RNA Editing Activity Is Associated with Splicing Factors in Lnrnp Particles: the Nuclear Pre-Mrna Processing Machinery

RNA editing activity is associated with splicing factors in lnRNP particles: The nuclear pre-mRNA processing machinery

Oleg Raitskin*†, Dan-Sung C. Cho†‡, Joseph Sperling§¶, Kazuko Nishikura‡¶, and Ruth Sperling*

*Department of Genetics, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; ‡The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104; and §Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

Communicated by Roger D. Kornberg, Stanford University School of Medicine, Stanford, CA, March 29, 2001 (received for review August 11, 2000) Multiple members of the ADAR (adenosine deaminases acting on ribonucleoproteins (snRNPs) required for pre-mRNA splicing, RNA) gene family are involved in A-to-I RNA editing. It has been as well as several non-snRNP protein splicing factors, including speculated that they may form a large multicomponent protein U2AF and the SR protein family (23–25) are present within the complex. Possible candidates for such complexes are large nuclear lnRNP particles (19, 21, 22). ribonucleoprotein (lnRNP) particles. The lnRNP particles consist Three-dimensional image reconstruction of isolated lnRNP mainly of four spliceosomal subunits that assemble together with particles by automated electron tomography revealed a model the pre-mRNA to form a large particle and thus are viewed as the that is composed of four major subunits of similar dimensions, naturally assembled pre-mRNA processing machinery. Here we which are connected to each other (26, 27). An additional investigated the presence of ADARs in lnRNP particles by Western domain is sometimes observed toward the center of the particle blot analysis using anti-ADAR antibodies and by indirect immuno- (26, 28). Each repeating subunit has a mass of 4.8 Ϯ 0.5 mDa, precipitation. Both ADAR1 and ADAR2 were found associated with close to the estimated mass of the fully in vitro-assembled 60S the spliceosomal components Sm and SR proteins within the lnRNP spliceosome, whereas the tetrameric lnRNP particle has a mass particles. The two ADARs, associated with lnRNP particles, were of 21.1 Ϯ 1.6 mDa (28). The mass determination studies also enzymatically active in site-selective A-to-I RNA editing. We dem- revealed a discrepancy of 1.9 mDa between the lnRNP particle’s onstrate the association of ADAR RNA editing enzymes with mass (21.1 mDa) and the cumulative mass of its four subunits physiological supramolecular complexes, the lnRNP particles. (4 ϫ 4.8 mDa), which was consistent with the frequent presence of an additional domain (28). This additional domain may be adenosine deaminases acting on RNA ͉ double-stranded RNA adenosine attributed to additional RNA processing activities such as cap deaminase ͉ nucleic acid–protein interactions ͉ large nuclear binding and 3Ј-end processing, as components involved in these ribonucleoprotein processes have been detected also in lnRNP particles (O.R., J.S., and R.S., unpublished observations). DAR (adenosine deaminases acting on RNA) deaminates The A-to-I editing of certain substrates such as GluR-B and Amultiple adenosines to inosines, specifically in double- 5-HT2CR RNAs must occur before or simultaneously with stranded RNA (dsRNA) (1, 2). In humans as well as rodents, splicing, because the dsRNA structure essential for the editing three separate ADAR genes (ADAR1–ADAR3) have been mechanism forms between the exonic editing site and the identified, revealing an ADAR gene family (1, 3–10). Both downstream intron sequences (12, 13, 15, 29, 30). Thus, it is ADAR1 and ADAR2 appear to be expressed ubiquitously (3–6, anticipated that ADARs involved in the A-to-I RNA editing 8), whereas ADAR3 is expressed at low levels only in several