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Interaction Between VA RNA and the Lupus Antigen La: Formation of A

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Interaction Between VA RNA and the Lupus Antigen La: Formation of A Proc. Natl. Acad. Sci. USA Vol. 79, pp. 6772-6776, November 1982 Biochemistry Interaction between VA RNA and the lupus antigen La: Formation of a ribonucleoprotein particle in vitro (systemic lupus erythematosus/antigen/transcription/adenovirus) A. MICHtLE FRANCOEUR AND MICHAEL B. MATHEWS Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, New York 11724 Communicated byJ. D. Watson, July 14, 1982 ABSTRACT The small adenovirus-encoded VA RNAs occur La-specific RNPs are distinct, there is some evidence that Ro- as ribonucleoprotein (RNP) particles in association with a cellular specific RNPs may carry La antigens (16). In contrast, these two protein antigen, La, recognized by the anti-La class of lupus sera classes of RNPs appear to be quite distinct from those recog- [Lerner, M. R., Boyle, J. A., Hardin, J. A. & Steitz, J. A. (1981) nized by two other lupus specificities: anti-RNP recognizes Science 211, 400-402]. We have tentatively identified the La an- complexes containing cellular U1 RNA, and anti-Sm recognizes tigen as a HeLa cell phosphoprotein of Mr "z'45,000, present in complexes containing the same Ul RNA as well as RNAs U2, infected and uninfected cells. The antigen appears not to be re- U4, U5, and U6 (19). All these RNAs are capped (20) and, in quired for the transcription of VA RNAs in vitro. RNP particles some cases at least, have been shown to be products of RNA that contain newly synthesized VA RNAs assemble rapidly in tran- scription extracts making VA RNA and also can be reconstituted polymerase II. from purified VA RNA and a source of La antigen. Variant forms In this paper we examine the proteins associated with these ofVA RNA, with sequence deletions and substitutions bind to the four lupus specificities and focus on the interaction between the La antigen, suggesting that the recognition site includes the RNA La antigen and VA RNA. The La antigen appears to be a single termini or the sequences corresponding to the internal control phosphoprotein of Mr =45,000 and can complex with VA RNA region (promoter), or both. Upon reconstitution with fragments synthesized in vivo or in vitro. Using mutant forms ofthe gene ofVA RNA,, oligonucleotides from both the 5' and 3' termini bind for VA RNA,, we show that the La antigen also recognizes ex- to the antigen, but those from the control region do not. The ter- tensively altered VA RNA, molecules. Combining these results minal oligonucleotides of wild-type VA RNA can form a base- with others obtained by reconstitution of RNPs with frag- paired stem, but structures of comparable stability cannot be mented wild-type VA RNA,, the La antigen binding site is iden- formed by the chimeric variant molecules. Therefore, the rec- tified as the termini of the RNA. Furthermore, we show that, ognition site is probably the terminal nucleotides themselves unlike the 5S RNA transcription factor that binds to the RNA rather than the stem structure. molecule and is also required for transcription (21, 22), the La antigen is not required for the transcription of VA RNA genes Adenovirus synthesizes two small noncoding RNA species, VA in vitro. RNA, and VA RNA,, (1-4). They have been well characterized as have their genes (5, 6), which are transcribed by polymerase MATERIALS AND METHODS III (1, 7-10). The VA RNAs bind to adenovirus mRNA and to Cells and Viruses. HeLa cells and adenovirus stocks were a cDNA copy of one of these but not to uninfected HeLa cell handled as described (23). For preparation oflabeled proteins, mRNA (11). However, their function remains speculative; monolayers were incubated with [3S]methionine (0.2 mCi/ models have been proposed for a role in the processing of ad- 1 Ci = x in medium. enovirus late mRNA (12) and, recently, in translation (T. Shenk, ml; 3.7 1010 becquerels) methionine-free personal communication). Protein extracts for immunoprecipitation or RNP reconstitution The VA RNAs, as well as analogous RNAs synthesized by were prepared by lysing cells in 2 vol of buffer A (10 mM Epstein-Barr virus, occur in vivo as ribonucleoprotein (RNP) Tris-HCl, pH 7.5/140 mM NaCl/1.5 mM MgCl2/0.5% Noni- particles (13, 14). The protein moiety of the RNP bears deter- det P-40 (19). minants recognized by a class of sera, called anti-La (also, Ha Plasmids. Derivatives ofpBR322 containing segments ofad- or SS-B), from patients with autoimmune disorders such as sys- enovirus DNA were kindly supplied by S. Berget (pBalM, 28.5- temic lupus erythematosus or Sjogren syndrome (reviewed in 29.4 map units; VA RNA, gene), P. Thomas (pD*, 29.0-31.5 ref. 15). In uninfected HeLa cells, anti-La sera recognize RNPs map units; VA RNA,, gene), and R. Guilfoyle and T. Shenk (VA containing mouse 4.5S RNA, precursor tRNA, and 5S RNA- RNA, gene deletions). pA2-dl6. 