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 transcription 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 dithiothreitol. 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 bovine 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). ml) for 1 hr at 37°C, then extracted with phenol, precipitated, and denatured by heating at 100°C for 5 min in 10mM Tris-HCl, teinized RNAs were not themselves precipitable, and digestion pH 7.4/1 mM EDTA. After complete RNase Ti digestion, oli- of the RNPs with trypsin (followed by trypsin inhibitor) ren- gonucleotides were separated in two dimensions by electro- dered the RNAs nonprecipitable (data not shown). phoresis and chromatography with "homomix c" (31). Identification of the La antigen was corroborated by using the electrophoretic blotting technique described by Towbin et RESULTS at (34). Proteins from unlabeled HeLa cells and from an anti- Proteins Precipitated by Lupus Antibodies. The polypep- La precipitate were separated in a polyacrylamide gel, together tides immunoprecipitated by the four classes of lupus antibod- with 3S-labeled marker proteins, and transferred to nitrocel- ies from [3S]methionine-labeled HeLa cell extracts were ana- lulose. The position of the La antigen was determined by in- lyzed on gels (Fig. 1). Ten major proteins with approximate Mr cubation with anti-La serum and then with "WI-labeled protein 110,000, 29,000, 25,000, 23,000, 22,000, 16,000, 15,000, A. The autoradiogram (Fig. 1A) showed a gredominant band of 13,000, 12,500, and 12,000 were precipitated with anti-Sm an- Mr =45,000, which comigrated with the S-labeled immuno- tibodies. The same proteins were also precipitated with anti- precipitated protein. Detection of the La antigen by this RNP serum, except that the proteins of=110,000 and =25,000 method confirms the conclusion that RNA is not required for were missing and the Mr :15,000 protein was present in lower its antigenicity. abundance. Anti-Ro serum precipitated two major proteins of Thus, the RNPs precipitated with anti-La sera appear to dif- Mr =90,000 and -94,000. All anti-La sera tested precipitated fer from those precipitated with the anti-Sm and anti-RNP sera, a single major protein OfMr :45,000 although additional bands in both RNA (13, 19) and protein constituents. Despite the re- unique to individual sera were also detected. The Mr 45,000 port (16) that the La- and Ro-specific RNPs share a common protein appeared to be the only phosphoprotein precipitated antigen, no common protein was detected by immunoprecipi- with anti-La sera (data not shown; ref. 32). The same proteins tation. However, the Sm- and RNP-specific RNPs exhibit con- were precipitated from extracts that had been pretreated with siderable protein overlap. micrococcal nuclease, suggesting that the antibodies recognize VA RNA Synthesized in Vitro Binds to the La Antigen. To protein rather than nucleic acid. In agreement with recent re- examine the interaction between the La antigen and the VA ports (13, 33), though not with some older findings, the depro- RNAs, we devised means of assembling the complex in vitro. 6774 Biochemistry: Francoeur and Mathews Proc. Natl. Acad. Sci. USA 79 (1982)

1 2 3 4 5 6 7 8 9 10

r XX00000 r0; f0CM00 :0 H ? +a' iL_:iiiio A iAELSHCML 6

;;;# FIG. 2. Immunoprecipitation of RNPs containing VA RNA syn- t :'SifatV~fiS' ...... ; u:ffV0000cof ' thesized in vitro. Transcription reactions programed with cloned VA i s i s_Fxt -;Rf (j tD RNA genes were incubatedfor 2 hr. Samples were immunoprecipitated i Sti s i41b~~~~~~'s with the sera specified and were analyzed by gel electrophoresis. The postprecipitation supernatants were analyzed on a parallel gel (not shown) to allow estimation of the efficiency of VA RNA precipitation (percentages are in parentheses). Lane 1, pD* (VA RNA11) template, precipitated with anti-La(T) serum (79%). pBalM (VA RNA,) template, precipitated with: anti-La(T) (lane 2; 65%); no antibody (lane 3; 0.2%); anti-La(B) (lane 4; 64%); anti-Ro(2) (lane 5; 2.1%); anti-Ro(l) (lane 6; 0.3%); anti-RNP (lane 7; 0.3%); anti-Ro(2) (3%) (not shown); followed by precipitation with anti-La(T) (lane 8; 52%); anti-Sm (lane 9; 0.2%); anti-La(B) no template (lane 10; 0.2%). o I- to N - On (9 +- 3:: :6 The first method involved transcription cloned VA RNA - of l I I -I Iv- I genes in a HeLa cell extract containing RNA polymerase III N cl- - or 4 41 activity. As shown in Fig. 2, both VA RNA, (lanes 2, 4, and 8) 4 0. 