mechanism may be included in the lnRNP complexes that regions of the brain (7, 10). The members of ADAR gene family constitute the natural pre-mRNA processing machinery. Here are involved in A-to-I RNA editing of transcripts of certain genes we report that enzymatically active ADAR1 and ADAR2 pro- such as glutamate receptor (GluR) subunits (11, 12) and sero- teins are indeed associated with spliceosomal Sm and SR tonin receptor 2C subtype (5-HT2CR) (13). Editing of the proteins within the 200S lnRNP particles as physiologically ‘‘Q͞R’’ site of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropi- significant supramolecular complexes. onic acid GluR-B subunit dramatically decreases Ca2ϩ perme- ability of the channel (11), whereas significant changes in the Materials and Methods G-protein coupling efficiency of 5-HT2CR by A-to-I RNA edit- Oligonucleotides. The following oligonucleotides used for PCR ing also have been reported (13–15). In view of the ubiquitous were synthesized at the University of Pennsylvania Cancer BIOCHEMISTRY expression of ADAR1 and ADAR2, the A-to-I RNA editing by Center Nucleic Acid Facility and, if necessary, purified in ADAR is likely a widespread phenomenon (16, 17). RNA editing 20% acrylamide-7 M urea gels. All ADAR oligonucleotides studies in vitro, using purified recombinant ADAR proteins, correspond to the human sequence (4, 5). The restriction have indicated a significantly different site selectivity displayed recognition site contained at the 5Ј end is shown in bold: by different ADAR gene family members. For instance, DR1PEP1, 5Ј-CGTGGATCCGGCCCCTCAAAAGCAGGG- ADAR2a, a splicing isoform of ADAR2, edits GluR-B RNA 3Ј; DR1PEP2, 5Ј-AGTCACGATGCGGCCGCTGGGGAC- very efficiently at the Q͞R site, whereas ADAR1 barely edits CTTGAGAGGA-3Ј; DR1PEP3, 5Ј-CGTGGATCCGGCACT- this site but edits an intronic ϩ60 site preferentially (3, 5, 7, 18). Although RNA editing has been shown to affect only selected gene transcripts, the posttranscriptional processing of most Abbreviations: ADAR, adenosine deaminases acting on RNA; lnRNP, large nuclear ribonucleoprotein; dsRNA, double-stranded RNA; GluR, glutamate receptor; 5-HT R, serotonin pre-mRNAs requires several common steps such as 5Ј-end 2C Ј receptor 2C subtype; snRNP, small nuclear ribonucleoprotein. capping, 3 -end processing, and splicing. These processes have †O.R. and D.-S.C.C. contributed equally to this work. been proposed to be carried out within large nuclear ribonucle- ¶To whom reprint requests should be addressed. E-mail: [email protected] or oprotein (lnRNP) particles that sediment at 200S in sucrose [email protected]. gradients. The composition of the lnRNP particles (19–22) is The publication costs of this article were defrayed in part by page charge payment. This very similar to that of the in vitro-assembled spliceosome (re- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. viewed in refs. 23 and 24). Specifically, all of the U small nuclear §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.111153798 PNAS ͉ June 5, 2001 ͉ vol. 98 ͉ no. 12 ͉ 6571–6576 Downloaded by guest on September 26, 2021 GTGGATGGGCCA-3Ј; DR1PEP4, 5Ј-AGTCACGATGCG- mAb 104 (36) according to Zahler et al. (37). ADAR1 was GCCGCATACTATACTGGGCAG-3Ј; DR2PEP1, 5Ј-CGTG- probed with mAb 15.8.6 or 4G3B9 (see above) diluted 1:2,000 GATCCAAGCACGCGTTGTACTGT-3Ј; and DR2PEP2, 5Ј- and visualized with horseradish peroxidase conjugated to affin- AGTCACGATGCGGCCGCCCGGGTCAGGGCGTGA-3Ј. ity-pure goat anti-mouse IgG F(ab)Ј fragment diluted 1:3,000. ADAR2a was probed with affinity-purified polyclonal Ab Preparation of ADAR-Specific Antibodies. Different regions of hu- P00381 (Bionostics) or mAb HBI-HYB7 diluted 1:1,000 and man ADAR1 (amino acids 440–826, dsRNA binding domain probed with horseradish peroxidase-conjugated