Alu RNA-protein complexes formed in vitro react with a novel lupus autoantibody.
ABSTRACT We have screened sera from patients with systemic lupus erythematosus for reactivity with RNA transcribed in vitro using HeLa whole cell extracts. Sera from 14 out of 114 patients precipitated an RNA transcribed by RNA polymerase III from a plasmid containing an Alu family sequence (i.e. the repetitive DNA sequence that is cut by the Alu restriction enzyme) located upstream from the human gamma G-globin gene. These Alu transcripts were not precipitated by anti-La, anti-Sm, anti-RNP or anti-Ro antibodies, suggesting that Alu RNA was precipitated by a previously undescribed lupus specificity. Analysis of [35S]methionine-labeled immunoprecipitates indicated that Alu RNA binds a protein of about 68 kDa. This protein may be Alu specific since three different Alu transcripts were precipitated by the anti-Alu sera whereas another RNA polymerase III transcript, adenovirus VA I RNA, was not precipitated by the same sera.
- SourceAvailable from: Wesley H Brooks
Article: Autoimmune diseases and polyamines.[Show abstract] [Hide abstract]
ABSTRACT: Genetics and environmental factors have important roles in autoimmune diseases but neither has given us sufficient understanding of these mysterious diseases. Therefore, we are now looking closer at epigenetics, an interface between genetics and environmental factors. Epigenetics can be defined as reversible heritable changes to chromatin that can alter gene expression without altering the gene's DNA sequence. Methylation of DNA and histones are primary means of epigenetic control. By adding methyl groups to DNA and histones, it can limit accessibility of the underlying gene thereby altering the amount of gene expression. The methyl group is derived from an essential molecule in the cell, S-adenosylmethionine (SAM). However, a group of small molecules called polyamines also require SAM for their synthesis. Polyamines are essential for many cellular functions and polyamine activity is increased in many autoimmune diseases. Presented here is the "polyamine hypothesis" in which increased polyamine synthesis competes with cellular methylation (epigenetic control) for SAM. It is proposed that increased polyamine activity can cause disruption of cellular methylation, which can lead to abnormal expression of previously sequestered genes and disruption of other methylation-dependent cellular processes.Clinical Reviews in Allergy & Immunology 11/2011; 42(1):58-70. · 4.73 Impact Factor
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ABSTRACT: We describe autoantibodies reactive with the signal-recognition particle (SRP) in serum of a patient with polymyositis. The serum specifically immunoprecipitated the SRP from human erythroleukemia (K562) cell extracts. Analysis of immunoblots revealed that the serum contained autoantibodies specific for the 54-kDa protein of the SRP but had little or no reactivity with its other five proteins. Indirect immunofluorescence of human laryngeal carcinoma (HEp-2) cells confirmed that the ribonucleoprotein immunoprecipitated by this serum is found mainly in the cytoplasm. This autoimmune serum may be useful for studying the function of the 54-kDa subunit of the SRP in binding to signal sequences or in interacting with other components of the translational machinery.Proceedings of the National Academy of Sciences 01/1987; 83(24):9507-11. · 9.81 Impact Factor
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ABSTRACT: The convergence of studies in the clinical and basic sciences has resulted in the definitive identification of many intracellular antigens which are the targets of autoantibodies in patients with systemic lupus erythematosus, scleroderma, dermatomyositis/polymyositis, Sjogren's syndrome, mixed connective tissue disease, and drug-induced autoimmunity. Some of this new knowledge includes the identification of the Sm and RNP antigens as ribonucleoprotein particles involved in splicing of precursor messenger RNA, Scl-70 as DNA topoisomerase I, proliferating cell nuclear antigen as auxiliary protein of DNA polymerase, δ, and certain antigens in myositis as aminoacyl transfer RNA synthetases. This information confirms, at a molecular level, the presence of specific profiles of autoimmune responses so that autoantibodies can be used in clinical medicine as diagnostically useful immune markers. In addition the data give compelling reasons to consider that certain autoimmune diseases are antigen-driven. Many autoantibodies have the interesting feature of recognizing epitopes on the antigens which are active or functional sites of the molecule. It is suggested that the data provide clues to the nature of the intracellular particle initiating the immune response and may help to elucidate some of the early mechanisms of the autoimmune process.Clinical Immunology and Immunopathology 06/1988;
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
The structure of the plasmid is shown in Fig. 1. This DNA
clone allows simultaneous transcription of the Alu RNA
(RNA polymerase I11 transcript) and the human yc-globin
gene (polymerase I1 transcript). In vitro transcribed RNA was
precipitated with sera from lupus patients essentially as de-
scribed by Lerner and Steitz (2) and analyzed by polyacryl-
amide gel electrophoresis (24). Fig. 2 shows the results of
RNA immunoprecipitation with a series of sera from lupus
and scleroderma patients.