1-1 was made by deleting all also transcribed by RNA polymerase III (14, 16, 17). These pBR322 DNA between the BamHI and Hind III sites of pA2- species, and other cellular transcripts, exhibit a number ofcom- dl6. Plasmid DNAs were isolated as described (24, 25). mon features, including internal and terminal sequence homol- Transcription Assay. HeLa cell extracts were prepared in ogies presumably reflecting their polymerase III heritage. buffer B (40 mM Hepes, pH 7.9/150 mM KC1/4.5 mM MgCl2/ One of these common features, the 5' terminal ppp-purine 0.5 mM dithiothreitol) essentially as described (26, 27) but with sequence, is shared with RNAs present in complexes recog- the hypotonic wash and dialysis steps omitted. Standard reac- nized by another class of lupus sera, anti-Ro (16). Clinically, tions contained (in 50 ,ul): 25 ,ul of cell extract, 50 mM Hepes anti-Ro antibodies are often found in association with anti-La at pH 7.9, 600 ,uM ofthree unlabeled nucleoside triphosphates, antibodies (18), and although the RNAs found in Ro-specific and 24 p.M of the labeled nucleoside triphosphate, and 2.5 p.M di- thiothreitol. Optimal activity was usually obtained with addition The publication costs ofthis article were defrayed in part by page charge of 40 mM KCl and 2.5 ,ug of DNA. [a-32P]GTP (New England payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: RNP, ribonucleoprotein. 6772 Biochemistry: Francoeur and Mathews Proc. Natd Acad. Sci. USA 79 (1982) 6773 Nuclear) was found to give the best labeling with the lowest A Bc M, x background and :20 tkCi was used per reaction. 10-3 2 3 2 3 4 5 2 Antibodies. Sera representing different lupus specificities 120 -i were generously provided by M. Reichlin (Oklahoma City) and were enriched for IgG by chromatography on DEAE-Sephadex 85-i. .I~~~~~~ A-50 columns run in 0.01 M Na phosphate at pH 7.6 (28). Frac- 62.5 e tions with A2N > 1 were pooled, dialyzed against 0.01 M Tris HCI, pH 7.2/0.15 M NaCl, and stored at -200C. 48- --ftir Immunoprecipitation. All procedures were carried out at 0- Al 4°C with constant mixing and an excess of antibody and im- .- munoadsorbent over antigen. Typically, to a standard transcrip- s b 6 tion reaction was added 100 1.d of buffer C (50 mM Tris HCl, b~ _ pH 7.4/150 mM NaCl/5 mM EDTA/0.5% Nonidet P-40/ ...... s 0.5% sodium deoxycholate/0.1% NaDodSO4) containing bo- vine serum albumin at 2 mg/ml and 20 pL ofantibody (2-3 mg/ 28.5- ml). After incubation for 15-30 min, 200 1Ld of IgGSORB (En- zyme Center, Boston,- MA) or 80-100 ,ul ofprotein A-Sepharose (Pharmacia), both suspended in 10 vol of buffer C with bovine 23- serum albumin, was added and incubation was continued for 10 min. The precipitates were washed six times with 1 ml of buffer C and analyzed for protein (after boiling in sample buffer) or RNA (after phenol extraction and ethanol precipitation) in 15% or 20% NaDodSO4 polyacrylamide gels, respectively (29). Radioactivity was detected by autoradiography or fluorography 8- (30). Immunoaffinity Chromatography. To deplete transcription 14.5 - extracts for specific antigens, 100 ,ul of IgG was mixed for 30 min at 4°C with 500 ,ul of protein A-Sepharose equilibrated in buffer B. After washing five times with 200 ,u1 ofbuffer B, the beads were mixed with 200 ,u1 oftranscription extract for 30 min at 4°C and then were removed by filtration. FIG. 1. Identification of lupus antigens. (A) Autoradiogram of ni- Reconstitution ofRNPs. 32P-Labeled VA RNA, isolated from trocellulose after transfer of HeLa cell proteins and markers and in- infected cells (5) was added to HeLa cell extracts prepared in cubation with anti-La antibody which was followed by incubation with C. 125I-labeled protein A. Lanes: 1, [3Slmethionine-labeled adenovirus buffer A, as described above, together with 2 vol of buffer marker proteins; 2, unlabeled HeLa cell extract; 3, immunoprecipitate After mixing at room temperature for 15-20 min, RNP assembly from [3S]methionine-labeled HeLa cell extract (La antigen marker). was assayed by immunoprecipitation. The reaction required (B and C) Two autoradiograms of [355]methionine-labeled HeLa cell Mg2' (2.5 mM) and it occurred at 0-37°C but was optimal at proteins immunoprecipitated with various lupus sera. B, Lane 1, ad- room temperature (20°C). Reconstitution of RNPs with frag- enovirus marker proteins. Proteins precipitated with anti-La (patient mented VA RNA was performed in the same way except that B; B, lane 2); anti-La (patient T; B, lane 3); anti-Ro(l) (B, lane 4); anti- the RNA was first partially digested with RNase Ti (0.008 unit/ Sm (B, lane 5 and C, lane 2); anti-RNP (C, lane 1).
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