RESEARCH COMMUNICATION

catalytic center of the . In later steps, multi- The anti-tumor drug E7107 ple complex rearrangements occur, resulting in binding of reveals an essential role U4/U5/U6 to form the B complex, and finally the catalytically active C complex. for SF3b in remodeling U2 Although a great deal has been learned about spliceo- some assembly and splicing, it has been difficult to achieve snRNP to expose the branch a detailed understanding of the numerous steps in this point-binding region pathway without the aid of small molecule inhibitors. Recently, an anti-tumor drug, E7107 (E7), was identified Eric G. Folco, Kaitlyn E. Coil, and Robin Reed1 that blocks splicing by targeting the U2 snRNP-associ- ated complex SF3b (Kotake et al. 2007). SF3b associates Department of Cell Biology, Harvard Medical School, Boston, with both the 59 end of U2 snRNA and the BPS region of Massachusetts 02115, USA the (Gozani et al. 1996; Kramer et al. 1999; Will et al. 2001; Dybkov et al. 2006). Thus, SF3b is positioned Duplex formation between the branch point-binding re- at the critical site of the spliceosome, where the BBR–BPS gion (BBR) of U2 snRNA and the branch point sequence duplex forms during establishment of the catalytic center (BPS) in the intron is essential for splicing. Both the BBR of the spliceosome. However, the precise function of SF3b and BPS interact with the U2 small nuclear ribonucleo- is not known. Here, we show that E7 results in formation (snRNP)-associated SF3b complex, which is the of a defective spliceosome in which U2 snRNP fails to target of the anti-tumor drug E7107. We show that E7107 bind tightly to pre-mRNA. Our data indicate that E7 does blocks spliceosome assembly by preventing tight binding not disrupt U2 snRNP or its association with SF3b, but of U2 snRNP to pre-mRNA. E7107 has no apparent effect does block an ATP-dependent remodeling event that ex- on U2 snRNP integrity. Instead, E7107 abolishes an ATP- poses the BBR of U2 snRNA. We conclude that SF3b is required to remodel U2 snRNP to expose the BBR, and this dependent conformational change in U2 snRNP that event may allow the SF3b- and ATP-dependent tight exposes the BBR. We conclude that SF3b is required for binding of U2 snRNP to the pre-mRNA. These results this remodeling, which exposes the BBR for tight U2 provide important new insights into the mechanism of snRNP binding to pre-mRNA. action of E7, and should aid in elucidating the novel anti- Supplemental material is available for this article. tumor properties of this drug. Received November 3, 2010; revised version accepted January 18, 2011. Results and Discussion

E7 results in formation of a defective, labile Most pre-mRNAs in higher eukaryotes contain multiple spliceosome that are excised by the spliceosome to generate To determine how E7 blocks splicing, we used a coupled mature mRNA (Konarska and Query 2005; Smith et al. RNA polymerase II (RNAP II) transcription/splicing sys- 2008; Wahl et al. 2009). The spliceosome, composed of tem (Das et al. 2006). When a CMV-Ftz DNA template five snRNAs (U1, U2, U4, U5, and U6) and many , encoding one intron and two (Fig. 1A) was incubated undergoes a series of dynamic changes during the splicing in the presence of E7, transcription occurred normally, but pathway (Staley and Guthrie 1998; Smith et al. 2008; splicing was blocked in a dose-dependent manner (Fig. 1B, Wahl et al. 2009). In the early steps, U1 small nuclear lanes 1–8). Splicing was not affected by a negative control ribonucleoprotein (snRNP) binds to the pre-mRNA, and compound, pladienolide F (PlaF), and was the same as U1 snRNA base-pairs to the 59 splice site to form the with no drug treatment (Fig. 1B, lanes 9–18). Significantly, ATP-independent early (E) complex (Reed 1996; Smith however, when CMV-Ftz was first transcribed by RNAP et al. 2008; Wahl et al. 2009). U2 snRNP is also recruited II, followed by addition of the drug, splicing was not to the E complex, but is not tightly bound (Das et al. 2000; affected by E7 (or PlaF) (Fig. 1C). Previous work showed Donmez et al. 2004, 2007). Subsequently, in an ATP- that the spliceosome assembles immediately on pre- dependent step, the A complex is assembled, in which U2 mRNA when synthesized in the coupled system (Das snRNP binds tightly. At this time, a duplex is formed et al. 2006). Thus, the data in Figure 1C indicate that E7 between the branch point sequence (BPS) in the intron inhibits spliceosome assembly. We obtained the same and the branch point-binding region (BBR) located near results with CMV-AdML, indicating that the effects of E7 the 59 end of U2 snRNA (Konarska and Query 2005; are general (Supplemental Fig. 1). Smith et al. 2008; Wahl et al. 2009). This tight binding of We next directly assayed for the spliceosome by tran- U2 snRNP to the pre-mRNA and formation of the BBR– scribing CMV-Ftz in the coupled system and analyzing BPS duplex are critical early steps in establishing the the reactions on a native gel. Surprisingly, the spliceo- some had the same mobility whether assembled in E7 or PlaF (Fig. 2A). Moreover, the spliceosome assembled in E7 [Keywords: small molecule splicing inhibitor; spliceosome assembly; was splice site-dependent, as it did not form on a CMV- pladienolide derivative E7107; 17S U2 snRNP; SF3b; branch point-binding Dss transcript, which lacks functional splice sites and region; anti-tumor drug] 1 assembles into a nonspecific hnRNP complex (Fig. 2B; Corresponding author. E-MAIL [email protected]; FAX (617) 432-3091. Das et al. 2006). We conclude that E7 results in formation Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.2009411. of a complex that requires splicing signals and comigrates

