Association of U2 snrnp with the spliceosomal complex E

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1 Nucleic Acids Research, 1997, Vol. 25, No Oxford University Press Association of U2 snrnp with the spliceosomal complex E Wanjin Hong, Maria Bennett 1, Yan Xiao, Rebecca Feld Kramer 1, Changyu Wang 1 and Robin Reed 1, * Membrane Biology Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore and 1 Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA Received September 12, 1996; Revised and Accepted November 14, 1996 ABSTRACT In metazoans, the E complex is operationally defined as an ATP-independent spliceosomal complex that elutes as a single peak on a gel filtration column and can be chased into spliced products in the presence of an excess of competitor pre-mrna. The A complex is the first ATP-dependent functional spliceosomal complex. U1 snrnp first binds tightly to the 5 splice site in the E complex and U2 snrnp first binds tightly to the branch site in the A complex. In this study, we have generated and characterized a monoclonal antibody (mab 4G8) directed against SAP 62, a component of U2 snrnp and a subunit of the essential mammalian splicing factor SF3a. We show that this antibody is highly specific for SAP 62, detecting only SAP 62 on Western blots and immunoprecipitating only SAP 62 from nuclear extracts. The anti-sap 62 antibody also immunoprecipitates U2 snrnp and the A complex. Significantly, however, we find that the E complex is also efficiently immunoprecipitated by the anti-sap 62 antibody. This antibody does not cross-react with any E complex-specific components, indicating that SAP 62 itself is associated with the E complex. To determine whether other U2 snrnp components are associated with the E complex, we used antibodies to the U2 snrnp proteins B and SAP 155. These antibodies also specifically immunoprecipitate the E complex. These observations indicate that U2 snrnp is associated with the E complex. However, we find that U2 snrnp is not as tightly bound in the E complex as it is in the A complex. The possible significance of the weak association of U2 snrnp with the E complex is discussed. INTRODUCTION The pre-mrna splicing reaction requires the formation of a series of highly dynamic spliceosomal complexes. These complexes are composed of U1, U2, U4, U5 and U6 small nuclear RNAs (snrnas) and >50 proteins (reviewed in 1). The E, A and B complexes contain unspliced pre-mrna and the C complex contains the products of catalytic step I of the splicing reaction (exon 1 and lariat exon 2). Two complexes, one containing the excised lariat intron (i complex) and the other containing the spliced exons (D complex), are the products of catalytic step II of the splicing reaction. The ATP-independent E complex was originally identified as a spliceosomal complex that elutes in a single peak on a gel filtration column and can be efficiently chased into spliced products in the presence of excess competitor pre-mrna (2 4). The E complex is thought to be the mammalian counterpart of the yeast commitment complex, which was identified prior to the E complex and shown to be a functional intermediate in the yeast spliceosome assembly pathway (5). The E complex is also likely the same as a mammalian complex identified on density gradients and shown to commit pre-mrnas to the splicing pathway (6). Highly purified mammalian E complex contains U1 snrnp, U2AF 35, U2AF 65 and several spliceosome-associated proteins (SAPs) (4; reviewed in 7). U2AF 65 binds to the pyrimidine tract at the 3 splice site in the E complex and U1 snrna base pairs to the 5 splice site. Both U1 snrnp and U2AF are destabilized from the pre-mrna during the E B complex transition, possibly as early as A complex assembly (8). The ATP-dependent A complex contains U2 snrnp and an essential duplex is formed between U2 snrna and the branch point sequence (BPS). This duplex is thought to specify the branch site adenosine as the nucleophile for the first transesterification reaction (9). U2 snrnp can also bind stably to the BPS in the A3 complex, which assembles on a pre-mrna lacking the 5 splice site (10). Two multimeric splicing factors, SF3a and SF3b, are components of U2 snrnp and are required for A complex assembly. SF3a consists of the spliceosome-associated proteins SAPs 61, 62 and 114 and SF3b is thought to consist of SAPs 49, 130, 145 and 155 (11,12; reviewed in 7). All of these proteins, except SAP 130, UV crosslink to pre-mrna in the A complex to a 25 nt region upstream of the branch site (13,14). The interactions of SF3a/b with this region, designated the anchoring site, are thought to play a central role in the tight binding of U2 snrnp to pre-mrna (14). U2 snrnp remains stably bound to pre-mrna in the B complex and the U4/U5/U6 tri-snrnp first binds to pre-mrna at this time (10; reviewed in 1). U6 snrna interacts with intron *To whom correspondence should be addressed. Tel: ; Fax: ; rreed@warren.med.harvard.edu