affinity-pure and amino acids 1,144–1,226, C terminal) and ADAR2a (amino rabbit anti-IgG (H ϩ L) diluted 1:5,000. acids 629–701, C-terminal) were constructed in pGEX-4T-3 vector (Amersham Pharmacia) as glutathione S-transferase Immunoprecipitations. Indirect immunoprecipitations were per- (GST) fusion protein (31). The inserts of all constructs were formed as described (22) by binding gradient fractions to anti-Sm prepared by PCR amplification of human ADAR1 (4) and mAb Y12 coupled to protein G Sepharose beads (Sigma). As ADAR2a cDNA plasmids (5) using a set of oligonucleotide controls, (i) gradient fractions were incubated with beads that primers designed to create NotI and XbaI restriction sites. had not been treated with mAb Y12, and (ii) beads carrying mAb DR1PEP1 and DR1PEP2 primers were used for PCR amplifi- Y12 were analyzed without prior incubation with gradient cation of the ADAR1 dsRNA binding domain, whereas fractions. The immunoprecipitated proteins were released by DR1PEP3 and DR1PEP4 primers were used for amplification of treatment with SDS-containing gel sample buffer. Western the ADAR1 C-terminal region. DR2PEP1 and DR2PEP2 were blotting revealed ADAR proteins as described. The relative used for PCR amplification of the ADAR2a C-terminal region. quantities of ADAR proteins in the pellet and supernatant The GST-ADAR fusion proteins were expressed in Escherichia fractions were determined by densitometric scanning (model coli BL21 and purified by glutathione Sepharose 4B column SL-TRFF, Biomed Instruments, Fullerton, CA). chromatography (31). mAbs were generated against the purified GST-ADAR fusion proteins. Selected mAbs 15.8.6 (ADAR1 Results dsRNA binding domain specific), 4G3B9 (ADAR1 C-terminal Identification of Different ADARs in lnRNP Particles by Immuno- specific), and HBI-HYB7 (ADAR2a C-terminal specific) were chemistry. We previously have shown that when HeLa cell screened for specific binding to ADAR1 and ADAR2a and nuclear supernatants were fractionated in sucrose gradients and confirmed not to cross-react with different members of ADAR probed for a specific pre-mRNA (e.g., ␤-actin), a relatively broad or GST proteins (D.-S.C.C., S. Kang, T. Sanford, and K.N., but distinct peak of the specific RNA was observed at the 200S unpublished results). Affinity-purified polyclonal antibody region of the gradient (38). All of the nucleoplasmic phosphor- P00381 specific to the N-terminus region (amino acids 6–66) of ylated SR proteins also sedimented as one peak at 200S and were ADAR2a was obtained from Bionostics (Ontario, Canada). associated exclusively with 200S lnRNP particles (22). Other components of the lnRNP particles, such as the U snRNPs, In Vitro RNA Editing Assay. Editing of a synthetic GluR-B RNA U2AF, and PTB (19, 22) gave a broader distribution across the B11 was assayed in vitro by using lnRNP fractions and control sucrose gradients and were particularly abundant in smaller recombinant ADAR1 and ADAR2a proteins as described (5, complexes that sedimented closer to the top of the gradient. For 18). The standard editing reactions containing 20 fmol of B11 the latter splicing factors, however, a specific association with RNA, cell-equivalent samples of total lnRNP proteins (10 ␮g 200S lnRNP particles was unambiguously demonstrated by re- from 200S fractions or 20 ␮g from 70S fractions), or 10 ng of fractionation of the combined 200S peak fractions on a second purified recombinant ADAR proteins were incubated at 30°C sucrose gradient, which gave rise to a single peak at the 200S for4h. region of the gradient (19, 22). Such experiments indicated the existence of at least two populations of U snRNPs, U2AF, and ADAR Enzyme Activity Assay. The dsRNA-specific A-to-I deami- PTB. One sediments at 200S in association with lnRNP particles. nation