06 C0 06 and VA RNA,, (lane 1) were efficiently and specifically precipitated with anti-La sera. About 50% and 80% ofthe transcripts, FIG. 3. Precipitation of RNPs containing mutant forms of VA respectively, were precipitated. Two different anti-La sera gave RNA,. Transcription reactions programed with equal amounts (2 pug similar results (lanes 2 and 4). Because purified VA RNAs are of DNA) of the cloned VA RNA gene constructs were incubated for 2 not precipitable in the absence of the HeLa extract (ref. 13; hr. Anti-La immunoprecipitates ("i") and half of the supernatants ("s") from the precipitation reactions were analyzed. From left to right, unpublished data), these results indicate that the La antigen is theplasmids usedwere (percentages of VA RNAthatwere precipitated present in the extract and can interact with the transcripts to are in parentheses): dl5'+ 10 (95%); wild-type VA RNA, (90%); pA2-dl6 form RNPs in vitro. The RNPs so formed exhibited the expected (13%); pA2-dl2 (56%); pA2-dll (46%); pA5-dl9 (96%); pA2-d16.1-1 reactivity towards the four classes of lupus sera tested: anti-RNP (95%). The structure of the transcripts are depicted: solid lines, se- and anti-Sm sera precipitated 0.3% of the VA RNA synthesized quences present in wild-type VA RNA, of Ad2 or Ad5; wavy lines, se- (Fig. 2, lanes 7 and 9); one anti-Ro serum gave a similar negative quences of linker and pBR322 origin; dashed lines, sequence deletions. result, whereas a second was weakly positive, precipitating 2- nts, Nucleotides. (Compiled from refs. 6, 35, and 36.) 3% of the VA RNA (lanes 6 and 5). Controls without antibody or VA RNA genes were negative (lanes 3 and 10). A further con- poorly precipitable (13%), possibly because ofits large size (580 trol, shown in lane 8, demonstrated that prior immunoprecip- bases): decreased precipitability of longer RNAs also has been itation with anti-Ro serum did not significantly affect the effi- noted by Shen and Maniatis (37). ciency of a subsequent precipitation with anti-La serum. These results indicate that nucleotides 69-122 are not re- The kinetics of RNP assembly, measured by immunoprecip- quired for La antigen binding. At the 5' end, nucleotides 1-8 itation, were compared with the kinetics of VA RNA synthesis can be substituted by foreign sequence without abolishing an- (data not shown). Over the period studied-1 min to S hr-both tigen recognition, and at the 3' end, nucleotides beyond base parameters were linear, indicating that a fixed proportion ofthe 122 also can be substituted. Furthermore, the 5' terminal RNA that was synthesized associated with the La antigen. This pppA-G-C extension on the A start form (38) of VA RNA, is not suggests that the antigen associates with the transcripts at the an essential recognition feature; RNAs starting with either an time of synthesis or very soon afterwards. A or G residue are precipitable. Thus, the La antigen must rec- Altered Forms of VA RNA, Form RNPs. The VA RNAs ognize the sequence corresponding to the internal control re- made in vitro by several different adenovirus serotypes (Ad2, gion or a common feature of the RNA termini (5' ppp-purine Ad5, Ad7, Adl2) and by two variants (Ad2 var-3 and AdS d1309) and 3' Un-OH). were all efficiently precipitated by anti-La sera (data not The La Antigen Is Not Required for VA RNA Synthesis. In shown). For a more systematic survey, we exploited VA RNA, view of these results and of the rapid kinetics of complex for- gene constructs containing internal deletions or sequence