440 & DEVELOPMENT 25:440–444 Ó 2011 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/11; www.genesdev.org SF3b remodels U2 snRNP

3A). U2 snRNA was efficiently cleaved in the presence of this oligo, whereas a negative control oligo (cntl) had no effect (Fig. 3B). When CMV-Ftz was incubated in U2 snRNA or cntl extracts in the coupled system, the spli- ceosome was detected in the PlaF-treated cntl extract, but was disrupted in the U2 snRNA extract (Fig. 3C, cf. lanes 1 and 2). In contrast, the heparin-disrupted E7 spliceosome had the same mobility in cntl and U2 snRNA extracts (Fig. 3C, lanes 3,4). These data indicate that U2 snRNP is not a stable component of the heparin-disrupted, E7 spliceo- some. In contrast, in low heparin, both the E7 and normal (PlaF) migrated with faster mobility in the U2 snRNA extract (Fig. 3D, lanes 2,4) versus the cntl ex- tract (Fig. 3D, lanes 1,3), indicating that U2 snRNP is a component of the low-heparin E7 (and PlaF) spliceosome. To further verify that the low-heparin E7 spliceosome contains loosely bound U2 snRNP, we next asked whether this snRNP could be competed off of the pre-mRNA in the presence of normal nuclear extract. To do this, we carried out a chase assay in which 1 mM E7 or PlaF was added prior to transcription for 5 min to allow formation of the E7 or PlaF spliceosome, followed by a 10-fold dilution of these reactions into normal nuclear extract. At this dilution Figure 1. E7 blocks splicing when added prior to but not after RNAP (100 nM), E7 has no effect on splicing (Fig. 3E, lanes 5–8). II transcription. (A) Schematic of CMV-Ftz DNA template showing However, when the E7 spliceosome was chased in the the CMV promoter and sizes of the exons and intron. (B) CMV-Ftz normal extract, splicing was inefficient (Fig. 3E, lanes 1,2). DNA was incubated in coupled RNAP II transcription/splicing re- In contrast, splicing was efficient when the PlaF spliceo- action mixtures for 5 min in the presence of the indicated amounts of E7, PlaF, or no drug. a-Amanitin was then added, and incubation was some was chased in normal nuclear extract (Fig. 3E, lanes continued for 45 min. (C) CMV-Ftz DNA was incubated for 5 min, 3,4). We conclude that the E7 spliceosome contains non- followed by addition of a-amanitin. (Lanes 1–6,11,12) After transcrip- functional U2 snRNP that is loosely bound to pre-mRNA tion, the indicated amounts of E7 or PlaF were added, and incubation but cannot exchange for the U2 snRNP in the normal was continued for 45 min. In lanes 7–10, 13, and 14, the indicated nuclear extract. Finally, antibodies to the U2 snRNP protein amounts of E7 or PlaF were added at the beginning of the coupled reaction as in B. No drug was added in lanes 15 and 16.TotalRNA was fractionated on a 5% denaturing polyacrylamide gel and detected by PhosphorImager. The splicing intermediates and products and endogenous U6 snRNA and tRNA are indicated. with the spliceosome, but is incapable of splicing. To determine why the E7 spliceosome is defective, we ex- amined it using different amounts of heparin, which af- fects spliceosome stability. Significantly, in the presence of high heparin, the E7 spliceosome was disrupted, whereas the PlaF spliceosome had similar mobility in low and high heparin (Fig. 2C). These data indicate that the E7 spliceosome is an aberrant, labile complex. To determine when along the spliceosome assembly pathway E7 exerts its effect, we made use of the uncoupled splicing system and a T7 transcript, because the kinetics of spliceosome assembly are so fast with the coupled system that the different spliceosomal complexes (e.g., A and B) are not detected. When T7 AdML was incubated in an un- coupled splicing reaction for 0, 4, or 25 min, the non- specific H, A, and B complexes were detected with no drug (Fig. 2D) or PlaF (data not shown). In contrast, only the H Figure 2. E7 results in formation of a labile spliceosome and blocks complex was detected with E7 (Fig. 2D). We conclude that the A complex assembly. (A) CMV-Ftz was incubated in coupled transcription/splicing mixtures in the presence of E7 or PlaF for 8 E7 blocks assembly of the spliceosomal A complex. min. CMV-Ftz was omitted in lane 3. Heparin (0.5 mL) was added to the reaction mixtures, and then an aliquot was fractionated on E7 results in weak binding of U2 snRNP to pre-mRNA a 1.2% native low-melting agarose gel and detected by Phosphor- Imager. The spliceosome (ssome) is indicated. (B) Same as A, except As E7 targets U2 snRNP, and this snRNP first binds tightly using CMV-Ftz and CMV-Dss DNA templates. The spliceosome and in the A complex, we next asked whether U2 snRNP was nonspecific Dss complexes are indicated. (C) Same as A, except 0.5 present in the defective, heparin-sensitive E7 spliceosome. mL and 1.5 mL of heparin was added. The white box to the right of lane 4 indicates the heparin-sensitive E7 spliceosome. (D)T7-AdML To do this, we inactivated U2 snRNP using the endoge- was incubated under standard splicing conditions in E7 or no drug nous RNase H in the nuclear extract with an oligonucle- for the indicated times, heparin was added, and spliceosomal otide complementary to the 59 portion of U2 snRNA (Fig. complexes were separated on a 1.5% native low-melting agarose gel.

