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Differential Binding of Heterogeneous Nuclear MOLECULAR AND CELLULAR BIOLOGY, JUlY 1992, P. 3165-3175 Vol. 12, No. 7 0270-7306/92/073165-11$02.00/0 Copyright (© 1992, American Society for Microbiology Differential Binding of Heterogeneous Nuclear Ribonucleoproteins to mRNA Precursors prior to Spliceosome Assembly In Vitro MARIA BENNETT, SERAFIN PINOL-ROMA,t DAVID STAKNIS, GIDEON DREYFUSS,t AND ROBIN REED* Cellular and Molecular Physiology, Program in Cell and Developmental Biology, Harvard Medical School, Boston, Massachusetts 02115 Received 3 March 1992/Accepted 23 April 1992 We have investigated the composition of the earliest detectable complex (H) assembled on pre-mRNA during the in vitro splicing reaction. We show that most of the proteins in this complex correspond to heterogeneous nuclear ribonucleoproteins (hnRNP), a set of abundant RNA-binding proteins that bind nascent RNA polymerase II transcripts in vivo. Thus, these studies establish a direct parallel between the initial events of RNA processing in vitro and in vivo. In contrast to previous studies, in which total hnRNP particles were isolated from mammalian nuclei, we determined the hnRNP composition of complexes assembled on individual RNAs of defined sequence. We found that a unique combination of hnRNP proteins is associated with each RNA. Thus, our data provide direct evidence for transcript-dependent assembly of pre-mRNA in hnRNP complexes. The observation that pre-mRNA is differentially bound by hnRNP proteins prior to spliceosome assembly suggests the possibility that RNA packaging could play a central role in the mechanism of splice site selection, as well as other posttranscriptional events. During transcription, nascent heterogeneous nuclear plex that commits pre-mRNA to the splicing pathway (30). E RNAs (hnRNAs) associate with a distinct set of abundant complex is a precursor to the first specific ATP-dependent nuclear proteins, known as heterogeneous nuclear ribonu- complex (A), which contains Ul and U2 snRNPs (20, 23, cleoproteins (hnRNP) to form hnRNP complexes (see refer- 30). U4, U5, and U6 snRNPs then join A complex to form ences 10, 12, and 14 for reviews). The major hnRNP proteins the mature spliceosome, or B complex, which contains Ul, detected by sodium dodecyl sulfate (SDS)-gel electrophore- U2, U4, U5, and U6 snRNPs (4, 20, 24, 30). sis of isolated mammalian hnRNP complexes (also referred Despite extensive characterization of the structure and to as hnRNP particles) include a group of 35- to 45-kDa composition of hnRNP complexes isolated from mammalian proteins and groups of 68- and 120-kDa proteins (7, 12). cells, the specific functions of these complexes in nuclear However, at least 20 distinct hnRNP proteins have been metabolism of pre-mRNAs have not been established (12). identified by two-dimensional gel electrophoresis (36). On However, at least some of the hnRNP proteins have been the basis of the observations that hnRNP proteins bind to shown to be associated with the splicing machinery. For nascent pre-mRNA and that splicing can be visualized on example, antibodies against hnRNP C proteins specifically these transcripts in the electron microscope (2, 32), hnRNP immunoprecipitate spliceosomes assembled in vitro and complexes are thought to be the substrate for posttranscrip- inhibit the splicing reaction (9, 42). Furthermore, binding tional processing events (12). studies showed that the hnRNP proteins A, C, and D interact In vitro studies have shown that pre-mRNA splicing takes specifically with the 3' splice site and that mutations in this place within spliceosomes, large multicomponent complexes element disrupt binding (45). In addition to demonstrating an containing Ul, U2, U4, U5, and U6 small nuclear RNPs association between hnRNP proteins and pre-mRNA, these (snRNPs) (see references 21 and 28 for reviews) and a and other studies indicated that hnRNP proteins bind RNA number of non-snRNP proteins (26, 27, 38). Spliceosome in assembly occurs by means of a stepwise pathway. Pre- a sequence-dependent manner (6, 45). Moreover, different mRNA is first assembled into an ATP-independent complex hnRNP proteins show high binding affinities for different which lacks snRNPs (15, 19, 24, 38). This complex (H) does ribohomopolymers, such as poly(U) or poly(G) agarose (46). not appear to be specific to splicing because it assembles as However, other studies have shown that hnRNP proteins efficiently on natural pre-mRNAs as on RNAs lacking func- can also bind RNA nonspecifically (10, 11). The conclusions tional splice sites (19, 24, 38). However, H complex assem- that hnRNP proteins can bind RNA both specifically and bly precedes that of any splicing-specific complexes (30), nonspecifically are not necessarily contradictory (see refer- indicating that this complex is either a spliceosome precur- ence 14 for discussion). Nevertheless, the actual organiza- sor or that it disassembles prior to spliceosome formation. tion of hnRNP proteins on natural pre-mRNAs remains to be Following H complex assembly, the first complex specific established. to splicing, E (early) complex, is