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p54nrb associates with the 5′ splice site within large transcription/splicing complexes

2004; Springer Nature; Volume: 23; Issue: 8 Linguagem: Inglês

10.1038/sj.emboj.7600187

1460-2075

Sei Kameoka, Paula Duque, Maria M. Konarska,

RNA and protein synthesis mechanisms

Article1 April 2004free access p54nrb associates with the 5′ splice site within large transcription/splicing complexes Sei Kameoka Sei KameokaPresent address: Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Ave., Cambridge, MA 02138, USA Search for more papers by this author Paula Duque Paula Duque Search for more papers by this author Maria M Konarska Corresponding Author Maria M Konarska The Rockefeller University, New York, NY, USA Search for more papers by this author Sei Kameoka Sei KameokaPresent address: Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Ave., Cambridge, MA 02138, USA Search for more papers by this author Paula Duque Paula Duque Search for more papers by this author Maria M Konarska Corresponding Author Maria M Konarska The Rockefeller University, New York, NY, USA Search for more papers by this author Author Information Sei Kameoka‡, Paula Duque‡ and Maria M Konarska 1 1The Rockefeller University, New York, NY, USA ‡These authors contributed equally to this work *Corresponding author. Laboratory of Molecular Biology & Biochemistry, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. Tel.: +1 212 327 8432; Fax: +1 212 327 7147; E-mail: [email protected] The EMBO Journal (2004)23:1782-1791https://doi.org/10.1038/sj.emboj.7600187 Present address: Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Ave., Cambridge, MA 02138, USA PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The functional coupling of transcription and splicing has been reported both in vivo and in vitro, but the molecular mechanisms governing these interactions remain largely unknown. Here we show that p54nrb, a transcription/splicing factor, associates with the 5′ splice site (SS) within large complexes present in HeLa cell nuclear extracts, in which the hyperphosphorylated form of RNA polymerase II (RNAPIIO) is associated with U1 or U1 and U2 snRNPs. These RNAPIIO–snRNP complexes also contain other transcription/splicing factors, such as PSF and TLS, as well as transcription factors that interact with RNAPIIO during elongation, including P-TEFb, TAT-SF1 and TFIIF. The presence of these factors in functional elongation complexes, demonstrated using an immobilized DNA template assay, strongly suggests that the RNAPIIO–snRNP complexes reflect physiologically relevant interactions between the transcription and splicing machineries. Our finding that both p54nrb and PSF, which bind the C-terminal domain of the largest subunit of RNAPII, can interact directly with the 5′ SS indicates that these factors may mediate contacts between RNAPII and snRNPs during the coupled transcription/splicing process. Introduction Splicing of mRNA precursors is an important step in eukaryotic gene expression, in which introns are removed from primary RNA polymerase II (RNAPII) transcripts. The majority of metazoan genes are interrupted by multiple introns, which comprise more than 95% of primary transcripts. Pre-mRNA splicing is executed by the spliceosome, whose assembly requires a complex series of binding and rearrangement events involving small nuclear ribonucleoprotein particles (snRNPs) (reviewed in Staley and Guthrie, 1998; Burge et al, 1999). In the first step of this assembly pathway, U1 snRNP binds the 5′ splice site (SS), in part due to the complementarity of the 5′ end of U1 snRNA to the 5′SS. Next, U2 snRNP binds to the branch site upstream of the 3′SS, generating pre-spliceosome complex A, which is subsequently converted into complex B upon joining of the U4/5/6 tri-snRNP. Additional rearrangements, involving significant changes in RNA:RNA interactions, yield the catalytically competent complex C, currently estimated to include five snRNAs and ∼150–300 proteins (Makarov et al, 2002; Rappsilber et al, 2002; Zhou et al, 2002). The early steps of spliceosome assembly can be bypassed in a simplified trans-splicing system, in which a DNA oligonucleotide complementary