Supplementary Information Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA Daisuke Kaida, Hajime Motoyoshi, Etsu Tashiro, Takayuki Nojima, Masatoshi Hagiwara, Ken Ishigami, Hidenori Watanabe, Takeshi Kitahara, Tatsuhiko Yoshida, Hidenori Nakajima, Tokio Tani, Sueharu Horinouchi, Minoru Yoshida
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Daisuke Kaida, Hajime Motoyoshi, Etsu Tashiro, Takayuki ...€¦ · Supplementary Information Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA
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Supplementary Information
Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of
Hagiwara, Ken Ishigami, Hidenori Watanabe, Takeshi Kitahara, Tatsuhiko Yoshida,
Hidenori Nakajima, Tokio Tani, Sueharu Horinouchi, Minoru Yoshida
Cyclin A
CDK2
CDK4
CDK6
FR901464
Supplementary Fig. 1
Supplementary Fig. 1 Detection of proteins in the extract from HeLa cellstreated with drugs. (a) Detection of cell cycle regulaters in the extract fromHeLa cells treated with FR901464 (100 ng ml–1, 14 h) using anti-cyclin A, anti-CDK2, anti-CDK4 and anti-CDK6 antibodies. (b) Detection of β-actin and β-tubulin in the extract from HeLa cells treated with SSA (100 ng ml–1, 14 h)using β-actin and β-tubulin antibodies.
β-actin β-tubulin
SSAa b
Supplementary Fig. 2
FRMG132
p27p27*
Supplementary Fig. 2 p27* is not the product of proteolytic processing.(a) Effect of a proteasome inhibitor MG132 (10 mM, 14 h) on the p27*production in the cells treated with FR901464 (100 ng ml–1, 14 h). (b)Detection of FLAG-p27. HEK293T cells were transfected with FLAG-p27cDNA and treated with FR901464 (100 ng ml–1, 14 h). FLAG-p27 wasdetected using an anti-FLAG antibody.
Anti-FLAG
FLAG-p27
Vec. FLAG-p27
FR
a
b
Supplementary Fig. 3
Supplementary Fig. 3 Stability of FR901464 and spliceostatin A, and biologicalactivity of biotinylated spliceostatin A. (a) Stability of FR901464 and spliceostatinA. FR901464 (FR, blue) and spliceostatin A (SSA, red) were incubated in culturemedium for 0, 1, 3 and 6 h and the amount of the remaining compounds wasmeasured by HPLC. t1/2 values were 45 min for FR and over 6 h for SSA,respectively. (b) Detection of p27* in HeLa cells treated with biotin-SSA at theconcentration of 0, 1, 3, 10, 30, 100, 300 and 1000 nM.
Time (h)
Resid
ual a
mou
nt (%
)
b-SSA
p27p27*
a
b
Supplementary Fig. 4
0 12 (h)
mature
Supplementary Fig. 4 Splicing inhibition by spliceostatin A is partial in vivo.(a) RT-PCR analysis of β-globin mRNA in the cells transfected with the FLAG-β-globin plasmid in the presence or absence of SSA treatment (100 ng ml–1, 12h). Primer sets were designed to amplify the sequence between exon 1 and exon2. (b, c) Effects of a high concentration of SSA on in vivo splicing. mRNAspecies in the cells treated with a high concentration of SSA (1 mg ml–1) for 12 hwere analyzed by RT-PCR using primer sets designed for the sequences betweenp27 exon 1 and exon 2 (b, upper), p27 exon 1 and intron 2 (b, lower), and IκBαexon 1 and exon 4 (c).
a
b
MeOH SSA
pre-mRNA
mature
β-globin mRNA
pre-mRNApartially spliced
p27 mRNA
c0 12 (h)
IκBα mRNA
mature
SAP155 DAPI
control
SAP155KD
SAP145 DAPI
control
SAP145KD
Supplementary Fig. 5
a b
Supplementary Fig. 5 Subcellular localization of SAP155 (a) and SAP145(b). HeLa cells were transfected with siRNA for SAP155 and SAP145, andthen SAP155 and SAP145 were observed using the antibodies 3 days afterthe transfection.
Supplementary Fig. 6 Amino acid sequence and localization of p27*. (a) C-tarminal amino acid sequence of p27 and p27*. Red letters indicate residuescommon to p27 and p27*. A green letter indicates the site of phosphorylation byCDKs. (b) Subcellular localization of p27 and p27*. Cos7 cells were transfectedwith pcDNA3.1-FLAG-p27 (0.3 µg) or pcDNA3.1-FLAG-p27* (0.3 µg). The cellsexpressing FLAG-p27 were treated with leptomycin B (10 ng ml–1, 6 h). Theprotein localization was detected by immunofluorescence.
Supplementary Fig. 6
total b-SSA
SF1
SAP155
Supplementary Fig. 7
a
b
SF1
HA
Myc
SF1Con
trol
*
Supplementary Fig. 7 Effect of SF1 knockdown on pre-mRNA retention. (a)Translation of unspliced mRNA in SF3b knockdown cells. HeLa cells that hadbeen transfected with the reporter plasmid p27-int-HA were knocked down withSF1 siRNA and production of p27*-HA was detected. (b) Biotinylated SSAdoes not bind to SF1. Proteins bound to biotinylated SSA were analyzed bywestern blotting using an anti-SF1 antibody. Asterisks indicate a non-specificband.
*
control
β-globin
Supplementary Fig. 8
MeOH 6 h FR 6 h WT NS WT NS WT NS
CHX 3 h
Supplementary Fig. 8 FR901464 does not inhibit NMD. (a) HeLa cells weretransfected with either β-globin WT or β-globin NS39, together with an internalcontrol plasmid β-globin wt+300+e3. The transfected cells were treated withMeOH (6 h), FR901464 (6 h, 10 ng ml–1) or cycloheximide (6 h, 100 ng ml–1).Cyclohexamide that inhibits translation is known to block NMD. mRNAsprepared from transfected cells were analyzed by Northern blotting. (b) Relativesignal intensities in (a).