enzyme activities of lnRNP fractions were assayed in vitro The other, most probably, represents smaller particles or free by using 10 fmol of [␣-32P]ATP-labeled c-myc synthetic dsRNA species that are present in excess of what is required for lnRNP as a substrate RNA as described (32). particles assembly or resulted from dissociation of lnRNP particles during preparation. Preparation of lnRNP Particles. LnRNP particles were prepared As a first step in analyzing the association of ADARs with from HeLa cells as described (19, 21). Briefly, nuclear super- lnRNP particles, we examined, by Western blotting, the distri- natants enriched in lnRNP particles were prepared from clean bution of different ADAR gene family members in fractionated cell nuclei and fractionated on 15–45% sucrose gradients in 100 lnRNP particles. HeLa cell nuclear supernatants enriched in mM NaCl, 10 mM Tris⅐HCl (pH 8.0), 2 mM magnesium chloride, lnRNP particles were fractionated on sucrose gradients, and the and 2 mM vanadyl ribonucleoside. Centrifugations were carried distribution of ADAR proteins was probed with Abs recognizing out at 4°C in an SW41 rotor run at 41 krpm for 90 min (or an two different regions of ADAR1 (4, 8, 9) or ADAR2 (3, 5, 6). equivalent ␻2t). When a second fractionation was required, 2–3 The full-length 150-kDa form of ADAR1 was found in a broad gradient fractions corresponding to the 200S region of the peak around the 200S region of the gradient (Fig. 1A Upper), gradient were combined, dialyzed, concentrated, and refraction- where specific nuclear pre-mRNAs have been shown to sediment ated on a second sucrose gradient (19). Peaks of 200S tobacco (19, 38–40). The more abundant shorter (110 kDa) form of mosaic virus and 70S bacterial ribosomes, sedimented in parallel ADAR1 had a broader distribution, with significant level sedi- gradients, were used as sedimentation references. menting at the 200S and 70S regions of the gradient (Fig. 1A Lower). The 110-kDa ADAR1, another enzymatically active Western and Slot Blot Analyses. For slot blot analysis, aliquots from form of the protein, is most likely the translation product sucrose gradient fractions were spotted directly onto a nitrocel- initiated from the downstream methionine codon (9). The lulose membrane. For Western blot analysis, aliquots were 90-kDa form of ADAR2 protein also showed a broad distribu- applied directly to the wells of a 10% or 8% polyacryl- tion, with significant levels sedimenting as large complexes at the amide͞SDS gel as described (33). Sm proteins were probed with 200S and 70S regions of the sucrose gradient (Fig. 1B). The anti-Sm polyclonal antibodies (34) diluted 1:100 and revealed bands detected at the top fractions of the gradient are likely due with protein A horseradish peroxidase conjugate diluted 1:3,000 to ADAR proteins disassembled from lnRNP particles during or with anti-Sm mAb Y12 (35). SR proteins were probed with cell disruption and gradient sedimentation, or due to free

6572 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.111153798 Raitskin et al. Downloaded by guest on September 26, 2021 Fig. 1. The distribution of ADAR proteins in a sucrose gradient. Nuclear supernatants of HeLa cells, enriched in lnRNP particles, were fractionated in a 15–45% sucrose gradient and collected (bottom to top) in 20 fractions. Aliquots from each fraction were analyzed by Western blotting. (A) Probing with anti-ADAR1 mAb 15.8.6. (Upper) The distribution of the 150-kDa form of ADAR1 protein (20-sec exposure). (Lower) The distribution of the 110-kDa form of ADAR1 protein (5-sec exposure). (B) Probing with anti-ADAR2 Abs P00381. The sedimentation position of the 200S TMV and 70S bacterial ribosomes size markers are indicated on the top. Molecular masses of the ADAR1 species are indicated on the left.