sub- mation, it seemed possible that the La antigen plays a role in stitutions derived by Fowlkes and Shenk (35) and Guilfoyle and VA RNA transcription. A precedent for this exists in the Xen- Weinmann (36). These authors defined a control region, cor- opus oocyte, in which a protein factor required for the tran- responding to bases 6-69 within the VA RNA, gene, which scription of5S rRNA binds to the RNA product as well as to its serves as the "promoter" for transcription in vitro. The plasmids gene (21, 22). To test this possibility, the transcription system tested by us contained alterations on the 5' side of the control was depleted for the La, Ro, Sm, or RNP antigen by adsorption region or insertions, deletions, or substitutions on its 3' side. on immunoaffinity columns prepared with the individual sera. In all cases, the RNA synthesized in vitro was precipitable As shown in Fig. 4, all the depleted extracts were competent with anti-La serum, albeit to variable extents (Fig. 3). For RNAs for VA RNA, synthesis. VA RNA synthesized in the La antigen- with internal deletions (pA2-dll, pA2-dl2), the efficiency of depleted extract was not precipitable with anti-La antibodies precipitation was similar to that observed with wild-type VA (Fig. 4, lanes 2 and 7), indicating efficient removal of the La RNA,. Higher efficiencies were observed with RNAs containing antigen. As expected, the RNA synthesized in extracts that had substitutions at their 5' ends (dl5'+10, pA5-dl9) or 3' ends been depleted for the other lupus antigens (lanes 3-5 and 8-10) (pA2-dl6. 1-1). Mutant pA2-dl6 RNA is unusual in being only or had been passed over a blank column (lanes 1 and 6) was Biochemistry: Francoeur and Mathews Proc. NatL Acad. Sci. USA 79 (1982) 6775

2 3 4 5 6 7 8 9:O 3', LILJI 4

CG C -G U sG .o C-G G-C d Mb _ _a_ AG (149-150) :C A| 150-G - C A-U G-C Gnu GNU G-C G -c C -G FIG. 4. Transcription with extracts depleted for lupus antigens. _CUCCUUU-OH A U , ccu~(151-159) A G Transcription reactions containing antigen-depleted extracts and 2 Aug 14 -C G of pBalM (VA template were incubated for 2 hr. After precip- UCAG AU RNAM) G -C itation with anti-La serum, the immunoprecipitates (lanes 1-5) and (135-138) AU half of the supernatants (lanes 6-10) were analyzed. Lanes 1 and 6, 13 C G-20 C 21 fiX2CGAIG U _G A control column, no antibody; lanes 2 and 7, anti-La(B) column; lanes 120-C uU~ U-A A 3 and 8, anti-Ro(1) column; lanes 4 and 9, anti-Sm column; lanes 5 and AAG 110-CC "AC-GA CGCUU G C-G 30 10, anti-RNP column. Similar results were obtained by using 1-5 /g C G UOG of template per reaction. ACAACG (139-144) CAUU A-U-40 go -G -C U -A (40-46) Ge U - C-G -=55% precipitable with anti-La serum. Similarly, addition of (65-71) C -G C G -C- C the lupus antibodies to the transcription system failed to inhibit C-G CACUCUUCCG C -G -50 VA RNA synthesis (data not shown). U-A * (4-13) G -C These results suggest that the La antigen as well as the other 8O- C -G C A lupus antigens are not required for synthesis ofVA RNA. Par- G -C G -c allel experiments showed that the lupus antigens, including La, (25-33) C-G 2nd C -G were not required for the synthesis of VA RNA,,, Drosophila ueG -t A 0-00G -60 and Xenopus 5S RNA, or a Drosophila tRNA species, although u ,,GGo* uv a fraction of these transcripts binds the La antigen (data not 70-C C shown). Thus, it would appear that the La antigen is not anal- -) 1St CCCA AG ogous to the 5S RNA transcription factor. FIG. 5. Reconstitution of RNP with fragmented VA RNA. La an- The La Antigen Binds to VA RNA Termini. To define more tigen-bound oligonucleotides (and their nucleotide numbers) are precisely the RNA sequences that are recognized by the La marked on the two-dimensional oligonucleotide fractionation and are antigen, a system was developed in which added VA RNAwould boxed on the secondary structure model (6, 39). Arrowheads identify complex with the antigen. Under suitable conditions, 10-25% the positions of three oligonucleotides from