GENES & DEVELOPMENT 441 Folco et al.

dence that SF3b/U2 snRNP is associated with the de- fective E7 spliceosome. We conclude that E7 causes the formation of a labile spliceosome containing weakly bound U2 snRNP. In recent work, another small mole- cule splicing inhibitor (spliceostatin A), which also tar- gets SF3b (Kaida et al. 2007), was shown to inhibit spliceosome assembly (Roybal and Jurica 2010). More- over, in a study published in this issue of Genes & Development, Corrionero et al. (2011) found that spli- ceostatin A, like E7, inhibits tight binding of U2 snRNP to pre-mRNA by interfering with mechanisms that en- sure proper base-pairing interactions between U2 snRNA and the pre-mRNA. Together, these results show that both drugs disrupt stable U2 snRNP recruitment, indi- cating that this step in spliceosome assembly is a key target for anti-tumor drugs.

E7 blocks ATP-dependent remodeling of U2 snRNP that exposes the BBR We next sought to determine why E7 inhibits tight bind- ing of U2 snRNP to pre-mRNA. We first examined the integrity of U2 snRNP, which exists as a 17S particle con- taining a 12S core U2 snRNP particle associated with SF3b, and another multiprotein complex known as SF3a (Wahl et al. 2009). When U2 snRNP was immunoprecip- itated from nuclear extract after treatment with E7, PlaF, or no drug, the full set of 17S U2 snRNP proteins, inclu- ding the SF3a/b proteins, was detected on a Coomassie- stained gel and by mass spectrometry of total proteins (Supplemental Fig. 2). Consistent with these results, West- ern analyses showed that the levels of SF3a/b components were the same in immunopurified E7-U2 snRNP and PlaF- U2 snRNP (Supplemental Fig. 2). Together, these data in- Figure 3. E7 results in weak binding of U2 snRNP to pre-mRNA. dicate that E7 has no apparent effect on the integrity of U2 (A) Schematic of the 59 end of U2 snRNA. The line indicates the 20- snRNP or its association with SF3a/b. nucleotide (nt) oligonucleotide complementary to U2 snRNA used We next asked whether E7 alters U2 snRNP conforma- for RNase H cleavage. See the Materials and Methods for sequences tion. The 59 region of U2 snRNA, which associates with of the U2 oligonucleotide and the cntl. (B) Analysis of RNase H-cleaved U2 snRNA. Total RNA from U2 snRNA or cntl extracts SF3b, plays a critical role early in spliceosome assembly was fractionated on a 5% denaturing polyacrylamide gel, and RNA (Kramer et al. 1999; Dybkov et al. 2006). As E7 targets was detected by ethidium bromide. The snRNAs and cleaved U2 SF3b, we focused on this region of U2 snRNP. Using native snRNA are indicated. (C,D) CMV-Ftz DNA template was incubated gels, we assayed for binding of a 32P-labeled 29OMethyl in coupled RNAP II transcription/splicing reaction mixtures con- oligonucleotide complementary to nucleotides 27–42 of taining either PlaF (lanes 1,2) or E7 (lanes 3,4) for 5 min in U2 U2 snRNA (designated BBR oligo) (Fig. 4A). Consistent snRNA (DU2) or cntl extracts. An aliquot of each sample was mixed with 1.5 mL (Fig. 2C) or 0.5 mL (Fig. 2D) of heparin and fractionated with previous work in yeast (Abu Dayyeh et al. 2002), the on a 1.2% native agarose gel. (E) Chase assay showing that non- BBR oligonucleotide (Fig. 4B, lanes 1,3), but not a cntl functional U2 snRNP remains loosely bound to pre-mRNA after E7 (Fig. 4B, lanes 2,4), binds to 17S U2 snRNP in an ATP- treatment. CMV-Ftz DNA template was transcribed for 5 min in the dependent manner. In striking contrast, when nuclear coupled transcription/splicing system containing 1 mM E7 (lanes 1,2) extract is treated with E7, ATP-dependent binding of or PlaF (lanes 3,4). An aliquot (2 mL) of each sample in lanes 1 and 2 was diluted into a new 25-mL reaction mixture in the presence of the BBR oligonucleotide to 17S U2 snRNP is abolished a-amanitin and incubated for an additional 40 min. For cntl samples (Fig. 4C, lanes 5–8). We conclude that E7 potently inhibits in lanes 5–8, CMV-Ftz was transcribed for 5 min in the presence of an ATP-dependent conformational change that exposes 100 nM E7107 (lanes 5,6) or 100 nM PlaF (lanes 7,8), a-amanitin was the BBR of U2 snRNA (Fig. 4D). added, and incubation was continued for an additional 40 min. Total To further investigate the remodeling of U2 snRNP, we RNA was fractionated on a 5% denaturing polyacrylamide gel and carried out an order-of-addition experiment in which nu- detected by PhosphorImager. (F) CMV-Ftz DNA template was in- cubated in coupled transcription/splicing reactions for 5 min in the clear extract was first preincubated in ATP, and then E7 or presence of E7 or PlaF. Immunoprecipitations were carried out in PlaF was added. Significantly, binding of the BBR oligo- low salt (250 mM) using a monoclonal antibody to the U2 snRNP B0 nucleotide to 17S U2 snRNP was now completely resis- protein, a rabbit polyclonal to SF3b155, and monoclonal or poly- tant to E7 (Fig. 4E). These data, together with the data in clonal antibodies to nonrelated proteins as negative controls. Total Figure 4C, suggest that, once the ATP-dependent, SF3b- RNA was run on a 5% denaturing polyacrylamide gel, and CMV-Ftz pre-mRNA was detected by PhosphorImager. dependent remodeling of U2 snRNP exposes the BBR (Fig. 4D), then binding of the BBR oligonucleotide can occur in an SF3b-independent manner (i.e., E7-resistant manner) B0 or an SF3b protein (SF3b155) specifically immunopre- (Fig. 4F). We conclude that SF3b and ATP, but not the BBR cipitate both the E7 and PlaF spliceosomes assembled on oligonucleotide, are required to trigger remodeling of U2 the CMV-Ftz transcript (Fig. 3F), providing further evi- snRNP to expose the BBR.