2 355 Nucleic Acids Research, 1994, 1997, Vol. Vol. 22, 25, No. No sequences at the 5 splice site, replacing U1 snrna (15 20). Base pairing interactions between U2 and U6 snrnas, in conjunction with the U6 snrna 5 splice site duplex, are thought to position the BPS near the 5 splice site for catalytic step I of the splicing reaction (21; for a review see 22). U5 snrnp also interacts with exon sequences at both the 5 and 3 splice sites and this interaction may hold the exons together for ligation (for reviews see 22,23). In this study, we have generated a monoclonal antibody (mab 4G8) that is highly specific for the SF3a subunit SAP 62. Unexpectedly, we found that this antibody, as well as antibodies to other U2 snrnp components, efficiently immunoprecipitates the E complex. These data, together with other observations, indicate that U2 snrnp is weakly bound to pre-mrna in the E complex and tightly bound in the subsequent spliceosomal complexes. The possible functional significance of the loose association of U2 snrnp with the E complex is discussed. MATERIALS AND METHODS Isolation of mab 4G8 Rat liver nuclei were isolated as described (24). The resulting nuclear pellet was extracted with 10 mm EGTA in 10 mm HEPES, ph 7.4, and the EGTA-extracted proteins were used as the antigen source for the preparation of monoclonal antibodies (25). The resulting hybridoma supernatants were screened by immunofluorescence for nuclear labeling and selected hybridomas were cloned. mab 4G8 is of the IgG1 subtype (ELISA class test kit, Gibco BRL). Ascitic fluid of mice was collected as described (25). IgG was purified using an antibody purification kit (Pierce) and the eluted antibody was dialyzed into phosphate-buffered saline (PBS). Immunofluorescence and in situ cell fractionation Cells grown to 50 80% confluency on coverslips were washed twice with PBSCM (PBS containing 1 mm CaCl 2 and 1 mm MgCl 2 ) and fixed in 3% paraformaldehyde, PBSCM for 30 min at room temperature. After washing with 0.3% NH 4 Cl, PBSCM and permeabilization in PBSCM containing 0.3% Triton X-100, cells were incubated with antibody as indicated, washed with 0.2% Triton X-100, PBSCM and incubated with sheep anti-mouse IgG conjugated to FITC (or rhodamine) (26). After washing with 0.2% Triton X-100, PBSCM, cell were mounted with 0.1% phenylenediamine and 80% glycerol in PBS or fixed by other methods as indicated. HeLa cells grown on poly(l-lysine)-coated coverslips were permeabilized for 3 min at 4 C by incubation with hypotonic buffer (10 mm HEPES KOH, ph 7.9, 10 mm KCl, 1.5 mm MgCl 2, 0.5 mm DTT, 0.1% Triton X-100). After washing with the same buffer, KCl was added to a final concentration of 0.15, 0.3 or 0.4 M by stepwise application from a stock solution of 2 M KCl in hypotonic buffer. Cells were fixed in methanol for 5 min on ice and processed for immunofluorescence. For Western blot analysis, cells grown in suspension were extracted as described above, centrifuged at g for 5 min and the supernatant and pellets were subjected to SDS PAGE and Western blot analysis. Spliceosomal complex assembly and purification padml, pad5 and pad3 were as described previously (3,4,27). Assembly of the H complex and the spliceosomal complexes E, E3, E5, A3 and B was carried out as described (2 4). Reaction mixtures were incubated for 5 min for E or A complex assembly and for 15 min for B complex assembly. Reaction mixtures for assembly of the E complex lacked ATP and MgCl 2. Complexes were isolated by gel filtration and, where indicated, also by affinity purification (2,27). For gel shift assay, reaction mixtures were adjusted to 0.5 mg/ml heparin and separated on a 4% native gel (10). Western analysis and immunoprecipitation Western analysis of proteins from cellular extracts was