to the 5′ end of U1 snRNA (U1-blocking DNA) is used to block binding of a short 5′SS RNA substrate to U1 and promote its interaction with U4/5/6 snRNP and U2/4/5/6 splicing complexes (Konforti et al, 1993; Konforti and Konarska, 1994). Early biochemical and cytological studies indicated that splicing can occur co-transcriptionally (Beyer and Osheim, 1988; Bauren and Wieslander, 1994) and that splicing factors are recruited to active transcription sites (Huang and Spector, 1991; Zhang et al, 1994). Subsequent functional studies showed that the identity of the RNAPII promoter affects splicing patterns of the nascent transcript (Cramer et al, 1997, 1999), further connecting transcription and splicing. Reciprocally, splicing appears to stimulate transcription, as both splicing factors (Fong and Zhou, 2001) and splicing signals (Furger et al, 2002) can enhance gene transcription. The C-terminal domain (CTD) of the largest subunit of RNAPII appears to provide a platform on which to link physically transcription to splicing and other gene expression steps, such as capping, polyadenylation and termination of transcription (reviewed in Hirose and Manley, 2000; Bentley, 2002; Proudfoot et al, 2002). The mammalian RNAPII CTD is composed of 52 tandem repeats of the YSPTSPS consensus peptide (Corden et al, 1985). Phosphorylation of Ser2 and/or Ser5 of this heptapeptide is a key event during the transcription cycle, with the hypophosphorylated form of RNAPII (RNAPIIA) being found in pre-initiation complexes and the hyperphosphorylated form (RNAPIIO) in elongation complexes (ECs). Several studies have reported the association of RNAPIIO with snRNP particles (Mortillaro et al, 1996; Vincent et al, 1996; Kim et al, 1997), as well as the direct interaction of the CTD with some splicing factors (Morris and Greenleaf, 2000; Emili et al, 2002). Yet, the functional specificity of these interactions has not been demonstrated. Moreover, both RNAPIIO (Hirose et al, 1999) or the CTD alone (Zeng and Berget, 2000) can enhance splicing, but the mechanisms underlying this stimulation are not known. Polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF) and p54nrb are two closely related human nuclear proteins implicated in transcription and splicing, whose specific functions remain unclear. Several studies indicate that both factors are involved in transcriptional control (Basu et al, 1997; Urban et al, 2000; Mathur et al, 2001). However, while PSF has been shown to be essential for splicing in vitro (Patton et al, 1993), the direct involvement of p54nrb in pre-mRNA splicing has not been demonstrated. These multifunctional proteins have also been implicated in RNA editing and DNA recombination (reviewed in Shav-Tal and Zipori, 2002). Importantly, PSF and p54nrb were recently shown to interact with each other (Peng et al, 2002) and with the CTD (Emili et al, 2002). Using protein–RNA crosslinking assays, we have identified a direct interaction between p54nrb and the 5′SS. The crosslink resides within large complexes, which contain RNAPII and snRNPs and are present in HeLa cell nuclear extracts (NE) independently of the ongoing transcription or splicing. One of these complexes contains RNAPIIO and U1 snRNP, while another additionally includes U2 snRNP. Besides p54nrb, both RNAPII–snRNP complexes contain other transcription/splicing factors (e.g. PSF and TLS), as well as those typically found in RNAPII ECs (e.g. P-TEFb and TAT-SF1). These factors are also present in functional ECs isolated using an immobilized DNA template assay, strongly suggesting that the analyzed complexes reflect physiologically relevant interactions between the transcription and splicing machineries. Furthermore, we confirmed the previously observed crosslinking of PSF to the 5′SS within the U2/4/5/6 spliceosome formed in the absence of ATP (Ismaili et al, 2001) and show that this complex also associates with RNAPIIO. The observed direct association of p54nrb and PSF with the 5′SS within snRNP complexes suggests their direct role in linking transcription to splicing. Results Crosslinking patterns of 5′SS RNA associated with U1 snRNP To detect spliceosomal components interacting with the 5′SS, we previously analyzed crosslinking