442 GENES & DEVELOPMENT SF3b remodels U2 snRNP

tightly to the pre-mRNA at a region known as the an- choring site, which is located immediately upstream of the BPS (Gozani et al. 1996). More recently, Query and colleagues (Newnham and Query 2001) showed that the anchoring site mediates the ATP requirement for stable U2 snRNP binding, and suggested that this could be due to remodeling of SF3a/b. Our studies using E7 provide di- rect evidence for this proposal, showing that SF3b is re- quired for tight binding of U2 snRNP to pre-mRNA and for remodeling U2 snRNP. The identification of specific roles for SF3b via the use of E7 and spliceostatin A, as well as other recent work using small molecules to examine splicing, underscores the utility of these compounds for elucidating specific functions of the highly complex, dynamic factors in the spliceosome (Pilch et al. 2001; Muraki et al. 2004; Kaida et al. 2007; Kotake et al.2007; O’Brien et al. 2008; Stoilov et al. 2008; Sumanasekera et al. 2008; Kuhn et al.2009; Younis et al. 2010). Although the binding affinity of SF3b to E7 is highly correlated with its cell growth inhibition Figure 4. E7 blocks ATP-dependent remodeling of U2 snRNP to activity (Kotake et al. 2007), the mechanistic basis for the expose the BBR. (A) Schematic of the 59-end region of U2 snRNA. anti-tumor effect of E7 is not known. If the effect is due to The 29 OMethyl BBR oligonucleotide is complementary to nucleo- splicing inhibition, then E7 may, for example, differen- tides 27–42 of U2 snRNA. The gray line above the U2 snRNA tially affect splicing of pre-mRNAs containing weak splic- sequence indicates the BBR. (B) Nuclear extracts were incubated in ing signals, and these pre-mRNAs may encode proteins the presence or absence of ATP for 5 min, followed by a 5-min incubation with 32P-labeled BBR or cntl 29 OMethyl oligonucleotide. involved in tumorogenesis. Our insights into the mech- Samples were fractionated on a 1.2% agarose gel and detected by anism of action of E7 and its effects on SF3b/U2 snRNP PhosphorImager. The 17S and 12S U2 snRNPs are indicated. (C) will contribute to establishing the therapeutic potential Same as B, except that nuclear extract was preincubated with 1 mM of the drug, as well as the development of second-gener- E7 or PlaF for 5 min. (D) Model showing that E7 blocks the ATP- ation derivatives. dependent remodeling of U2 snRNP that exposes the BBR. The circle indicates the ATP-independent form of U2 snRNP, and the curved line within the circle indicates the inaccessible BBR in U2 Materials and methods snRNA. The square indicates remodeled U2 snRNP, and the straight lines indicate U2 snRNA base-paired to the BBR oligonucleotide. (E) Same as C, except that nuclear extract was preincubated in ATP for Plasmids 5 min, followed by addition of the drugs for 5 min, and then the BBR The plasmid encoding the 755-nucleotide (nt) CMV-Ftz transcript was or cntl oligonucleotide for 5 min. The 17S and 12S U2 snRNPs are described (Das et al. 2006). The plasmid encoding the 695-nt CMV-Dss indicated. (F) Model showing that the BBR oligonucleotide binds to transcript was a generous gift from H. Lei and encodes the naturally remodeled 17S U2 snRNP in an E7-resistant manner. The square on the left indicates remodeled U2 snRNP, and the line within the intronless HSBP3 . CMV-DNA templates used in the coupled RNAP square indicates U2 snRNA with an exposed BBR. In the square on II transcription/splicing system were amplified by PCR using a forward the right, the lines indicate U2 snRNA and U2 snRNA base-paired to primer (59-TGGAGGTCGCTGAGTAGTGC-39) and reverse primer (59- the BBR oligonucleotide. TAGAAGGCACAGTCGAGGCT-39). AdML pre-mRNA was transcribed with T7 RNA polymerase from a PCR product using the same primers and the DoA plasmid (Das et al. 2006). In this study, we showed that E7 results in formation of defective spliceosomes in which U2 snRNP cannot bind Coupled transcription/splicing tightly to pre-mRNA. Consistent with this conclusion, In vitro RNAP II transcription/splicing reactions were performed at 30°C our data show that E7 abolishes the A complex assembly, in 25-mLreactionmixturescontaining200ngofCMV-DNAtemplate, in which U2 first binds tightly to pre-mRNA. We did not 1 mLofa-32P-UTP (800 Ci/mmol; Perkin Elmer Life Sciences catalog detect any effect of E7 on the integrity of U2 snRNP, but no. BLU507x), 0.5 mM ATP, 3.2 mM MgCl2, 20 mM creatine phosphate showed that E7 does potently block an ATP-dependent re- (di-Tris salt), and 15 mL of HeLa nuclear extract (Das et al. 2006). The modeling event that exposes the BBR of U2 snRNA. As E7 indicated amounts of E7 or PlaF were added to the reaction mixtures targets SF3b, our data lead to a model in which SF3b is before or after RNAP II transcription, as designated. a-Amanitin (10 ng/25 required for the ATP-dependent remodeling of U2 snRNP mL) was added after transcription, and incubation was continued for the indicated times. to expose the BBR, and this event may be required for the SF3b- and ATP-dependent tight binding of U2 snRNP to pre-mRNA. Previous studies in yeast suggested that splic- Spliceosome assays ing factor Prp5p, which is an ATPase/helicase, facilitates an ATP-dependent alteration in U2 structure that is re- For spliceosome assembly, CMV-DNA templates were incubated under RNAP II transcription/splicing conditions for 8 min. G-50 columns (GE quired for the BBR of U2 snRNA to become accessible to 32 the BPS region in the intron (Ruby et al. 1993; Abu Dayyeh Healthcare Life Sciences) were used to remove unincorporated a- P-UTP. m et al. 2002). Our work showing that E7 blocks ATP- For low- or high-heparin samples, 0.5 or 1.5 L of heparin (6.5 g/L), re- spectively, was added to 10 mL of G-50 column-purified reactions prior to dependent remodeling of U2 snRNP to expose the BBR loading on 1.2% low-melting-point agarose gels (Das and Reed 1999). For strongly suggests that E7 exerts its effects at this Prp5p- assembly of spliceosomal complexes using the uncoupled system, AdML dependent remodeling step. Another study showed that, pre-mRNA (2 ng) was incubated for 0, 4, and 25 min under standard splic- during the A complex assembly, SF3a/b components bind ing conditions (0.5 mM ATP, 3.2 mM MgCl2, 20 mM creatine phosphate

GENES & DEVELOPMENT 443 Folco et al.