Fig. 2. ADAR proteins cosediment with spliceosomal Sm and SR proteins at nuclear ADAR proteins (also see Discussion). This distribution the 200S region in a sucrose gradient. HeLa cell nuclear supernatant, enriched is reminiscent of that of U snRNPs, U2AF, and PTB as detailed in lnRNP particles, was fractionated in a 15–45% sucrose gradient. The 200S peak fractions were combined and refractionated in a second sucrose gradi- above. Thus, three fractions corresponding to the 200S region of ent. Aliquots from each fraction were analyzed by slot blotting (A) or Western the gradient (Fig. 1, fractions 10, 11, and 12) were combined and blotting (B). The distributions of SR proteins, Sm antigens, and ADAR1 and refractionated in a second sucrose gradient. Probing the gradient ADAR2 proteins were revealed by their cognate antibodies. Shown are the fractions with the respective antibodies revealed a single peak of results of two independent experiments. Similar results were obtained in ﬁve ADAR1 and ADAR2 proteins that comigrated with the 200S different experiments. The sedimentation position of the 200S TMV and 70S lnRNP particles represented by the Sm and SR proteins (Fig. bacterial ribosomes size markers are indicated. 2A). These slot blot results were further confirmed by Western blot analyses of ADAR1 and Sm proteins (Fig. 2B). The distinct cipitated by the anti-Sm mAbs (Fig. 3, lanes 2 and 5), whereas peak at 200S and the low abundance of ADAR proteins in the only a small amount (Ͻ5%) of the protein was precipitated from top and bottom fractions of the second gradient suggest their the top fraction (Fig. 3, lane 7), where free ADAR1 presumably specific association with 200S particles. sediments. Similar results were obtained by indirect immunoprecipitation of lnRNP particles by anti-SR mAbs and probing ADAR Proteins Are Integral Components of lnRNP Particles. The with anti-ADAR1 or anti-ADAR2 mAbs (data not shown). association of the ADAR proteins with 200S lnRNP particles was These results demonstrate specific association of ADAR1 within confirmed by indirect immunoprecipitation experiments. The lnRNP particles where the pre-mRNA, spliceosomal U snRNPs, efficacy of this approach already has been demonstrated by SR proteins, and other splicing factors are assembled together. immunoprecipitating lnRNP particles using antibodies directed against the Sm antigens or the SR proteins (22, 34). Here, we Reconstitution of ADAR Proteins into lnRNP Particles. We previously immunoprecipitated sucrose gradient-fractionated 200S lnRNP have shown that the stability of the lnRNP complex depends on particles with Y12 anti-Sm mAbs (35) and probed both the immunoprecipitate (Fig. 3, lane 2) and supernatant (Fig. 3, lane BIOCHEMISTRY 1) with anti-ADAR1 mAbs. In parallel, fractions from the 70S and top regions of the gradient were analyzed in the same manner (Fig. 3, lanes 5 and 7). The absence of adventitious adhesion of complexes to protein G Sepharose was confirmed by analyses of beads that were incubated with gradient fractions but were not treated with the antibody, which showed no signal of ADAR1 (Fig. 3, lane 3 for the 200S fraction; identical results were obtained for the 70S and top fractions). In a second control, Fig. 3. ADAR proteins are associated with spliceosomal proteins in 200S beads were incubated only with the antibody, but not with the lnRNP particles. HeLa cell nuclear supernatant, enriched in lnRNP particles, antigen, to ensure that no bands resulting from the antibody was fractionated in a 15–45% sucrose gradient. Aliquots from the 200S, 70S, comigrated with ADAR1 proteins (Fig. 3, lane 8). The relative and top fractions were immunoprecipitated by anti-Sm Y12 mAbs bound to amount of ADAR1 that was indirectly immunoprecipitated from protein G Sepharose beads. The precipitated proteins (lanes 2, 5, and 7) as well as the proteins left in the supernatant (lanes 1, 4, and 6) were analyzed by each of the 200S, 70S, and top fractions of the gradient provides Western blotting with anti-ADAR1 mAb 15.8.6. Controls: Lane 3, analysis of confirmation of the association of the ADAR1 proteins with beads incubated with the 200S fraction without Y12 mAb. Lane 8, analysis of RNP particles. We found that 40% and 15% of the ADAR1 beads incubated with Y12 mAb alone (no antigen added). Similar results were content of the 200S and 70S fractions, respectively, were pre- obtained in ﬁve different experiments.