performed as described (26). Filters containing immobilized proteins were incubated in mab 4G8 tissue culture supernatant or a 1:500 dilution of ascitic fluid. Rabbit anti-mouse IgG and [ 125 I]protein were used for detection (Fig. 1B). For Western analysis of spliceosomal proteins, total protein from affinity purified spliceosomes assembled on AdML pre-mrna was separated by SDS or twodimensional gel electrophoresis and transferred to nitrocellulose. Blots were probed with mab 4G8 ascitic fluid at 1:1000 dilution, with anti-b antibody (mab 4G3) tissue culture supernatant (undiluted) or with anti-sap 155 polyclonal antibodies at 1:1000 dilution. Detection was with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (Amersham). For immunoprecipitation of metabolically labeled SAP 62, subconfluent monolayers of cells were labeled with [ 35 S]methionine and/or [ 35 S]cysteine (28). Cell extracts were then used for immunoprecipitations with mab 4G8. Immunoprecipitation of in vitro translated SAP 62 To map the mab 4G8 epitope, derivatives of SAP 62 (29) were produced by in vitro translation. psap 62(1 257) was constructed by deleting the sequence between the StuI and BamHI sites in psap 62. psap 62( ) was constructed by deleting the sequence in psap 62 between the NcoI and RsrII sites. For immunoprecipitations, mab 4G8 or mab 2G2 was coupled to protein A Trisacryl and rotated with 1 µl of in vitro translation reaction mixture in 100 µl PBS for 1 h at 4 C and washed with cold buffer (10 mm Tris HCl, ph 7.4, 500 mm NaCl, 1% NP40). Immunoprecipitation of spliceosomal complexes and U2 snrnp Aliquots (200 µl) of gel filtration isolated complexes were rotated at 4 C with 3 µl purified mab 4G8 coupled to protein A Trisacryl. For immunoprecipitation of the E complex in high salt, the buffer was adjusted to 300 mm NaCl. After washing, total RNA in the bound and unbound fractions was extracted and fractionated on 8% denaturing gels. Immunoprecipitation of complexes using the anti-sap 155 antibody or the anti-b antibody (mab 4G3) was performed as above except that 25 µl αsap 155 rabbit serum or 500 µl mab 4G3 from tissue culture supernatant respectively were used. Preimmune serum or rabbit secondary antibody were used as negative controls. To immunoprecipitate U2 snrnp, mab 4G8 was coupled to beads as above and rotated for 3 h at 4 C with either 100 µl nuclear extract or 100 µl nuclear extract that had been pre-incubated under splicing conditions (+ATP, +MgCl 2 ) for 5 min at 30 C. The secondary antibody alone was used as a control. After washing in PBS, 300 mm NaCl, 1% NP40, total RNA was extracted, fractionated on an 8% denaturing gel and visualized by ethidium bromide staining.

3 356 Nucleic Acids Research, 1997, Vol. 25, No. 2 Figure 1. SAP 62 is present in nuclear speckles and co-localizes with splicing factors. (A) Double labeling immunofluorescence microscopy showing co-localization of the 4G8 antigen (panels A and C) with SC35 (panel B) and Sm antigens (panel D) in HeLa (panels A and B) and C6 glioma (panels C and D) cells. The same speckled pattern was observed with the 4G8 antibody when HeLa cells were fixed on ice with 2.7% paraformaldehyde or at 20 C with methanol, acetone or 1:1 methanol and acetone (data not shown). (B) The 4G8 antigen is 60 kda and is associated with nuclear speckles. (Top panel) Immunofluorescence localization of the 4G8 antigen in HeLa cells after different salt extractions. Monolayers of HeLa cells were extracted with hypotonic buffer (panel b) and then by stepwise addition of 2 M KCl to final concentrations of 0.15 (panel c), 0.3 (panel d) or 0.4 M (panel e). Non-extracted cells are shown in panel a. Cells in panel b were incubated with mab 4G8 and then processed for indirect immunofluorescence; the others were fixed and then processed. (Bottom panel) HeLa cells were extracted as in (A) and the extracted fractions (S) and pellets (P) were separated by SDS PAGE; Western analysis was then performed using mab 4G8 (the high molecular weight material detected at the gel origin is likely due to aggregation of proteins). Each preparation started from the same number of cells. Molecular size markers (kda) are indicated. RESULTS mab 4G8 recognizes the splicing factor SAP 62 During a screen of a panel of monoclonal antibodies generated against nuclear components (see Materials and Methods), we identified