[di-Tris salt]) in a 25-mL reaction mixture containing 7.5 mL of nuclear Kotake Y, Sagane K, Owa T, Mimori-Kiyosue Y, Shimizu H, Uesugi M, extract and 7.5 mLofSplicingDilutionBuffer(20mMHepesatpH7.6,100 Ishihama Y, Iwata M, Mizui Y. 2007. Splicing factor SF3b as a target of mM KCl). No drug or 1 mM E7 was included in the reactions. Aliquots (10 the antitumor natural product pladienolide. Nat Chem Biol 3: 570–575. mL) of each reaction were mixed with 1 mL of heparin (6.5 g/L) and run on Kramer A, Gruter P, Groning K, Kastner B. 1999. Combined biochemical 1.5% low-melting-point agarose gel. For preparation of U2 snRNA and and electron microscopic analyses reveal the architecture of the control extracts, 250 ng of oligonucleotide targeting U2 snRNA (59-CCA mammalian U2 snRNP. J Cell Biol 145: 1355–1368. AAAGGCCGAGAAGCGAT-39) or cntl (59-GGGGTGAATTCTTTGCCA Kuhn AN, van Santen MA, Schwienhorst A, Urlaub H, Luhrmann R. A-39) was added to 15 mL of nuclear extract, followed by incubation for 5 2009. Stalling of spliceosome assembly at distinct stages by small- min at 30°C for endogenous RNase H cleavage of the snRNA. Total RNA molecule inhibitors of protein acetylation and deacetylation. RNA 15: was prepared from these samples and fractionated on a 5% denaturing gel, 153–175. and snRNAs were detected by ethidium bromide. The DU2 and cntl ex- Muraki M, Ohkawara B, Hosoya T, Onogi H, Koizumi J, Koizumi T, Sumi tracts were used for spliceosome assembly assays as described above. K, Yomoda J, Murray MV, Kimura H, et al. 2004. Manipulation of by a newly developed inhibitor of Clks. JBiol Chem 279: 24246–24254. U2 snRNP conformation assay Newnham CM, Query CC. 2001. The ATP requirement for U2 snRNP The BBR 29OMethyl oligonucleotide complementary to the BBR addition is linked to the pre-mRNA region 59 to the branch site. RNA (59-mCmAmGmAmUmAmCmUmAmAmCmAmCmUmUmGmA-39)and 7: 1298–1309. the cntl (59-mAmCmUmGmUmAmCmUmAmAmCmUmGmAmCmU O’Brien K, Matlin AJ, Lowell AM, Moore MJ. 2008. The biflavonoid mG-39) were 32P-labeled using g-ATP (6000 Ci/mmol) and T4 polynucle- isoginkgetin is a general inhibitor of Pre-mRNA splicing. JBiolChem otide kinase. For all assays, ATP was depleted from nuclear extract by 283: 33147–33154. incubation for 20 min at room temperature. Reaction mixtures (25 mL) Pilch B, Allemand E, Facompre M, Bailly C, Riou JF, Soret J, Tazi J. 2001. containing the depleted nuclear extract (15 mL) were incubated in the Specific inhibition of serine- and arginine-rich splicing factors phos- phorylation, spliceosome assembly, and splicing by the antitumor presence or absence of 0.5 mM ATP, 3.2 mM MgCl2, and 20 mM creatine phosphate (di-Tris salt) for the indicated times. BBR or control 29OMethyl drug NB-506. Cancer Res 61: 6876–6884. oligonucleotide (34 ng of each) and 1 mM E7 or PlaF were added to the Reed R. 1996. Initial splice-site recognition and pairing during pre-mRNA reaction mixtures in the orders indicated in Figure 4. For reactions carried splicing. Curr Opin Genet Dev 6: 215–220. out in the absence of ATP, water was used to bring the volume up to 25 mL. Roybal GA, Jurica MS. 2010. Spliceostatin A inhibits spliceosome assem- bly subsequent to prespliceosome formation. Nucleic Acids Res 38: 6664–6672. Acknowledgments Ruby SW, Chang TH, Abelson J. 1993. Four yeast spliceosomal proteins (PRP5, PRP9, PRP11, and PRP21) interact to promote U2 snRNP We