Raitskin et al. PNAS ͉ June 5, 2001 ͉ vol. 98 ͉ no. 12 ͉ 6573 Downloaded by guest on September 26, 2021 Fig. 4. Reconstitution of ADAR proteins into 200S lnRNP particles. EDTA- dissociated spliceosomal Sm proteins (Sm) and ADAR1 protein (ADAR1) were reconstituted into 200S lnRNP particles after readdition of Mg2ϩ. Nuclear supernatant of HeLa cells was sedimented in a standard Mg2ϩ-containing sucrose gradient and the 200S peak fractions were combined. One-third was refractionated in the presence of Mg2ϩ (Top); another third was incubated Fig. 5. Detection of the A-to-I base conversion activity in 200S lnRNP particles with EDTA and refractionated in the presence of EDTA (Middle); and the and 70S fractions made from HeLa cells. The A-to-I conversion activity of 200S ␮ ␮ remaining third was incubated with EDTA, then with Mg2ϩ and fractionated lnRNP particles (10 g) and 70S fractions (20 g) was determined by a base 32 in the presence of Mg2ϩ (Bottom). Sm and ADAR1 proteins were analyzed, in modiﬁcation assay using 10 fmol of [ P]ATP-labeled c-myc dsRNA as a sub- aliquots taken from the same gradients, by slot immunoblotting. The sedi- strate (32). For comparison, puriﬁed recombinant ADAR1 and ADAR2a pro- mentation positions of the 200S TMV and 70S bacterial ribosomes size markers teins (10 ng each) were tested in the same assay. are indicated. Detection of the ADAR Enzymatic Activity in lnRNP Particles. The Mg2ϩ concentration. The complex dissociates into 70S particles A-to-I conversion activity of ADARs present in lnRNP particles when Mg2ϩ is removed by chelating agents. Under these con- was examined by using a previously described base modification ditions the family of essential splicing factors, the SR proteins in assay with a synthetic long c-myc dsRNA substrate (32). As their phosphorylated form, stay associated with the pre-mRNA positive controls, we analyzed purified recombinant ADAR1 and sediment at 70S. Some of the U snRNPs, such as U1, almost and ADAR2a proteins simultaneously. The results clearly indi- completely dissociate and migrate at the top of the gradient, cated that both 200S and 70S fractions of lnRNP preparation whereas other U snRNPs, such as U2 and U5, only partially contain enzymatically active ADARs (Fig. 5). The A-to-I base dissociate and partially stay associated with the pre-mRNA in modification assay is useful to identify the activity of ADAR 70S particles (19). The dissociated complex can be reconstituted proteins, but it cannot distinguish the activity of different ADAR by readdition of Mg2ϩ into a complex that is indistinguishable gene family members. The different ADARs display very dis- from the original one. The reconstituted complex thus regains tinctive site selectivity for editing of GluR-B or 5-HT2CR RNA the original biochemical composition, and morphology as seen in at certain sites when they are tested in an in vitro editing assay. the electron microscope (19). We thus examined whether Therefore, we tested both 200S and 70S lnRNP fractions by using ADAR1 can be dissociated from and then reconstituted back as substrate GluR-B B11 RNA harboring the Q͞R site and the into 200S lnRNP particles. Gradient fractions containing 200S intronic hot spot ϩ60 site (18). ADAR2a and ADAR2b, enzy- lnRNP particles (analogous to fractions 10–12 of Fig. 1) were matically active splicing isoforms of ADAR2, selectively edit the combined and concentrated, and one-third was dissociated by Q͞R site, whereas ADAR1 preferentially selects the ϩ60 site (3, addition of EDTA and reconstituted by readdition of Mg2ϩ. 5, 6, 18). Fig. 6 shows that both 200S and 70S fractions contained Another third was just dissociated by addition of EDTA. The the enzymatic activities that edit Q͞R and ϩ60 sites, confirming remaining untreated third and the two treated thirds were the presence of both ADAR1 and ADAR2 activities in these fractionated in parallel sucrose gradients and analyzed for the lnRNP particles as indicated by our Western blotting and