an antibody, mab 4G8, that specifically labels subnuclear regions in HeLa cells (Fig. 1A, panel A). Significantly, the 4G8 antigen co-localizes with the splicing factor SC35 (30), as well as with snrnp core proteins which bear the Sm epitope (31) (Fig. 1A, panels A D). The structures labeled by these splicing factors are referred to as nuclear speckles and are thought to be sites of storage and/or assembly of spliceosomal components (30,32 34). In order to identify the 4G8 antigen and determine whether a single antigen is responsible for the speckled immunoflouresence staining pattern, mab 4G8 was used to stain cells extracted in increasing concentrations of salt after permeabilization in Triton X-100 (Fig. 1B, top panel). Total protein was prepared from the cell pellets and supernatants after the salt extractions and then used for Western analysis with the 4G8 antibody (Fig. 1B, bottom panel). At salt concentrations up to 0.15 M KCl, a speckled immunostaining pattern similar to that obtained after conventional permeabilization in isotonic buffer was observed (Fig. 1A, panels A C). Much less 4G8 antigen was detected in the speckled pattern after incubation with 0.3 or 0.4 M KCl (Fig. 1B, panels d and e). As shown in Figure 1B, mab 4G8 detects a single 60 kda protein on a Western blot and the majority of this protein is associated with the cell pellet at salt concentrations up to 0.15 M (lanes 1 4); in contrast, the 60 kda protein is largely extracted at 0.3 M KCl and above (lanes 5 10). These data indicate that the mab 4G8 antigen is a 60 kda protein. In addition, the correlation between the immunofluorescence patterns and the Western analysis at the different salt concentrations indicates that the 60 kda protein and not a cross-reacting protein(s) is the antigen responsible for the speckled staining pattern. To determine whether the 60 kda mab 4G8 antigen corresponds to a splicing factor, a Western blot of spliceosomal proteins

4 357 Nucleic Acids Research, 1994, 1997, Vol. Vol. 22, 25, No. No Figure 2. mab 4G8 recognizes the proline-rich C-terminal portion of the splicing factor SAP 62. (A) Purified spliceosomes assembled on 300 ng AdML pre-mrna were separated by two-dimensional gel electrophoresis, immobilized on nitrocellulose and probed with mab 4G8. The immunoreactive spot was identified as SAP 62 by examination of an ink stain of the blot (data not shown). Molecular size markers (kda) are indicated. (B) The proline-rich domain and the conserved zinc finger in SAP 62 are indicated in the schematic of the SAP 62 protein, with amino acid positions shown on the right. SAP 62 or the indicated derivatives of SAP 62 were produced by in vitro translation using [ 35 S]methionine; the translation products are designated by arrows (lanes 1 3). SAP 62 or SAP 62 derivatives as indicated were immunoprecipitated with mab 4G8 (lanes 4, 7 and 10), a negative control antibody (mab 2G2) (lanes 5, 8 and 11) or the secondary rabbit antibody alone (lanes 6, 9 and 12). Molecular size markers (kda) are indicated on the right. fractionated on a two-dimensional gel was carried out. As shown in Figure 2A, 4G8 detects a single protein and an ink stain of this two-dimensional blot (data not shown) showed that this protein is the U2 snrnp component SAP 62. In vitro-translated SAP 62 (Fig. 2B, lane 1) is also specifically immunoprecipitated by mab 4G8 (Fig. 2B, lane 4). In vitro-translated SAP 62 is not immunoprecipitated by a control monoclonal antibody (2G2) or by the secondary antibody alone (R) (Fig. 2B, lanes 5 and 6 respectively). Thus, mab 4G8 specifically recognizes both the native as well as the denatured form of SAP 62. To identify the epitope recognized by mab 4G8, N-terminal (residues 1 257) and C-terminal (residues ) portions of SAP 62 (see schematic, Fig. 2B) were generated by in vitro translation (Fig. 2B, lanes 2 and 3). Immunoprecipitation assays were then carried out. mab 4G8 immunoprecipitates the C-terminal proline-rich portion of SAP 62 (Fig. 2B, lane 7) as efficiently as full-length SAP 62 (Fig. 2B, lane 4). In contrast, the N-terminal region is not immunoprecipitated by mab 4G8 (Fig. 2B, lane 10). None of the SAP 62 in vitro translation products are immunoprecipitated by the control antibodies (Fig. 2B, lanes 5, 6, 8, 9, 11 and 12). We conclude that the 4G8 epitope resides in the proline-rich C-terminal domain