the control region that are ofVA RNA, that was purified from a transcription reaction be- greatly decreased in or absent from the immunoprecipitate. came specifically immunoprecipitable with anti-La serum upon reconstitution in this system. For reasons that are not clear, but (21, 22)-does not appear to be involved in transcription. may be related to the low efficiency of VA RNA immunopre- The VA RNPs are identified by immunoprecipitation with cipitation from infected cells (2% in our hands), reconstitution the anti-La class of lupus sera. By this criterion, the particles was less efficient with VA RNA synthesized in vivo. VA RNA assembled in vitro cannot be distinguished from those existing synthesized in Xenopus oocytes behaved like the RNA made in vivo, but we have not applied any more stringent test of in vivo (unpublished data). identity. To a minor extent, the VA RNPs also were recognized Using this assay, we examined the portions offragmented VA by some samples of anti-Ro sera-in view of the clinical cor- RNA, molecules that interact with the La antigen. VA RNA, was relation between these two lupus specificities (15, 18), most partially digested with ribonuclease TI, incubated with a source likely as a result of contamination with traces of anti-La anti- of La antigen, and the resultant complexes immunoprecipi- bodies. Making due allowance for the likelihood that these hu- tated. The RNA fragments in the precipitate were characterized man sera are not monospecific, we have tentatively identified by RNase T1 digestion and two-dimensional oligonucleotide the La antigen as a HeLa cell phosphoprotein of Mr =45,000. fractionation (Fig. 5) and subsequent redigestion with different This is the only protein consistently precipitated by all our anti- enzymes. The bound oligonucleotides come exclusively from La sera, obtained from six different patients. It is found in both the RNA termini and comprise the 5'-terminal 13 nucleotides adenovirus-infected and uninfected HeLa cells and also is rec- and 3'-terminal 26 nucleotides. Further experiments (not ognized by anti-La sera by using the electrophoretic blotting shown) have shown that removal ofthe 5'-terminal triphosphate technique (34). This result, and others mentioned above, re- group does not interfere with RNP formation, whereas blocking affirms the conclusion that the antigen is protein in nature (13, of the 3' end eliminates La antigen binding. 33). Further, VA RNA can be crosslinked to a cellular protein of similar antigenicity (unpublished data) and size (ref. 40; also unpublished data). Taking all the data together, it is likely that DISCUSSION the Mr =45,000 protein is the La antigen and it is entirely pos- RNP complexes containing the lupus antigen La and the ad- sible that it is the only protein component of the VA RNP. In enovirus RNAs VA RNA, and VA RNA,, are efficiently assem- contrast, the particles precipitated with other lupus sera (anti- bled in cell-free transcription reactions. The RNP complexes Ro, anti-Sm, and anti-RNP) contain proteins that differ in size are stable and form quickly, but their formation is not depen- and number from the La antigen. The failure of anti-Ro sera to dent on transcription because similar complexes also can be precipitate the Mr =45,000 La-specific protein lends no support reassembled, though less efficiently, by using purified VA to the idea (16) that Ro-specific RNPs contain the La antigen. RNA. Conversely, VA RNA synthesis is not inhibited by the Using mutant VA RNAs, we have shown that the sequences addition of anti-La antibodies to the transcription reaction or recognized by the La antigen must lie near the RNA termini by depletion of the antigen content of the extract by affinity or in the site corresponding to the intragenic control region. chromatography. Thus, the antigen-unlike the 5S RNA factor Analysis of fragmented VA RNA molecules that bind the La 6776 Biochemistry: Francoeur and Mathews Proc. Natl. Acad. Sci. USA 79 (1982) antigen