distribution of spliceosomal Sm proteins (Fig. 4, Sm) and immunoprecipitation experiments. We conclude that the RNA ADAR1 (Fig. 4, ADAR1) by slot blotting. Addition of EDTA editing active ADAR1 and ADAR2 are complexed with lnRNP caused the migration of ADAR1 proteins as 70S complexes, as particles. was previously shown for the pre-mRNA, the SR proteins and part of the U snRNPs (19). ADAR1 proteins, as well as the Sm Discussion antigens, were reconstituted into lnRNP particles after addition Complex Formation of ADAR Enzymes with lnRNP Particles. Analysis of Mg2ϩ to the dissociated particles, indicating once again that of specific as well as general polyadenylated populations of the association of ADARs with lnRNP particles is specific and nuclear pre-mRNAs by sucrose gradient fractionation revealed is physiologically significant. a single peak at the 200S region of the gradient. These nuclear

6574 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.111153798 Raitskin et al. Downloaded by guest on September 26, 2021 factors Sm, U2AF, and PTB), which are present in the nucleus in excess of what is required for spliceosome assembly and splicing (ref. 22 and references cited therein). Alternatively, the ADAR proteins recovered at the top of the gradient may represent the proteins dissociated from lnRNP particles during sample preparation.

Presence of Both Editing and Splicing Machineries in lnRNP Particles. ADARs containing both substrate binding and catalytic domains appear to be self-sufficient for executing the site selective A-to-I RNA editing in vitro (5, 6, 18). However, the presence of regulatory factors as well as inhibitors in vivo also has been suggested (18, 42). It is possible that some of these putative regulators and inhibitors of the A-to-I RNA editing may be known components of lnRNP particles. The A-to-I RNA editing mechanism requires a critical dsRNA structure formed around editing site(s) (2). ADAR identifies and binds to the dsRNA structure through its dsRNA binding domain consisting of two or three dsRNA binding motifs (4, 43). Interestingly, the dsRNA Fig. 6. RNA editing site selectivity of lnRNP fractions. Editing site selectivity structure proven to be essential for editing of certain substrate of 200S lnRNP particles (10 ␮g) and 70S fractions (20 ␮g) was examined in vitro RNAs such as GluR and 5-HT2CR RNAs (12, 13, 29, 30), is at the Q͞R and intronic ϩ60 sites of GluR-B RNA. For comparison, purified formed between exon sequences around the editing site(s) and recombinant ADAR1 and ADAR2a proteins (10 ng) were tested simulta- downstream intron sequences. It contains the critical dinucle- neously. The RNAs were examined by primer extension analysis using 32P- otide GU, which is part of the 5Ј splice site consensus sequence labeled oligonucleotide primers specific for each editing site. The extension AG͞GURAGU predominantly found in most mammalian pre- primer used (P) and extended bands specific for edited (E) and unedited (U) mRNAs (24, 44, 45). Thus, the 5Ј splice site sequence serves as RNA are indicated. a component of the dsRNA structure required for editing of these RNAs as well as an essential element of the splicing Ј pre-mRNAs are packaged in 200S lnRNP particles in a supra- machinery. Binding of U1 snRNA to the 5 splice site is required spliceosome configuration (20, 26, 28, 38). All of the nucleo- for spliceosome assembly, and the same consensus sequence is plasmic phosphorylated SR proteins, essential for spliceosome subsequently base-paired with U6 snRNA during the activation assembly and splicing, are found associated exclusively with 200S of the spliceosome (46, 47). The formation of the dsRNA lnRNP particles (22). These previous studies have established structure essential for A-to-I RNA editing and spliceosome the 200S lnRNP particles as the naturally assembled pre-mRNA assembly may, therefore, interfere with each other. If both splicing machinery. Formation of the dsRNA structure essential splicing and RNA editing