of SAP 62. In addition to detecting a single band on a Western blot (Fig. 1B), mab 4G8 specifically immunoprecipitates a single major 60 kda band from human (HeLa and A431), monkey (Vero) and rat (C6) cell lines (Fig. 3A, lanes 1 4; the faint lower molecular weight bands are most likely breakdown products of the 60 kda protein). Thus, SAP 62 is phylogenetically conserved, both in size and in reactivity with 4G8. These data, together with the Western analysis, indicate that mab 4G8 is highly specific for SAP 62 and does not cross-react with other cellular proteins. Consistent with the observation that mab 4G8 recognizes SAP 62, we find that 4G8 also specifically immunoprecipitates U2 snrnp from HeLa cell nuclear extracts; this snrnp is not immunoprecipitated by the secondary antibody alone (Fig. 3B, lanes 2 and 3). SAP 62 remains associated with U2 snrnp when Figure 3. mab 4G8 immunoprecipitates a 60 kda protein from cellular lysates and U2 snrnp from nuclear extract. (A) Extracts from HeLa (lane 1), Vero (lane 2), A431 (lane 3) or C6 (lane 4) cells labeled with [ 35 S]methionine and [ 35 S]cysteine were used for immunoprecipitation with mab 4G8. Molecular size standards (kda) are indicated. (B) HeLa cell nuclear extract (lanes 2 and 3) or nuclear extract preincubated with ATP and MgCl 2 for 5 min at 30 C (lanes 4 and 5) was rotated for 2.5 h at 4 C with mab 4G8 or secondary antibody alone immobilized on protein A Trisacryl. After washing, RNA was extracted, fractionated on an 8% denaturing gel and visualized by ethidium bromide staining. An aliquot of nuclear extract was run as a marker; U1 and U2 snrnas are indicated. Ori indicates the origin of the gel. nuclear extracts are pre-incubated in the presence of ATP (Fig. 3B, lane 4). Thus, the association of SAP 62 with U2 snrnp is not significantly affected by ATP (11). As show in Figure 3B (lane 2), we find that a low level of U1 snrnp is immunoprecipitated by mab 4G8. This observation is consistent with previous work showing that a small fraction of the U2 snrnp in the nuclear extract is associated with U1 snrnp (35; see Discussion). We were unable to prepare splicing extracts specifically immunodepleted of

5 358 Nucleic Acids Research, 1997, Vol. 25, No. 2 Figure 4. Spliceosomal complexes E and B are immunoprecipitated by mab 4G8. (A) B complex (lanes 1 4), H complex (lanes 5 8) and E complex (lanes 9 and 10) were assembled on 32 P-labeled AdML pre-mrna, fractionated by gel filtration and immunoprecipitated with mab 4G8 (lanes 1, 2, 5, 6, 9 and 10) or secondary antibody alone (lanes 3, 4, 7 and 8). After washing, the pre-mrna present in the pellet (P) and supernatant (S) was extracted and fractionated on an 8% denaturing gel. (B) 32 P-Labeled AdML pre-mrna was incubated in splicing extracts in the absence (lanes 1 and 2) or presence of ATP and MgCl 2 (lanes 3 and 4) for the times indicated and then fractionated on a 4% native gel. The H, E, A and B complexes are indicated. Note that the E and H complexes co-fractionate. SAP 62 (or U2 snrnp) using mab 4G8 (data not shown). In addition, we were unable to detect specific inhibition of splicing when 4G8 antibodies were added to or pre-incubated with nuclear extracts. Thus, although mab 4G8 specifically recognizes SAP 62, the interaction is weak and/or is with a partially masked epitope on the protein. U2 snrnp components are weakly associated with the spliceosomal complex E To characterize the association of SAP 62 with spliceosomal complexes, we used the 4G8 antibody for immunoprecipitation studies (3) (see Materials and Methods). We found that mab 4G8 efficiently immunoprecipitates the B complex, but not the H complex [Fig. 4A, compare the relative levels of pre-mrna in the pellet (P) and supernatant (S); lanes 1 and 2 versus lanes 5 and 6]. The secondary antibody alone does not immunoprecipitate either complex (Fig. 4A, lanes 3, 4, 7 and 8). Unexpectedly, we found that mab 4G8 also efficiently immunoprecipitates the E complex (Fig. 4A, lanes 9 and 10; see below for additional controls with non-specific antibodies). The E complex is immunoprecipitated somewhat less efficiently than the B complex (Fig. 4A, compare the ratio of pre-mrna in lanes 9 and 10 with that in lanes 1 and 2), indicating that SAP 62 is bound less tightly to the E complex or is present in only a sub-population of these complexes (see below for