THE JOURNAL OF BIOLOGICAL CHEMISTRY
0 1985 by The American Society of Biological Chemists, InC.
Vol. 260, No. 21, Issue of September 25, pp. 11781-11786 1985
Printed in d.S.A.
Alu RNA-Protein Complexes Formed in Vitro React with a Novel
(Received for publication, March 11, 1985)
Ryszard Koles, Lucille D. FrescoQ, Jack D. KeeneQ, Philip L. Cohenll, Robert A. Eisenbergll, and
Phyllis Golden Andrews
From the Department of Pharmacology and Cancer Research Center, University of North Carolina, Chapel Hill,
North Carolina 27514, the $Department of Microbiology and Immunology, Duke University Medical Center, Durham,
North Carolina 27710, and the (IDivision of Rheumatology, University of North Carolina, Chapel Hill, North Carolina 27514
We have screened sera from patients with systemic
lupus erythematosus for reactivity with RNA tran-
scribed in vitro using HeLa whole cell extracts. Sera
from 14 out of 114 patients precipitated an RNA tran-
scribed by RNA polymerase 1 1 1 from a plasmid con-
taining an Alu family sequence (i. e. the repetitive DNA
sequence that is cut by the Alu restriction enzyme)
located upstream from the human rG-globin gene.
These Alu transcripts were not precipitated by anti-
La, anti-Sm, anti-RNP or anti-Ro antibodies, suggest-
ing that Alu RNA was precipitated by a previously
undescribed lupus specificity. Analysis of [36S]methio-
nine-labeled immunoprecipitates indicated that Alu
RNA binds a protein of about 68 kDa. This protein
may be Alu specific since three different Alu tran-
scripts were precipitated by the anti-Alu sera whereas
another RNA polymerase 1 1 1 transcript, adenovirus VA
I RNA, was not precipitated by the same sera.
Sera from patients with systemic lupus erythematosus and
related autoimmune diseases exhibit antibodies directed
against numerous cellular antigens (see Ref. 1 for review).
Since the original observation by Lerner and Steitz (2) that
principal classes of these antibodies (anti-RNP,’ anti-La,
anti-Ro, anti-Sm) recognize small nuclear ribonucleoprotein
particles, the sera from systemic lupus erythematosus patients
have been used to elucidate the structure of several RNP
particles (3, 4) as well as the potential role of U1-RNP in
mRNA splicing (5-7).
The anti-La class of autoantibodies is believed to recognize
RNPs formed by RNA polymerase 111 transcripts (3,8). These
transcripts include precursors to small cellular RNAs such as
tRNA, 5 S RNA, and 7 S RNA (9, 10) as well as several viral
RNAs (11-13). In addition the vesicular stomatitis virus
leader RNP, which is transcribed by a virus-specific RNA
polymerase, is also precipitated by anti-La sera (14, 15). The
La antigen seems to bind to the oligo-U sequence typically
present at the 3‘-end of the polymerase 111 transcripts (16).
The role of the La antigen in vivo is not known. There were
* This work was supported in part by Grant GM32994 to R. K.
from the National Institutes of Health. The costs of publication of
this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement” in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Recipient of the American Cancer Society Junior Faculty Award.
To whom reprint requests should be addressed.
The abbreviations used are: RNP, ribonucleoprotein particle;
SRP, signal recognition particle. Alu family sequence, the repetitive
DNA sequence that is cut by the Alu restriction enzyme.
suggestions that La antigen is a transcription factor (9) or
that it is complexed with a transcription factor (17) since sera
with anti-La specificity inhibited in vitro transcription of
RNAs transcribed by RNA polymerase 111 (17). However,
Francoeur and Mathews (13) reported that in vitro transcrip-
tion of adenovirus VA RNA, which is precipitated by anti-La
antibody, could not be inhibited by depletion of the extract
with anti-La sera.
We describe here a previously unidentified antibody speci-
ficity from certain lupus and scleroderma patients, distinct
from anti-La, anti-Ro, anti-RNP, and anti-Sm, that reacted
with in vitro synthesized RNPs containing Alu transcripts
but did not precipitate another RNA polymerase 1 1 1 transcript
synthesized in vitro. The antigen is a protein which appears
to bind to the 5‘ half of the Alu RNA.
Whole cell and nuclear extracts were obtained from HeLa cells as
described by Manley et al. (18) and Dignam et al. (19), respectively.
Sera were from a bank of frozen samples which had been sent for
characterization of possible antibodies to extractable nuclear antigens
and were from rheumatology patients of the North Carolina Memorial
Hospital. In most experiments, sera were used without further treat-
ment. In some instances purified IgG fraction (20) was used.
Plasmids A36 (21) and RBa-1 ((22) a kind gift of Dr. T. Maniatis,
Harvard University) have been described. Plasmid dl-313 which car-
ries genes coding for VA I and VA I1 RNA from adenovirus serotype
5 was a gift from Dr. Dana Fowlkes, Department of Pathology,
University of North Carolina. Plasmid U6-13, a subclone of a plasmid
described previously (23)’ carries a truncated copy of an Alu family
gene. All plasmids were linearized before transcription as indicated
in Fig. 1.