are grateful to R. Das, K. Dufu, C.S. Lee, and members of the Reed binding to pre-mRNA. Genes Dev 7: 1909–1925. laboratory for useful discussions, and to H. Lei and K. Dufu for critical Smith DJ, Query CC, Konarska MM. 2008. ‘Nought may endure but comments on the manuscript. HeLa cells were obtained from the Na- mutability’: spliceosome dynamics and the regulation of splicing. Mol tional Cell Culture Center (Minneapolis, MN). This work was supported Cell 30: 657–666. by a post-doctoral fellowship to E.F. from Eisai Pharmaceuticals and an Staley JP, Guthrie C. 1998. Mechanical devices of the spliceosome: motors, NIH grant to R.R. clocks, springs, and things. Cell 92: 315–326. Stoilov P, Lin CH, Damoiseaux R, Nikolic J, Black DL. 2008. A high- throughput screening strategy identifies cardiotonic steroids as alter- References native splicing modulators. Proc Natl Acad Sci 105: 11218–11223. Abu Dayyeh BK, Quan TK, Castro M, Ruby SW. 2002. Probing in- Sumanasekera C, Watt DS, Stamm S. 2008. Substances that can change teractions between the U2 small nuclear ribonucleoprotein and the alternative splice-site selection. Biochem Soc Trans 36: 483–490. DEAD-box protein, Prp5. JBiolChem277: 20221–20233. Wahl MC, Will CL, Luhrmann R. 2009. The spliceosome: design Corrionero A, Min˜ ana B, Valca´rcel J. 2011. Reduced fidelity of branch point principles of a dynamic RNP machine. Cell 136: 701–718. recognition and alternative splicing induced by the anti-tumor drug Will CL, Schneider C, MacMillan AM, Katopodis NF, Neubauer G, Wilm spliceostatin A. Genes Dev (this issue). doi: 10.1101/gad.2014311. M, Luhrmann R, Query CC. 2001. A novel U2 and U11/U12 snRNP Das R, Reed R. 1999. Resolution of the mammalian E complex and the protein that associates with the pre-mRNA branch site. EMBO J 20: ATP-dependent spliceosomal complexes on native agarose mini-gels. 4536–4546. RNA 5: 1504–1508. Younis I, Berg M, Kaida D, Dittmar K, Wang C, Dreyfuss G. 2010. Rapid- Das R, Zhou Z, Reed R. 2000. Functional association of U2 snRNP with response splicing reporter screens identify differential regulators of the ATP-independent spliceosomal complex E. Mol Cell 5: 779–787. constitutive and alternative splicing. Mol Cell Biol 30: 1718–1728. Das R, Dufu K, Romney B, Feldt M, Elenko M, Reed R. 2006. Functional coupling of RNAP II transcription to spliceosome assembly. Genes Dev 20: 1100–1109. Donmez G, Hartmuth K, Luhrmann R. 2004. Modified nucleotides at the 59 end of human U2 snRNA are required for spliceosomal E-complex formation. RNA 10: 1925–1933. Donmez G, Hartmuth K, Kastner B, Will CL, Luhrmann R. 2007. The 59 end of U2 snRNA is in close proximity to U1 and functional sites of the pre-mRNA in early spliceosomal complexes. Mol Cell 25: 399–411. Dybkov O, Will CL, Deckert J, Behzadnia N, Hartmuth K, Luhrmann R. 2006. U2 snRNA-protein contacts in purified human 17S U2 snRNPs and in spliceosomal A and B complexes. Mol Cell Biol 26: 2803–2816. Gozani O, Feld R, Reed R. 1996. Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A. Genes Dev 10: 233–243. Kaida D, Motoyoshi H, Tashiro E, Nojima T, Hagiwara M, Ishigami K, Watanabe H, Kitahara T, Yoshida T, Nakajima H, et al. 2007. Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA. Nat Chem Biol 3: 576–583. Konarska MM, Query CC. 2005. Insights into the mechanisms of splicing: more lessons from the ribosome. Genes Dev 19: 2255–2260.