further data indicating that the antigen detected by 4G8 in the E complex is SAP 62). One possible explanation for the detection of a U2 snrnp component in the E complex is that low levels of the A complex contaminate the E complex. To examine this question, we assembled the E or A complexes under the same conditions used for the immunoprecipitations and then fractionated the complexes on a native gel. As shown in Figure 4B (lanes 1 4), no A complex is detected under E complex conditions (i.e. in the absence of ATP), indicating that SAP 62 is present in the E complex. Further evidence that the E complex is not contaminated with the A complex is the observation that the SF3a/b subunits crosslink in the A complex, whereas no crosslinking of these proteins at all can be detected in the E complex (8). To further investigate the association of SAP 62 with the E complex, immunoprecipitations were carried out using mab 4G8 or a control antibody in the presence of low (60 mm) or high (300 mm) salt. Neither the E nor the H complex was immunoprecipitated by the control antibody in high or low salt (Fig. 5A, lanes 5, 6, 9, 10, 13, 14, 17 and 18). In addition, the H complex was not immunoprecipitated by mab 4G8 at either salt concentration (Fig. 5A, lanes 7, 8, 15 and 16). In contrast, the E complex was immunoprecipitated by mab 4G8 at low salt, but not at high salt (Fig. 5A, lanes 3, 4, 11 and 12). To ensure that immunoprecipitation of the E complex was specific for the 4G8 antibody, we carried out additional immunoprecipitations of the E complex in low salt (125 mm). In this case, the amount of immunoprecipitated E complex was determined by Cerenkov counting the 32 P-labeled pre-mrna in the immunoprecipitation pellets. We observed that 31% of the pre-mrna assembled into the E complex was immunoprecipitated by the 4G8 antibody, while significantly lower levels were immunoprecipitated with three non-specific antibodies (mab CD18, 3%; rabbit anti-mouse pab, 2%; LexA pab, 2%). Together these data indicate that SAP 62 is specifically, but loosely, associated with the E complex. The observation that SAP 62 is associated with the E complex raised the possibility that other U2 snrnp components may also be associated with this complex. To address this question, we used a monoclonal antibody to the U2 snrnp protein B (designated 4G3; 31) and a polyclonal antibody to another U2 snrnp protein, SAP 155 (C.Wang and R.Reed, unpublished results). As expected, the A complex was immunoprecipitated by these antibodies (Fig. 5B, lanes 11, 12, 15 and 16). Significantly, the E complex was also specifically immunoprecipitated (Fig. 5B, lanes 3, 4, 7 and 8). Control antibodies did not immunoprecipitate either the E or A complexes (Fig. 5B, lanes 5, 6, 9, 10, 13, 14, 17 and 18). These data indicate that B and SAP 155 are also specifically associated with the E complex (see below for data indicating that the antigens detected in the E complex by the anti-b and anti-sap 155 antibodies are B and SAP 155 respectively). We note that it is not possible to assay for U2 snrna levels in the immunoprecipitated E and A complexes because, while these complexes are enriched in the gel filtration fractions, the levels of endogenous U2 snrnp are vastly higher. Thus, it is not possible to distinguish between endogenous U2 snrnp and the U2 snrnp in the E or A complexes using immunoprecipitation as an assay. The observation that several U2 snrnp components are associated with the E complex indicates that U2 snrnp itself is most likely present in the E complex. To investigate the nature of this association, we asked whether the anti-b antibody could immunoprecipitate the E3 or E5 complexes (which are E-type complexes that assemble on RNAs lacking the 5 or 3 splice site respectively; 4). Surprisingly, the data reveal that both the E5 and E3 complexes are immunoprecipitated by the anti-b antibody (Fig. 5C, lanes 3 6). As expected, the A complex assembled on