machineries are kept together, the dsRNA structure critical for A-to-I RNA editing is likely to be for A-to-I editing of GluR and 5-HT2CR RNAs requires base pairing of exon and intron sequences and therefore is expected constantly involved in two functional interactions. One is binding of U1 snRNA to the 5Ј splice site, including the GU nucleotides, to occur before or concomitant with splicing. The association of followed by initiation of spliceosome assembly; and the other is ADAR1 with the RNP matrix on transcriptionally active Xeno- binding of ADAR followed by initiation of A-to-I RNA editing. pus lampbrush chromosomes and its cotranscriptional function It has been recently reported that resolution of the dsRNA has been suggested previously (41). In this study, we have structure involved in A-to-I RNA editing may be regulated by a demonstrated directly that enzymatically active ADAR proteins specific ATP-dependent dsRNA helicase (48). The mutation in involved in the A-to-I RNA editing mechanism are associated the helicase and thus delayed resolution of the dsRNA structure with 200S lnRNP particles. result in the occurrence of a ‘‘splicing catastrophe’’ of the In addition to their association with the 200S particles, Drosophila para Naϩ channel transcripts at the region surround- ADAR proteins and activity appear to associate with particles ing the editing sites (48). In addition, cryptic and alternative sedimenting in the 70S fractions of the sucrose gradient. The splicing of 5-HT2CR RNA have been detected at the region biological significance of the association of ADAR proteins surrounding the editing sites (15). These studies indicate an with the 70S fractions remains to be clarified due to our limited active interaction of the dsRNA structure, as well as the dsRNA understanding of the relationship of 70S and 200S particles.

helicase activity, with the A-to-I RNA editing and splicing BIOCHEMISTRY The 70S fractions most likely represent a population of machinery. Therefore, our direct demonstration that both dissociated particles similar to the complexes obtained upon ADAR1 and ADAR2 are present within 200S lnRNP complexes treatment of 200S lnRNP particles with EDTA. These particles is significant as it confirms spatial proximity of two different have been shown to contain intact pre-mRNA associated with posttranscriptional processes (spliceosome assembly and RNA only part of the components of the 200S lnRNP particles (19). editing) within the naturally assembled pre-mRNA processing Alternatively, they may represent degradation products of the machinery. 200S lnRNP particles in which the pre-mRNA is partially degraded. Finally, the 70S fractions could represent interme- We thank J. A. Steitz and I. Mattaj for anti-Sm Y12 mAbs, T. Sanford, diate complexes in the assembly of the 200S lnRNP particles. S. Kang, and A. Pecho for excellent technical assistance, and J. M. Further studies are required to elucidate whether the 70S Murray for critical reading of this manuscript. We also thank the Wistar fractions and their association with ADARs represent a phys- editorial services department for preparing the manuscript. This work iologically significant intermediate in the assembly pathway of was supported by grants from the National Institutes of Health the lnRNP particles. The ADAR proteins detected at the top (GM40536, CA72765, and AG10124) and the Doris Duke Charitable Foundation (to K.N.), the Israel-U.S. Binational Science Foundation (to of the gradient may indicate that they are usually present in R.S., J. S. and K. N.), and the Joseph and Ceil Mazer Center for excess of what is required for pre-mRNA processing com- Structural Biology at the Weizmann Institute of Science (to J.S.). plexes. This is similar to what was found for several spliceo- D.-S.C.C. was supported by a training grant from the National Cancer somal components (e.g., U snRNPs, and the splicing protein Institute (CA09171).

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