indicates that only terminal sequences are involved. In 11. Mathews, M. B. (1980) Nature (London) 285, 575-577. view of secondary structure models of VA RNA, in which the 12. Murray, V. & Holliday, R. (1979) FEBS Lett. 106, 5-7. termini pair to form a stem (6, 39), it is conceivable that the 13. Lerner, M. R., Boyle, J. A., Hardin, J. A. & Steitz, J. A. (1981) Science 211, 400-402. antigen binds to the double-stranded stem structure. However, 14. Lerner, M. R., Andrews, N. C., Miller, G. & Steitz, J. A. (1981) this seems unlikely because the fragmented RNAwas denatured Proc. Natl Acad. Sci. USA 78, 805-809. prior to binding in the experiment shown in Fig. 5 (although 15. Reichlin, M. (1981) Clin. Exp. Immunol. 44, 1-10. the possibility ofrenaturation cannot be ignored). Furthermore, 16. Hendrick, J. P., Wolin, S. L., Rinke, J., Lerner, M. R. & Steitz, mutant VA RNAs that have terminal sequence replacements J. A. (1981) Mol. Cell. Biol. 1, 1138-1149. with severely limited base-pairing potential are also competent 17. Rinke, J. & Steitz, J. A. (1982) Cell 29, 149-159. 18. Wasick, C. A. & Reichlin, M. (1982)J. Clin. Invest. 69, 835-843. for La antigen binding. Indeed, in some cases such hybrid mol- 19. Lerner, M. R. & Steitz, J. A. (1979) Proc. Natl. Acad. Sci. USA 76, ecules are more efficiently precipitated than wild-type VA 5495-5499. RNA, or molecules that contain simple internal deletions, so it 20. Reddy, R. & Busch, H. (1981) in The Cell Nucleus, ed. Busch, H. is possible that both ends of the VA RNA are separately rec- (Academic, New York), Vol. 8, pp. 261-306. ognized by the antigen and that the stem must be unpaired for 21. Pelham, H. R. B. & Brown, D. D. (1980) Proc. Natl. Acad. Sci. binding. Because both termini can be replaced by essentially USA 77, 4170-4174. 22. Engelke, D. R., Ng, S.-Y., Shastry, B. S. & Roeder, R. G. (1980) unrelated sequences, the precise recognition elements elude Cell 19, 717-728. definition at present. However, additional preliminary findings 23. Mathews, M. B. & Grodzicker, T. (1981)J. Virol 38, 849-862. emphasize the role of the 3' terminus. Crosslinking experi- 24. Tanaka, J. & Weisblum, B. (1975)J. Bacteriol. 121, 354-362. ments reveal intimate contact between the 3' end and the La 25. Holmes, D. S. & Quigley, M. (1981) Anal. Biochem. 114, 193- antigen, and destruction of the 3' end of VA RNA with snake 197. venom diesterase eliminates binding; on the other hand, re- 26. Wu, G.-J. & Zubay, G. (1974) Proc. Natl Acad. Sci. USA 71, 1803-1807. moval ofthe 5'-terminal phosphates has no effect (unpublished 27. Weil, P. A., Segall, J., Harris, B., Ng, S.-Y. & Roeder, R. G. data). The fact that many polymerase III transcripts bind the (1979)J. Biol. Chem. 254, 6163-6173. La antigen, at least transiently (16, 17), suggests that the role 28. Fahey, J. L. & Terry, E. W. (1978) in Handbook ofExperimental of this protein may relate to the run of uridylate residues that Immunology, ed. Weir, D. M. (Blackwell, London), Vol. 1, Ch. generally terminate such molecules. 8., pp. 8.1-8.16. 29. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 30. Bonner, W. M. & Laskey, R. A. (1974) Eur. J. Biochem. 46, 83- We thank Fred Asselbergs for useful discussions, Patty Reichel for 88. technical assistance, and Peter Wensink and Dan Bogenhagen forclones 31. Barrell, B. (1971) in Procedures in Nucleic Acid Research, ed. of Drosophila and Xenopus DNA, respectively. This work was sup- Cantoni, T. L. & Davies, D. R. (Harper & Row, New York), Vol. ported by a Cancer Center grant from the National Cancer Institute and 2, pp. 751-779. a fellowship to A. M. F. from the Medical Research Council ofCanada. 32. Pizer, L., Tan, E. M., Deng, J.-S. & Stenberg, R. (1982)J. Cell. Biochem. Suppl. 6, 324 (abstr.). 1. Reich, P. R., Rose, J., Forget, B. & Weissman, S. M. (1966)J. 33. Rosa, M. D., Gottleib, E., Lerner, M. R. & Steitz, J. A. (1981) Mol Biol 17, 428-439. Mol Cell. Biol 1, 785-796. 2. Ohe, K. 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