6 359 Nucleic Acids Research, 1994, 1997, Vol. Vol. 22, 25, No. No Figure 6. Detection of B, SAP 155 and SAP 62 in the E and A complexes. Affinity purified A (lanes 1, 3 and 5) or E complex (lanes 2, 4 and 6) were separated on a 9% SDS gel, transferred to nitrocellulose and probed with anti-b (lanes 1 and 2), anti-sap 155 (lanes 3 and 4) or anti-sap 62 (lanes 5 and 6) antibodies. in the E complex than in the A complex. Thus, both the Western analysis and the immunoprecipitation studies using U2 snrnpspecific antibodies support the conclusion that U2 snrnp is associated with the E complex. Figure 5. Immunoprecipitation of the E complex with antibodies to U2 snrnp. (A) The E complex (lanes 3 6 and 11 14) and the H complex (lanes 7 10 and 15 18) were assembled, isolated by gel filtration and then immunoprecipitated with mab 4G8 or secondary antibody alone (R) as indicated. The immunoprecipitations and washes were carried out in either 60 or 300 mm salt as shown. RNA present in the pellet (P) and supernatant (S) was extracted and analyzed on an 8% denaturing gel. The band corresponding to the pre-mrna is indicated. Lanes 1 and 2 are input RNA from the E and H complex peaks respectively. (B) The E complex (lanes 3 10) and the A complex (lanes 11 18) were assembled, isolated by gel filtration and immunoprecipitated with anti-sap 155 polyclonal antibodies or an anti-b monoclonal antibody (mab 4G3) as indicated. Preimmune serum (pre) or secondary antibody alone (R) were used as controls. Immunoprecipitations and washes were carried out in 60 mm salt. Lanes 1 and 2 are input pre-mrna isolated from the E and H complexes respectively. (C) The A3 (lanes 1 and 2), E3 (lanes 3 and 4), E5 (lanes 5 and 6) and H complexes (lanes 7 and 8) were assembled on 32 P-labeled Ad3 or Ad5 pre-mrna, fractionated by gel filtration and immunoprecipitated with the anti-b antibody. The pre-mrna in the pellet (P) and supernatant (S) was extracted and analyzed as described above. the 3 portion of the intron (A3 ; Fig. 5C, lanes 1 and 2) is also specifically immunoprecipitated, whereas H5 complex is not (Fig. 5C, lanes 7 and 8). These data indicate that B (and most likely U2 snrnp) can associate independently with both the 5 and 3 portions of the intron. One possible explanation for the observation that antibodies to B, SAP 62 or SAP 155 immunoprecipitate the E complex is that these antibodies are cross-reacting with E complex-specific proteins. To address this question, we carried out side-by-side Western blots of affinity purified E and A complexes using the anti-b antibody, the anti-sap 155 antibody and mab 4G8. As shown in Figure 6 (lanes 1 6), these antibodies do not cross-react with any E complex-specific proteins. Instead, the antibodies detect the expected antigens for each antibody, but at lower levels DISCUSSION In this study, we have characterized a monoclonal antibody (mab 4G8) that recognizes SAP 62, a U2 snrnp component and subunit of the essential splicing factor SF3a (11,12,29). SAP 62 is the only protein detected by mab 4G8 on immunoblots and is the only protein immunoprecipitated from total cell lysates, indicating that the antibody is highly specific for SAP 62. Our data indicate that the epitope recognized by mab 4G8 is located in the C-terminal portion of SAP 62, which consists of 23 repeats of a proline-rich sequence (GVHPPAP) (29). As was observed with other U2 snrnp components (31,32,36) and splicing factors (for a review see 37), SAP 62 is localized in nuclear speckle domains in vivo. U2 snrnp (and SAP 62) first binds stably to pre-mrna in spliceosomal complex A. Not unexpectedly, we found that mab 4G8 efficiently immunoprecipitates this complex. Surprisingly, however, we found that this antibody also efficiently immunoprecipitates the E complex. In previous work, analysis of the total protein composition of affinity purified spliceosomal complexes by silver staining revealed that U2 snrnp-specific proteins (A, B and SF3a/b subunits) are present in the E complex, but at very low levels (4,27). Consistent with this result, B, SAP 155 and SAP 62 are all detected at low levels on Western blots of affinity purified E complex. However, on the basis of these data alone, it was not possible to distinguish between the possibility that a low level of U2 snrnp is tightly associated with a very small population of the E complex or that U2 snrnp is present in most if not all of the E complexes in the population, but dissociates during affinity purification. Our new analysis, in which we were able to use a gentle immunoprecipitation assay, supports the latter conclusion. Further evidence that U2 snrnp is associated with the E complex comes from the observation that low levels of U2 snrna are detected in affinity purified E complex (3,4,27).