444 GENES & DEVELOPMENT Supplemental Materials & Methods

Plasmids.

The plasmid encoding the 386 nt CMV-AdML transcript was described (Das et al.

2006).

Coupled transcription/splicing.

In vitro RNAP II transcription/splicing reactions were performed at 30°C in 25 µl reaction mixtures containing 200 ng CMV-AdML DNA template, 1 µl a-32P-UTP (800

Ci/mmol; Perkin Elmer Life Sciences catalog #BLU507x), 0.5 mM ATP, 3.2 mM

MgCl2, 20 mM creatine phosphate (di-Tris salt), and 15 µl HeLa nuclear extract (Das et al. 2006). The indicated amounts of E7 or PlaF were added to the reaction mixtures before or after RNAP II transcription as designated. a-amanitin (10 ng/25

µl) was added after transcription, and incubation was continued for the indicated times.

Immunoprecipitations, Western analysis, and Mass Spectrometry

For immunoprecipitations, a monoclonal antibody to the U2 snRNP protein B”

(Mattaj et al. 1986), a rabbit polyclonal antibody to SF3b155 (Wang et al. 1998) or a negative control rabbit polyclonal antibody against DCP1a (generous gift of F.

McKeon) were coupled to protein A (polyclonal) or G (monoclonal) and then covalently crosslinked using dimethylpimelimidate (Sigma Aldrich). Nuclear extract reaction mixtures used for immunoprecipitations were incubated for 20 minutes at

30 degrees in 0.5 mM ATP, 3.2 mM MgCl2, 20 mM creatine phosphate (di-Tris salt), 2 mM PMSF, 2 ml protease inhibitor EDTA-free (Roche) and containing 1 µM E7 or PlaF as indicated. After incubation, reaction mixtures were spun at 4 degrees for 5 minutes at 14,000 rpm. Supernatants were then added to the antibody-crosslinked beads and immunoprecipitations were rotated for 2 hours at 4 degrees and washed five times with 1 ml of wash buffer (1x PBS, 0.1% Triton-X). A portion of the IPs was analyzed by SDS PAGE stained with Coomassie, and a portion was used for Westerns analysis. Westerns were carried out using a rabbit polyclonal antibody to SF3a

(which recognizes SF3a120, 66 and 60) (Das et al. 2000), SF3b130 (Das et al. 1999), or SF3b155 (Wang et al. 1998). For mass spectrometry, a portion of the IPs was precipitated with TCA, and the total samples were analyzed (Gygi et al. 1999).

Keratins and abundant cellular proteins that were likely contaminants, including desmoplakin, actin, tubulin, , and translation proteins were omitted from the tables.

Supplemental References

Das, B.K., Xia, L., Palandjian, L., Gozani, O., Chyung, Y., and Reed, R. 1999.

Characterization of a Protein Complex Containing Spliceosomal Proteins

SAPs 49, 130, 145, and 155. Mol Cell Biol 19(10): 6796-6802.

Gygi, S.P., Rochon, Y., Franza, B.R., and Aebersold, R. 1999. Correlation between

protein and mRNA abundance in yeast. Mol Cell Biol 19(3): 1720-1730.

Mattaj, I.W., Habets, W.J., and van Venrooij, W.J. 1986. Monospecific antibodies

reveal details of U2 snRNP structure and interaction between U1 and U2

snRNPs. Embo J 5(5): 997-1002.

Wang, C., Chua, K., Seghezzi, W., Lees, E., Gozani, O., and Reed, R. 1998.

Phosphorylation of spliceosomal protein SAP 155 coupled with splicing

catalysis. Genes Dev 12(10): 1409-1414.

