was incubatedwith E1 (150 ng), E2 (30 ng), and SUMO1(1 μg), either with or without GST-SF2/ASF (150 ng or 300 ng) for 60 min in sumoylation assay buffer. Reactions were stopped by addition of an equal volume of 2×Laemmli sample buffer, and analyzed by SDS/PAGE followed byWesternblottingwith an anti-Topo I antibody. (B) SF2/ASF stimulates SUMO1 conjugation to p53 invitro. GST-p53 (250 ng) was incubated with E1 (150 ng), E2 (30 ng), and SUMO1 (1 μg), either with or without GST-SF2/ASF (100 ng or 200 ng) for 60 min in su-moylation assay buffer. Reactions were stopped by addition of an equal volume of 2× Laemmli sample buffer, and analyzed by SDS/PAGE, followed by Westernblotting with an anti-p53 antibody. (C) SF2/ASF interacts with Topo I in whole-cell lysates as assessed by coimmunoprecipitation. HEK 293T cells were transfectedwith Myc-Topo I and T7-SF2/ASF when indicated (500 ng each). Cells were lysed as described inMaterial and Methods and lysates were immunoprecipitated withanti-T7 agarose beads. Afterwashing, precipitated proteinswere resuspended in 2× Laemmli sample buffer and subject toWestern blot, as indicated at the bottomof each panel. (D) SF2/ASF interacts with p53 in whole-cell lysates as assessed by coimmunoprecipitation. HEK 293T cells were transfected with HA-p53 and T7-SF2/ASF as indicated, and coimmunoprecipitation assaywas performed as in C. (E) SF2/ASF does not stimulate Sp100 sumoylation. A GST-Sp100 fragment encompassingamino acids 241 to 360 (Lys-297 is the physiological SUMO1 attachment site, 200 ng) was incubated with E1 (150 ng), E2 (30 ng), and SUMO1 (1 μg), either with orwithout GST-SF2/ASF (200 ng, 500 ng, 1 μg) or GST-RanBP2ΔFG (10 ng) for 30 min in sumoylation assay buffer. Reactions were stopped by addition of an equalvolume of 2× Laemmli sample buffer, and analyzed by SDS/PAGE, followed by Western blotting with an anti-SUMO1 antibody. (F, G) SF2/ASF regulates SUMO 1conjugation to Nop58 in living cells. HEK 293T cells were transfected with the indicated siRNAs and 24 h later with the indicated plasmids. After 48 h, cells wereharvested and lysates were subject to Ni-NTA agarose purification under denaturing conditions. His-tagged sumoylated proteins were eluted in buffer containing100mM imidazole and2%SDS,mixedwith an equal volumeof 2× Laemmli sample buffer and subject toWestern blot usingNop58 antibodies (F). A fraction of eachcell lysate (3%) was run in parallel as input control. (G) Lysates were analyzed for SF2/ASF expression levels and β-actin as a control.

Pelisch et al. www.pnas.org/cgi/content/short/1004653107 7 of 8

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GST

-PIA

S1

Inpu

t (20

%)

GST

WB: T7

37 SF2/ASF

SF2/ASF RRM2

WB: HA (input)

HA-PIAS1:

T7-SF2/ASF:

T7-SF2/ASF RRM2:

75

37

26

75

IP: T7 WB: T7

IP: T7 WB: HA

+

-

-

+

-

+

+

+

-

SF2/ASF

PIAS1

PIAS1

37

26IP: HA / WB: T7

SF2/ASF

D

A

E

100

75

150

GST-p53:GST-SF2/ASF:

GST-PIAS1:E1/E2/SUMO3:

+--+

+-++

++-+

++++

WB: p53

p53

p53-SUMO3

Cell

lysates

WB: T7

T7-SF2/ASF:

T7-SF2/ASF RRM2:

T7-SF2/ASF RS:

Pull-down:

+

-

-

-

+

-

-

-

+

+

-

-

-

+

-

-

-

+

+

-

-

-

+

-

-

-

+

37

26

20

Input (20%) GST GST-PIAS1

1 2 3 4 5 6 7 8 9

SF2/ASF

SF2/ASF RRM2SF2/ASF RS

C

SF2/ASF

Input GST-PIAS1 DNase/RNase: - +

20

37

WB: T7

GST

B

/

/

Fig. S9. (A) HEK 293T cells were transfected either with wild type T7-SF2/ASF ΔRRM2, or ΔRS, and lysates were prepared as described in Materials andMethods. Cleared lysates were incubated with 2 μg GST or GST-PIAS1 and pulled-down with glutathione sepharose beads. SF2/ASF binding was analyzed byWestern blotting with an anti-T7 antibody. (B) Purified recombinant T7-SF2/ASF (500 ng) was incubated with 2 μg GST or GST-PIAS1 and pulled down withglutathione Sepharose beads. SF2/ASF binding was analyzed by Western blotting with an anti-T7 antibody. (C) Pull-down assay was performed essentially asin A. Before addition of the recombinant GST fusion protein, the lysate was divided in two aliquots: one of them was mock-treated and the other was treatedwith DNase and RNase. (D) SF2/ASF interacts with PIAS1 in whole-cell lysates as assessed by coimmunoprecipitation. HEK 293T cells were transfected with HA-PIAS1 alone or with either wild-type T7-SF2/ASF or its ΔRRM2 mutant. Cells were lysed as described in Materials and Methods and lysates were im-munoprecipitated with anti-T7 agarose beads or an anti-HA antibody together with protein A/G plus agarose beads. After washing, precipitated proteins wereresuspended in Laemmli sample buffer and subject to Western blot, as indicated at the bottom of each panel. (E) GST-p53 (250 ng) was incubated with E1(150 ng), E2 (30 ng), and SUMO3 (1 μg), either with or without GST-SF2/ASF (400 ng) and GST-PIAS1 (100 ng) in sumoylation assay buffer for 30 min. Reactionswere stopped by addition of one volume of Laemmli sample buffer and analyzed by Western blotting with an anti-p53 antibody.

Pelisch et al. www.pnas.org/cgi/content/short/1004653107 8 of 8