In vitro transcription was carried out in a total volume of 50 ~l as
described (18). At the end of incubation samples were diluted with
150 pl of 50 m M Tris-HC1, pH 7.4, 150 m M NaCI, 0.05% Nonidet P-
40 (Sigma), immunoprecipitated as described by Lerner and Steitz
(2), and the isolated RNA was analyzed by polyacrylamide gel elec-
trophoresis (24). 5 ~1 of undiluted serum was used in each immuno-
HeLa cells grown in monolayers were labeled overnight with [%SI
methionine at 0.5 mCi/ml or with 13H]uridine at 100 pCi/ml. In vivo
labeled cell extracts were prepared and immunoprecipitated as de-
scribed (25). [3H]Uridine-labeled RNA was analyzed as above, and
[35S]methionine-labeled proteins were analyzed by electrophoresis on
sodium dodecyl sulfate containing 10% polyacrylamide gels (26) fol-
lowed by fluorography.
Immunoprecipitation of in Vitro Transcribed Alu RNA-A
plasmid carrying both an Alu family sequence and a fragment
of the 7”-globin gene (A36, Ref. 21) was used as a template
for in vitro transcription with HeLa whole cell extract (18).
R. Kole, unpublished data.
Immunoprecipitation of in Vitro Transcribed Alu RNA
8. Hendrick, J. P., Wolin, S. L., Rinke, J., Lerner, M. R., and Steitz,
J. A. (1981) Mol. Cell. Biol.
waran, P., Duncan, C. H., and Weissman, S. M. (1983) Cold
Spring Harbor Symp. Quant. Biol.
9. Rinke, J., and Steitz, J. A. (1982) Cell 29, 149-159
10. Chambers, J. C., Kurilla, M. G., and Keene, J. D. (1983) J. Biol.
Chern. 258, 11438-11441
11. Lerner, M. R., Boyle, J. A., Hardin, J. A., and Steitz, J. A. (1981)
Science 2 1 1,400-402
12. Lerner, M. R., Andrews, N. C., Miller, G., and Steitz, J. A. (1981)
Proc. Natl. Acad. Sci. U. S. A. 78,805-809
13. Francoeur, A. M., and Mathews, M. B. (1982) Proc. Natl. Acad.
Sci. U. S. A. 79,6772-6776
14. Kurilla, M. G., and Keene, J. D. (1983) Cell 34,837-845
15. Wilusz, J., Kurilla, M. G., and Keene, J. D. (1983) Proc. Natl.
Acad. Sci. U. S. A. 80,5827-5831
24. Donis-Keller, H. (1979) Nucleic Acids Res. 7, 179-192
25. Chambers, J. C., and Keene, J. D. (1985) Proc. Natl. Acad. Sci.
U. S. A. 82,2115-2119
26. Laemmli, U. K. (1970) Nature 227, 680-685
27. Hardin, J. A., Rahn, D. R., Shen, C., Lerner, M. R., Wolin, S. L.,
Rosa, M. D., and Steitz, J. A. (1982) J. Clin. Inuest. 70, 141-
28. Burnett, W. N. (1981) Anal. Biochern. 112, 195-203
29. Ullu, E., and Tschudi, C. (1984) Nature 312, 171-172
30. Duncan, C. H., Jagadeeswaran, P., Wang, R. C., and Weissman,
S. M. (1981) Gene 13,185-196
31. Stefano, J. E. (1984) Cell 36, 145-154
16. Mathews, M. B., and Francoeur, M. A. (1984) Mol. Cell. Biol. 4,
32. Akusjarvi, G., Mathews, M. B., Anderson, P., Vennstrom, B., and
Pettersson, U. (1980) Proc. Natl. Acad. Sci.
17. Gottesfeld, J. M., Andrews, D. L., and Hoch, S. 0.
Acids Res. 12,3185-3200
18. Manley, J. L., Fire, A., Cano, A., Sharp, P. A., and Gefter, M. L.
(1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3855-3859
19. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic
Acids Res. 11, 1475-1489
20. Fahey, J. L., and Terry, E. W. (1978) in Handbook ofEperimenta1
Immunology, (Weir, D. M., ed) Vol. 1, Chapter 8, pp. 8.1-8.16,
21. Duncan, C., Biro, P. A., Choudary, P. V., Elder, J. T., Wang, R.
R. C., Forget, B. G., deRiel, J. K., and Weissman, S. M. (1979)
Proc. Natl. Acad. Sci.
U. S. A. 76,5095-5099
22. Shen, C. K. J., and Maniatis, T. (1982) J. Mol. Appl. Genet. 1, 39. Zouali, M., and Eyquem, A. (1984) J. Clin. Irnmunol. 4,209-219
33. Walter, P., Gilmore, R., and Blobel, G. (1984) Cell 38, 5-8
34. Gundelfinger, E. D., Krause, E., Me& M., and Dobberstein, B.
(1983) Nucleic Acids Res. 11,7363-7374
35. Segall, J., Matsui, T., and Roeder, R. G. (1980) J. Biol. Chem.
36. Engelke, D. R., Ng, S.-Y., Shastry, B. S., and Roeder, R. G. (1980)
Cell 19, 717-728
37. Pelham, H. R. B., and Brown, D. D.
U. S. A. 77,4170-4174
38. Fritzler, M. J., Ali, R., and Tan, E. M. (1984) J. Irnrnunol. 132,
(1980) Proc. Natl. Acad.
23. Fukumaki, Y., Collins, F., Kole, R., Stoeckert, C. J., Jagadees-
U. S. A. 80, 4296-4300
U. S. A. 77,2424-
Economidis, I. V., and Pederson, T. (1983) Proc. Natl. Acad. Sci.