profiles of site-specifically modified, photoreactive 5′SS substrates (Ismaili et al, 2001). In the presence of U1-blocking DNA, the 5′SS RNA associates with U2, U4, U5 and U6 snRNPs (Konforti et al, 1993, and Figure 3A, lanes 1 and 2) and forms specific crosslinks with one of two splicing factors: PSF in the absence of ATP or hPrp28 in its presence (Ismaili et al, 2001, and Figure 1B, lanes 1 and 2). Figure 1.Crosslinking profile of the BP-derivatized 5′SS RNA. (A) Structure of the 32P-labeled, BP-derivatized 14-nt 5′SS RNA. (B) The 5′SS RNA was incubated with HeLa NE in the absence (lanes 1, 3) or presence (lanes 2, 4) of ATP and in the presence (lanes 1, 2) or absence (lanes 3, 4) of U1-blocking DNA. (C) Wild-type 5′SS RNA (WT) or 5′SS RNAs with single point mutations at intron pos. +1 or +2 was incubated with HeLa NE in the absence of ATP or U1-blocking DNA. (D) The 5′SS crosslinks were immunoprecipitated under denaturing conditions with α-Sm (Y12) antibodies. Crosslinked products were resolved in 11% SDS gels. Positions of the molecular weight markers and the 5′SS crosslinks are indicated. Download figure Download PowerPoint Here, we have used an analogous approach to analyze 5′SS interactions in the U1 snRNP complex. A 14-nt synthetic 5′SS RNA containing a 32P group between intron pos. +6 and +7 and a photoreactive benzophenone (BP) group between intron pos. +7 and +8 (Figure 1A) was incubated with HeLa NE in the absence of U1-blocking DNA and irradiated with 302-nm UV light. Under these conditions, the BP-5′SS RNA stably binds U1 snRNP in an ATP-independent manner (confirmed by native gel analysis; see Figure 3A, lanes 3–5). This binding yields a 5′SS:U1 snRNA crosslink (Figure 1B, lanes 3 and 4) identified by its sensitivity to RNase H digestion in the presence of U1-specific DNA oligos (data not shown). In addition, two major protein crosslinks, p18 and a 54/56-kDa doublet, were detected in these reactions (the sizes of all crosslinked proteins were corrected for the contribution of the RNA component). Consistent with their association with U1 snRNP, these two crosslinks were precipitated by α-U1A antibodies under native conditions (see Figure 2B, lane 6). The observed interactions are specific to the 5′SS, as the crosslinks were not detected using 5′SS RNAs with single point mutations at either intron pos. +1 or +2 (Figure 1C). Figure 2.The 5′SS:p54/56 crosslink corresponds to p54nrb. (A) The 5′SS:p54/56 crosslinks were treated with RNase A (lane 2) or RNase A and phosphatase (CIP) (lane 3) and resolved in an 8% SDS gel. (B) The 5′SS crosslinks were immunoprecipitated under denaturing (lanes 1–4) or native (lanes 5 and 6) conditions using the indicated antibodies and resolved in an 11% SDS gel. (C) The 5′SS:p54nrb and 5′SS:hPrp28 crosslinks were purified from an SDS gel, mixed (lane 1), immunoprecipitated with pre-immune serum (lane 2) or the indicated antibodies (lanes 3 and 4) and resolved in an 8% SDS gel. (D) Diagram of the human PSF and p54nrb sequences. The two RRMs, the proline/glutamine-rich regions (PQ), the 293-aa internal region of high similarity between p54nrb and PSF, the sequences of three Lys-C peptides and the mapped region of the 5′SS crosslink within p54nrb (aa 18–53) are indicated. Download figure Download PowerPoint Among the known U1 snRNP proteins, only U1C (17.5 kDa) and SmD (D1–13.5, D2–13.9 and D3–13.4 kDa) fall in the 18 kDa size range. p18 appears to represent one of the SmD proteins, as the 5′SS:p18 crosslink was efficiently recognized by the α-Sm antibody Y12 under denaturing conditions (Figure 1D). In yeast, the charged C-terminal tails of SmB, SmD1 and SmD3 directly contact the 5′SS bound to U1 snRNP, possibly stabilizing snRNP:intron interactions within the commitment complex (Zhang et al, 2001). This suggests that contacts between the 5′SS and Sm proteins within U1 snRNP are conserved between yeast and metazoans. Thus, crosslinking analysis using a photoreactive 5′SS RNA bound to U1 snRNP revealed the formation of two products (5′SS:U1 snRNA and 5′SS:SmD) expected of U1 snRNP interactions and a 54/56 kDa doublet crosslink of unknown origin. The p54/56 crosslinked to the 5′SS represents p54nrb, a transcription/splicing factor