an ATP-independent com- In this study, we investigated the protein composition of H complex assembled on different RNAs in vitro. Previous studies revealed that the predominant proteins in H complex * Corresponding author. assembled on ,B-globin pre-mRNA have molecular sizes t Howard Hughes Medical Institute and Department of Biochem- between 35 and 45 kDa and approximately 65 and 116 kDa istry and Biophysics, University of Pennsylvania School of Medi- (38). On the basis of the similarity in size to hnRNPs and the cine, Philadelphia, PA 19104-6148. observation that the proteins in H complex bind to diverse 3165 3166 BENNETT ET AL. MOL. CELL. BIOL. RNAs added to splicing extracts, we hypothesized that H avidin agarose was determined by counting by the Cerenkov complex may contain hnRNP proteins. Here we show that H method. Analysis of the protein composition of the purified complex contains the same set of hnRNP proteins as are complexes was carried out as described previously (38). associated with nascent hnRNA in vivo. We find that distinct Two-dimensional gel electrophoresis. Two-dimensional gel combinations of hnRNP proteins associate with pre-mRNAs electrophoresis was carried out essentially as described by of different sequences. Thus, our data show that hnRNP O'Farrell et al. (31). The first dimension was separated by complexes assemble on pre-mRNA in a sequence-dependent nonequilibrium pH gradient gel electrophoresis, and the manner. The implications of transcript-dependent binding of second dimension was resolved by SDS-12.5% polyacryl- pre-mRNA by hnRNP proteins are discussed. amide gel electrophoresis (PAGE) as described by Dreyfuss et al. (13). The separated proteins were visualized by silver MATERIALS AND METHODS staining. Immunopurification of hnRNP complexes. hnRNP com- Plasmids. pAdML and T7-H, have been described by plexes were immunopurified from the nucleoplasm of HeLa Michaud and Reed (30). pAd3' was constructed by ligating cells with the anti-C protein monoclonal antibody 4F4 (8) as the FspI-BamHI fragment from pAdML into the PvuII- previously described (7, 36). The immunopurified complexes BamHI sites of SP72 (Promega). pGC+DX, which encodes a were eluted from the protein A-Sepharose beads with SDS- portion of rat ao-tropomyosin (44), and pB3P3, which en- PAGE sample buffer or with nonequilibrium pH gradient gel codes the 3' portion of the rat ox-tropomyosin intron 2 (35), electrophoresis sample buffer for analysis of the protein were gifts from B. Nadal Ginard. pAdML, pAd3', and composition by SDS-PAGE or by two-dimensional gel elec- T7-H, were linearized with BamHI for in vitro transcription trophoresis, respectively. with T7 polymerase. pAdML was linearized with FspI to synthesize Ad5' RNA and with EcoRI to synthesize the antisense of AdML RNA (transcription was done with SP6 RESULTS polymerase). pGC+DX and pB3P3 were linearized with BamHI and AccI, respectively, for transcription with SP6 Several methods have been employed to fractionate spli- polymerase. ceosomes and intermediate complexes in the spliceosome Pre-mRNA synthesis and in vitro splicing reactions. Bio- assembly pathway, including density gradient sedimentation tinylated pre-mRNAs (4, 20, 38) were synthesized with SP6 (5, 15, 19), native gel electrophoresis (23, 24), and gel filtra- or T7 polymerase in standard transcription reactions (29), tion (30, 39). With any of these methods, the first complex which included 15 FiM biotinylated UTP (Enzo Biochemi- detected assembles with similar efficiencies on RNAs con- cals) and 100 ,uM UTP. In vitro splicing reactions were taining or lacking functional splice sites. This complex has carried out as described previously (25). For splicing com- been designated H (24) or nonspecific (48) complex, when plex purification, large-scale in vitro splicing reaction mix- detected by native gel electrophoresis, and appears to cor- tures (1.2 ml) contained 4.8 p.g of 32P-labeled biotinylated respond to a 30S complex on density gradients (3, 15, 19). RNA. For spliceosome purification, reaction mixtures were H complex forms immediately when pre-mRNA is added incubated at 30°C for 15 min. For purification of H com- to nuclear extracts in either the presence or absence of ATP plexes, reaction mixtures did not contain ATP, MgCl2, and and at 00 or 30°C (data not shown) (15, 19, 23, 30). Following creatine phosphate and were incubated for 5 min. For Fig. 2, H complex assembly, the kinetics and efficiency of spliceo- 5, 6, and 7, the HeLa cells used for nuclear extracts were some assembly vary significantly between different pre- obtained from the Massachusetts Institute of Technology, mRNA substrates and between different preparations of while HeLa cells from Endotronics were used for Fig. 1 and nuclear extracts (38a) (see below). With P-globin pre- 3. mRNA, H complex is detected after a 5-min incubation in Structure of RNAs. The AdML pre-mRNA (236 nucleo- the presence or absence of ATP (Fig. IA). Spliceosomes tides [nt]) contains exon 1 (92 nt), intron 1 (97 nt), and exon containing Ul, U2, U4, U5, and U6 snRNPs (designated B 2 (47 nt).
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