Supplemental Figure Legends

Supplemental Fig 1. E7107 blocks splicing when added prior to but not after

RNAP II transcription of CMV-AdML DNA template.

(A), Schematic of CMV-AdML DNA template (Das et al. 2006), indicating the CMV promoter and the sizes of the exons and intron. (B), CMV-AdML was incubated in coupled RNAP II transcription/splicing reaction mixtures for 5 minutes in the presence of the indicated amounts of E7107, PlaF, or no drug. α-amanitin was then added, and incubation was continued for 45 minutes total. (C), CMV-AdML was incubated for 5 minutes, followed by addition of α-amanitin. After transcription, the indicated amounts of E7107 or PlaF were added, and incubation was continued for a total of 45 minutes (lanes 1-6, 11, 12). In lanes 7-10, 13 and 14, the indicated amounts of E7107 or PlaF were added at the beginning of the coupled RNAP II transcription/splicing reaction as in panel B. No drug was added in lanes 15 and 16.

Total RNA was fractionated on a 5% denaturing polyacrylamide gel and detected by PhosphorImager. The splicing intermediates and products and endogenous U6 snRNA and tRNA are indicated.

Supplemental Fig 2. E7107 has no apparent effect on 17S U2 snRNP composition.

(A), U2 snRNP was immunopurified using an antibody against the U2 snRNP protein

B” from E7107-treated, PlaF- treated, or normal nuclear extract, and total proteins were analyzed on a Coomassie-stained gel. The U2 snRNP components and size markers (in kD) are indicated. (B), Western analysis of samples prepared as in panel

(A) using the antibodies against B”, SF3a, and SF3b components. (C), Mass spectrometry of total proteins as prepared in panel (A) from E7107- or PlaF-treated nuclear extract. HnRNP proteins and common contaminants are not listed in the table. Folco_Supplemental_Fig1 A CMV-AdML 133 130 CMV 123

B E7107 Pla F 10 nM 100 nM 300 nM 1 µM 10 nM 100 nM 300 nM 1 µM no drug time (min) 5 45 5 45 5 45 5 45 5 45 5 45 5 45 5 45 5 45

-pre-mRNA -mRNA

-U6 snRNA

-tRNA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

pla F pla F C E7107 after txn E7107 before txn after txn before txn 100 nM 300 nM 1 µM 100 nM 1 µM 1 µM 1 µM no drug time (min) 5 45 5 45 5 45 5 45 5 45 5 45 5 45 5 45

-pre-mRNA

-mRNA

-U6 snRNA

-tRNA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Folco_Supplemental_Fig2

A B” IP neg cntl IP B B’’ IP No No 12.5% No E7 PlaF drug E7 PlaF drug inpt E7 PlaF drug SF3a120-

SF3b155 SF3a66, SF3a60- SF3b145, SF3b130- -182 SF3b125- SF3a120 -116 B”-

-82 12.5% No SF3a66, SF3a60- inpt E7 PlaF drug SF3b49- SF3b130- ∗ -64 -49 B”- B” -37 A’- No 12.5% E7 PlaF drug inpt -26 SF3b155- -19 SF3b14a, b- -15 B”- SF3b10- -6 1 2 3 4 5 6 C Mass spectrometry of B” IPs from E7- or PlaF-treated nuclear extract unique peptides Reference Name Aliases E7107 PlaF 12S U2 snRNP RU2A_HUMAN U2snRNP A' RU2A, U2A' 15 15 RU2B_HUMAN U2snRNP B'' SNRPB2 9 6 SF3b complex SF3B1_HUMAN SF3B1 SAP155, SF3b155 57 48 SF3B3_HUMAN SF3B3 SAP130, SF3b130 45 39 SF3B2_HUMAN SF3B2 SAP145, SF3b145 41 35 SF3B4_HUMAN SF3B4 SAP49, SF3b49 5 4 PM14_HUMAN P14 SAP14, SF3B14a 6 4 PHF5A_HUMAN PHF5A SAP14b, SF3B14b 3 3 SF3a complex SF3A1_HUMAN SF3A1 SAP114, SF3a120 31 33 SF3A3_HUMAN SF3A3 SAP61, SF3a60 23 18 SF3A2_HUMAN SF3A2 SAP62, SF3a66 14 11 U2 snRNP-associated DDX46_HUMAN Prp5 PRPF5 42 33 Q99730_HUMAN TAT-SF1 31 25 U1 snRNP specific proteins RU17_HUMAN U1-70K U1AP, U1RNP 6 3 RU1C_HUMAN U1C 1 1 SNRPA_HUMAN U1A 1 1 TIA1_HUMAN TIA 7 7 TIAR_HUMAN TIAR 4 2 Sm core proteins AY894074_1 Sm D2 7 7 RSMB_HUMAN Sm B 6 5 Q5VYJ4_HUMAN Sm E 4 3 Q7Z5A3_HUMAN Sm D1 3 3 RUXF_HUMAN Sm F 3 4 SMD3_HUMAN Sm D3 2 3 M21258_1 sm E 1 1