There are no known U1 snRNP proteins in the 55 kDa size range, suggesting that the p54/56 crosslink represents a novel protein associated with this particle. The p54/56 doublet represents two differentially modified forms of a single protein, as digestion of the individually purified bands with several proteases yielded identical peptide patterns (data not shown). In addition, phosphatase treatment of the crosslink doublet generated a single product of ∼54 kDa (Figure 2A, lane 3), indicating that the two forms of the doublet differ in their phosphorylation state. In an attempt to identify p54, we carried out immunoprecipitations under denaturing conditions using a battery of antibodies against selected splicing factors. To disrupt snRNP integrity, crosslinking reactions were first treated with RNase A, yielding faster migrating products due to the removal of eight 5′-terminal nucleotides of the 5′SS RNA in the crosslink (Figure 2A). Under these conditions, the α-SR antibody 16H3 immunoprecipitated the 5′SS:p54/56 doublet, but not the 5′SS:SmD crosslink (Figure 2B, lane 2). In contrast, antibodies against PTB (Figure 2B, lane 3), SF1, SRp54, U2AF65, hUAP56 or the 60K subunit of U2-SF3a (data not shown) did not recognize the 5′SS:p54/56 crosslink. Similarly, an α-U1A antibody, which efficiently precipitated both crosslinks under native conditions (Figure 2B, lane 6), failed to recognize them after the RNase A treatment (Figure 2B, lane 4), confirming that U1 snRNP was largely disrupted under these conditions. To identify p54/56, complexes from a large-scale crosslinking reaction were RNase-digested and resolved by two-dimensional SDS gel electrophoresis. Positions of the labeled crosslinks were visualized by autoradiography and superimposed with signals of Western blot analysis using the α-SR antibody that immunoprecipitated the crosslink and the protein pattern detected by Coomassie staining (data not shown). Microsequencing analysis of the ∼54-kDa protein detected by these three methods yielded three peptide sequences (VELDNMPLRGK, LVIK and QQQDQVDRNIK) identified as fragments of p54nrb (Figure 2D), a protein originally identified due to its crossreactivity with an antibody raised against the yeast splicing factor Prp18 (Dong et al, 1993). p54nrb shows no significant homology to yPrp18, but is instead closely related to human splicing factor PSF, with which it shares a region of high similarity (Figure 2D). Analysis of the p54nrb primary sequence reveals an N-terminal P/Q-rich segment, followed by two RNA recognition motifs (RRMs) (Figure 2D). Although p54nrb does not contain RS repeats, it must contain SR-like epitopes, as both the 5′SS:p54nrb crosslink (Figure 2B, lane 2) and recombinant, bacterially expressed p54nrb (data not shown) were recognized by α-SR antibodies. Finally, crosslink identity was confirmed by specific precipitation of the 5′SS:p54nrb crosslink from a mixture of SDS gel-purified p54/56 and hPrp28 crosslinks using α-p54nrb antibody (Figure 2C). Preliminary characterization of the crosslink (data not shown) mapped its site within p54nrb to a 36-aa segment (aa 18–53) in the N-terminal region of the protein (Figure 2D). We have therefore identified the p54/56 doublet as the transcription/splicing factor p54nrb, which crosslinks to the 5′SS via its N-terminal region. This protein shows significant homology with the splicing factor PSF, which also crosslinks to the 5′SS but within the U2/4/5/6 spliceosome complex formed in the absence of ATP. The 5′SS:p54nrb crosslink resides in large complexes containing U1 and U2 snRNP particles Crosslinking of p54nrb to the 5′SS RNA does not require ATP and is inhibited by blocking the 5′ end of U1 snRNA with a complementary DNA (Figure 1B) but not by equivalent amounts of other oligos (data not shown), suggesting an association dependent on binding of the 5′SS by U1 snRNP. To better characterize snRNP complexes associated with the 5′SS, crosslinking reactions were resolved in a native polyacrylamide gel (Figure 3A, lanes 1–4). As we have shown before (Konforti and Konarska, 1994), in the presence of U1-blocking DNA the 5′SS RNA associates with U2/4/5/6 and U4/5/6 snRNP complexes (Figure 3A, lanes 1 and 2), whereas in