7 360 Nucleic Acids Research, 1997, Vol. 25, No. 2 However, U2 snrna appears to be less tightly bound in the E complex than in the A complex, as this snrna is detected at higher levels in E complex affinity purified in low (100 mm) than in high (300 mm) salt, whereas U2 snrna is present in the A complex at both salt concentrations (3). This salt labile association of U2 snrnp with the E complex was also seen in our study; we found that the anti-sap 62 antibody immunoprecipitates the E complex in low but not in high salt. In previous work, five U2 snrnp components (SF3a/b subunits) were shown to crosslink to pre-mrna upstream of the branch site to a 25 nt region called the anchoring site, and functional studies indicated that the interactions of these proteins with the anchoring site are required to anchor U2 snrnp to the pre-mrna in the A complex (14). Significantly, no crosslinking of any of the SF3a/b subunits to pre-mrna can be detected in the E complex (10). If SF3a/b pre-mrna interactions are not involved in the association of U2 snrnp with the E complex, how is this snrnp associated? One possibility is through interactions with U1 snrnp. Mattaj and co-workers (35) showed that antibodies to U2 snrnp A could co-immunoprecipitate low levels of U1 snrnp along with U2 snrnp from oocyte extracts. Consistent with this observation, we found that low levels of U1 snrnp are co-immunoprecipitated with U2 snrnp from HeLa extracts using the 4G8 antibody. The factors that mediate the interactions between U1 and U2 snrnps are not known, but changes to U2 snrna or U2 snrnp structure disrupt the interaction between the two snrnps (35). Another possible interaction that could be responsible for the association of U2 snrnp with the E complex is a protein protein interaction between U2AF and SAP 155. This interaction was detected in a Far Western assay and by yeast two-hybrid assays (O.Gozani and R.Reed, unpublished results). As U2AF is present in the E complex, U2 snrnp may associate with E complex via the SAP 155 U2AF interaction. Our data also show that U2 snrnp is associated with E5 and E3 complexes, which assemble on RNAs lacking a 3 or a 5 splice site respectively. It is possible that U2 snrnp binds via distinct interactions in these two complexes. U1 snrnp is also detected in both the E5 and E3 complexes (38) and the interactions in each complex involve distinct domains on U1 snrnp (39). The E complex has been operationally defined as a complex that elutes in a single peak on a gel filtration column and can be quantitatively chased into the A complex (2 4). Whether the E complex is actually a mixture of complexes that contain different combinations of U1 and U2 snrnps cannot be strictly ruled out on the basis of present information. However, considering the operational definition of the E complex, our data indicate that, at a minimum, 30% of the E complex population is associated with U2 snrnp (based on the efficiency of immunoprecipitation of the E complex using anti-u2 snrnp antibodies). Thus, at least a portion, if not all, of the E complex that can be chased into the A complex contains U2 snrnp. SR proteins are another example of a splicing factor that is detected in gel filtration isolated E complex but not in complexes purified under more stringent conditions (38). The SR proteins, though loosely associated with the E complex, nevertheless play a role in its assembly (38). The question of most obvious importance is whether U2 snrnp is bound in a functional manner to the E complex. We have used several approaches to address this question but, so far, have not been able to obtain a definitive answer. A straightforward approach to the problem is to inactivate U2 snrnp or deplete it from nuclear extracts and then determine whether pre-assembled E complex can be chased into the A complex. However, we are unable to obtain an extract specifically lacking U2 snrnp that is sufficiently active for these studies. In yeast, U2 snrnp was not found to associate with the commitment complex (5,40), even under conditions that should detect a weak association of this snrnp (41). Thus, it is possible that there are differences in the early steps of spliceosome assembly between yeast and mammals (2 4,39 41). However, these differences may be subtle and may only reflect quantitative differences rather than fundamental differences in the mechanism. Consistent with this view, U2 snrnp binding in yeast requires the 5 splice site and the U1 snrna 5 splice site duplex (5,41). In mammals, the efficiency of U2 snrnp binding is increased by the presence of an upstream 5 splice site (42) and by the presence of U1 snrnp (though the U1 snrna 5 splice site duplex is not required for this stimulatory effect) (39). In addition, there is one example in yeast in which ATP-independent binding of U2 snrnp to pre-mrna occurs (41). In this case, a mutation in U1 snrna that weakens the U1 snrna 5 splice site duplex results in a low level of ATP-independent U2 snrnp binding (41). Liao et al. (41) propose that this may occur because an ATP-dependent disruption of the U1 snrna 5 splice site duplex may be required for U2 snrnp binding. It is possible that the weak U2 snrnp binding that we detect in the E complex is in some way related to this observation in yeast. If U2 snrnp is not bound to the E complex in a functional manner, our detection of this association may nevertheless reflect an important interaction that occurs at some other point during the normal spliceosome assembly pathway. For example, an interaction between U1 and U2 snrnps may be involved in forming the association between the 5 and 3 splice sites in the A complex. If, on the other hand, U2 snrnp is functionally bound in the E complex, the early steps of spliceosome assembly may involve U2 snrnp binding weakly in the E complex and then undergoing an ATP-dependent conformational change that results in stable U2 snrnp binding. ACKNOWLEDGEMENTS We are grateful to W. J. van Venrooij (University of Nijmegen, The Netherlands), Tom Maniatis (Harvard University) and Ann Beyer for providing anti-sm, anti-sc35 and anti-b (4G3) monoclonal antibodies respectively. We also thank members of the Hong and Reed laboratories for critical reading of the manuscript. 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