the absence of U1-blocking DNA it forms a characteristic, abundant complex with U1 snRNP (Konforti et al, 1993). In addition, two slowly migrating complexes, X1 and X2, were detected under these conditions (Figure 3A, lanes 3 and 4). The superimposable signals generated in reactions containing labeled 5′SS RNA and by Northern analysis of endogenous NE complexes using a U1 snRNA-specific probe (Figure 3A, lanes 3–5) suggested that both X1 and X2 contain U1 snRNP. This was confirmed by immunoprecipitation of glycerol gradient fractions (see below). While U4, U5 or U6 snRNA-specific signals were not detected in either X1 or X2 (Figure 3A, lanes 4 and 7), the most slowly migrating form of U2 snRNP and X2 exhibited similar mobility in the gel (Figure 3A, lanes 4 and 6, and data not shown). The formation of X2 requires incubation at 30°C, whereas X1 forms even on ice (data not shown). Moreover, X2 formation requires ATP, whereas the formation of X1 does not (Figure 3A, lanes 3 and 4). These features resemble requirements for the formation of splicing complexes involving U1 and U2 snRNPs. The distribution of the 5′SS:p54nrb crosslink in these complexes was analyzed by resolving crosslinking reactions in a native gel in the first dimension and in an SDS gel in the second (Figure 3B). The 5′SS:p54nrb crosslink was detected exclusively in X1 and X2, but not in the most abundant 5′SS:U1 snRNP complex. In contrast, both the 5′SS:U1 snRNA and SmD crosslinks were present in all complexes, further confirming that they all contain U1 snRNP. To better characterize these large complexes, X1 and X2 were separated by sedimentation through a 10–30% glycerol gradient (Figure 4A and B). Gradient fractions containing X1 (fraction 4) and X2 (fraction 7) were subjected to immunoprecipitation under native conditions using selected snRNP-specific antibodies (Figure 4C). As a control of their functionality, all antibodies were confirmed to precipitate the targeted protein, as shown by Western analysis of material immunoprecipitated from crosslinking reactions (data not shown). Anti-U1 70K antibody precipitated the 5′SS:p54nrb, SmD and U1 snRNA crosslinks from both fractions (Figure 4C, lanes 3 and 4), confirming that both X1 and X2 contain U1 snRNP. In contrast, the two U2 snRNP-specific proteins U2B″ and SAP155 (an SF3b component) were detected predominantly in fraction 7 (Figure 4C, lanes 5–8). We therefore conclude that X1 contains U1 snRNP, whereas X2 contains both U1 and U2 snRNPs. Consistent with this, hPrp5, FBP11 and FBP21 were found in both these complexes (Figure 4C, lanes 9–14). The higher level of these factors associated with X2 is consistent with their role in the U1–U2 snRNP interaction during pre-spliceosome assembly. X1 and X2 complexes do not appear to be associated with U4, U5 or U6 snRNPs, as α-U5-116K and α-hPrp4 antibodies (specific for components of U5 and U4/6 snRNPs, respectively) did not efficiently recognize any of the crosslinks (Figure 4C, lanes 15–18). Figure 3.The 5′SS:p54nrb crosslink exists exclusively in large complexes containing U1 snRNP. (A) Standard binding reactions in the presence (lanes 1–4) or absence (lanes 5–7) of the 5′SS RNA, in the absence (lanes 1,3) or presence (lanes 2,4–7) of ATP and in the presence (lanes 1,2) or absence (lanes 3–7) of U1-blocking DNA were resolved in a native 4% gel. snRNP complexes were detected by autoradiography (lanes 1–4) or Northern hybridization with U1, U2 or U4 probes (lanes 5–7). Positions of complexes are indicated. (B) The 5′SS crosslinks were resolved in a native 4% gel in the first dimension and in an 11% SDS gel in the second. Positions of the free 5′SS and the crosslinks are indicated. Download figure Download PowerPoint Figure 4.X1 and X2 complexes can be separated in a glycerol gradient. A 150-μl crosslinking reaction performed in the presence of ATP and absence of U1-blocking DNA was separated in a 10–30% glycerol gradient. In all, 10 μl of each fraction was resolved in either a 4% native gel (A) or an 11% SDS gel (B). (C) Fractions 4 and 7 of the glycerol gradient were immunoprecipitated under native conditions using the indicated antibodies, and the bound material was resolved in an 11% SDS gel. The identity of the ∼70 kDa signal generated with the α-TLS antibody (lane 21) has not been determined. Positions of the 5′SS crosslinks are indicated. Download figure Download PowerPoint The splicing factor PSF was originally isolated in a stable interaction with PTB (Patton et al, 1993). Anti-PSF, but not α-PTB (data not shown), antibodies precipitated the U1 snRNA, p54nrb and SmD crosslinks from the gradient-separated X1 and X2 (Figure 4C, lanes 19 and 20), indicating that PSF is present in both complexes. X1 and X2 were also recognized by an antibody raised against TLS (Figure 4C, lanes 21 and 22), another transcription/splicing factor. As expected, α-SR antibodies precipitated the crosslinks from both fractions 4 and 7 (data not shown), possibly reflecting the reactivity of U1-70K, p54nrb or SR proteins associated with X1 and X2. In summary, we have identified two large complexes, X1 and X2, within which the 5′SS crosslinks to p54nrb. Immunoprecipitation experiments using snRNP-specific antibodies show that both complexes contain U1 snRNP, whereas X2 also contains U2 snRNP. In addition to p54nrb, both X1 and X2 contain other transcription/splicing factors, such as PSF and TLS. The 5′SS:p54nrb-containing snRNP complexes are associated with RNAPIIO and transcription factors The electrophoretic and sedimentation properties of X1 and X2 indicated high molecular weight of these complexes (Figures 3A and 4A). Furthermore, the presence of PSF, p54nrb and TLS proteins, all of which can act as transcription factors and bind to both RNA and DNA, suggested a connection between X1, X2 and transcription. To test this possibility, immunoprecipitation of crosslinking reactions was carried out using several antibodies against RNAPII specific for the N-terminal segment (N-20) or the CTD (8WG16 recognizes the hypophosphorylated form, whereas H5 and H14 recognize the Ser2- and Ser5-phosphorylated forms, respectively). The N-20, H5 and H14 antibodies all precipitated the p54nrb, SmD and U1 snRNA crosslinks (Figure 5A, lanes 3, 5 and 6), indicating the presence of RNAPIIO. Similar experiments using gradient-separated X1 and X2 confirmed that both complexes are associated with RNAPIIO (data not shown). In contrast, the 8WG16 antibody did not efficiently recognize X1 or X2 complexes (data not shown and Figure 5A, lane 4), although it did precipitate large amounts of U1 snRNA (Figure 5C, lane 4). Therefore, hypophosphorylated RNAPIIA can bind both p54nrb (Emili et al, 2002) and U1 snRNPs, but this association does not produce the 5′SS:p54nrb crosslink, suggesting that rearrangement of RNAPII-bound factors takes place following CTD phosphorylation. Figure 5.The p54nrb crosslink in X1 and X2 complexes is associated with RNAPIIO. A crosslinking reaction performed in the presence of ATP and in the absence (A) or presence (B) of U1-blocking DNA was immunoprecipitated under native conditions using the indicated α-RNAPII antibodies and the bound material resolved in an 11% SDS gel. The identity of the ∼65 kDa signal (e.g. lane 3 in panel A) has not been determined. (C) HeLa NE was incubated with ATP in the absence of the 5′SS RNA and immunoprecipitated as in (A). The bound RNA was analyzed by Northern hybridization with a mixture of U1, U2, U4, U5 and U6 snRNA probes. Positions of snRNAs are indicated. Although co-migrating with U6, the signal indicated by an arrow in lane 4 represents a degradation product of U1 snRNA. Download figure Download PowerPoint The preferential association with RNAPIIO is characteristic not only of X1 and X2 but also of the spliceosome. The U2/4/5/6 complex, formed in the presence of U1-blocking DNA and ATP, binds RNAPIIO as evidenced by the precipitation of the 5′SS:hPrp28 crosslink with α-RNAPIIO antibodies (Figure 5B). Similarly, spliceosome complexes formed in the absence of ATP, in which the 5′SS RNA crosslinks to PSF (see Figure 1B, lane 1), are also associated with RNAPIIO (data not shown). These findings are consistent with earlier reports demonstrating a connection between spliceosome assembly and RNAPIIO (Hirose et al, 1999). Additional immunoprecipitation experiments demonstrated the association of X1 and X2 with two basal transcription factors, TFIIF and TFIIH, as indicated by the presence of their RAP74 and p62 subunits, respectively (Figure 5A, lanes 7 and 8). In contrast, the initiation factor TFIIB was not detected in X1 or X2 (data not shown). Moreover, the transcription factor Sox6 and two elongation factors, P-TEFb (or its Cdk9 kinase subunit) and TAT-SF1, were detected in these complexes (Figure 6, lanes 7–12). Figure 6.X1 and X2 complexes contain several transcription elongation factors. A crosslinking reaction performed in the presence of ATP was separated in a glycerol gradient, and fractions containing X1 (fraction 4) and X2 (fraction 7) complexes were immunoprecipitated under native conditions using the indicated antibodies and the bound material was resolved in an 11% SDS gel. Download figure Download PowerPoint Both X1 and X2 are therefore associated with RNAPII preferentially in its hyperphosphorylated, elongating form. Consistently, X1 and X2 also contain transcription elongation factors, including P-TEFb or TAT-SF1, but lack factors typically involved only in the initiation phase of transcription, such as TFIIB. The RNAPIIO–snRNP complexes contain a subset of functional EC factors The association of X1 and X2 with RNAPIIO and multiple transcription/splicing and elongation factors suggests that these complexes reflect associations formed during transcriptional elongation. To compare X1 and X2 with functional complexes involved in active transcription, an immobilized DNA template assay (Ranish et al, 1999) was used to isolate ECs. A biotinylated, 889-bp DNA transcription template containing the adenovirus major late promoter (AdMLP) was linked to magnetic streptavidin beads (Ad22, Figure 7A). Negative controls included a 490-bp DNA template identical to Ad22 but lacking the AdMLP (Ad20, Figure 7A) and reactions without any DNA bound to beads. After incubation of the immobilized DNA with NE to allow transcription, the templates were washed and cleaved at a unique PvuII site 32 bp downstream of the RNAPII start site (Figure 7A). This step liberates the formed ECs from the beads, while retaining initiation complexes assembled on the promoter-containing DNA fragments bound to the beads. The composition of the released ECs was then analyzed by SDS–PAGE and Western blotting. The Ad22 immobilized template yielded the expected 398-nt RNAPII transcript, whereas no transcript was detected with Ad20 (see Figure 7C), indicating that the complexes formed on the Ad22 template were active in transcription. As PvuII digestion released similar amounts of Ad20 and Ad22 DNA fragments (data not shown), the absence of signal from the Ad20 control reflects the lack of EC formation on this template. Figure 7.X1 and X2 complexes contain a subset of factors present in functional transcription ECs. (A) Schematic representation of the immobilized Ad20 and Ad22 DNA templates, showing the AdMLP, the two leading exons of the adenovirus major late transcription unit (black boxes), the start site for RNAPII transcription and the unique PvuII restriction sites. (B) Western blot analysis of protein factors bound to template fragments released from beads lacking DNA or linked to the Ad20 or Ad22 templates using the indicated antibodies. (C) Northern blot analysis of template fragments released from beads lacking DNA or linked to the Ad20 or Ad22 templates using probes for all the five U snRNAs and the Ad22 transcript. Download figure Download PowerPoint Consistent with the formation of transcription ECs on the Ad22 template, the N-20 antibody detected RNAPII preferentially in the hyperphosphorylated form (Figure 7B). Moreover, the transcription/splicing factors p54nrb, PSF and TLS were detected at considerable levels. Other splicing factors, such as the U2 snRNP-associated hPrp5 or SAP155, were also associated with ECs formed on the Ad22, but not on Ad20 DNA or on beads without DNA. In addition, the U5 snRNP-associated hPrp8 and the U4/6-associated hPrp4 were detected, indicating the presence of U4, U5 and U6 snRNPs in functional ECs. This was confirmed by Northern blot analysis (Figure 7C), which detected all five snRNAs associated with the Ad22 template. Other EC-associated proteins (Figure 7B) included the transcription elongation factors TAT-SF1, P-TEFb