Certain sera which we have determined to have anti-RNP,
anti-Sm, anti-Ro, and anti-SCL-70 specificities precipitated
in vitro transcribed Alu RNA (Fig. 2, lanes 2, 3, 4, and lo),
suggesting that anti-Alu reactivity is present in these sera in
addition to, and is distinct from, the major classes of auto-
Eco RI EcoRl
E ] - ,
p i T r
FIG. 1. Structure of the DNA templates. Boxes represent tran-
scribed genes. Arrows represent the RNA transcribed in uitro. The
length of the transcripts in nucleotides is indicated. The size of the
genes and the distances between them are not to scale. The DNA
templates were linearized by cleavage at the indicated restriction
N AL JH VG EC MF CF KM JC JM ala aSmaRNP T
. . -_ , ." -
~ ~- ., ....
FIG. 2. Immunoprecipitation of Alu RNA transcribed in ui-
tro from A36 DNA template. RNA was synthesized in vitro and
immunoprecipitated as described under "Experimental Procedures."
Electrophoresis was on an 8% urea-polyacrylamide gel (24). The gel
was autoradiographed overnight at -70 "C with DuPont Lightning
Plus screen and Kodak XAR-5 film. Lane 1, serum from healthy
individual; Lanes 2-10, immunoprecipitates by patients' sera; Lanes
11-13, immunoprecipitates by standard reference lupus sera; specific-
ity of the sera is indicated at the top of the figure. Lane 14, in uitro
transcribed RNA loaded directly on a gel. Immunoprecipitated RNA
from whole 50-pl reaction mixture is loaded on a gel in lunes 1-13.
One-fourth of this amount is loaded in lune 14. Patients' initials are
indicated at the top of the figure. The position of yG-globin run-off
transcript and Alu RNA is indicated. The Alu RNA doublet is due to
the partial read-through of the RNA polymerase I11 past the termi-
nation site (30). N, normal human serum; T, in vitro transcribed
2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4
antibodies. This was also supported by the fact that the
reference anti-La, anti-Sm, and anti-RNP sera were anti-Alu
negative (Fig. 2, lanes 11-13).
The finding that some anti-La sera did not precipitate Alu
RNA was surprising, since La protein has been reported to be
associated with RNA polymerase I11 transcripts, including
Alu RNA (3, 8, 9, 10-12, 22). Moreover, it was previously
found that one anti-La serum (from patient Le, a generous
gift from Dr. J. Steitz, see also Ref. 22) precipitated Alu RNA
in our assay? These results indicate that sera from certain
lupus patients contain an anti-Alu antibody that is distinct
from the major classes of lupus autoantibodies. Furthermore,
the precipitation of Alu RNA by the La serum from patient
Le (22) may have been due to the presence of this anti-Alu
antibody rather than the anti-La
The amount of RNA precipitated with each patient anti-
Alu serum was variable. In our experimental conditions the
most active sera precipitated about 25% of the transcribed
Alu RNA. As shown in Fig. 2, the intensity of the bands in
lanes 2 and 10 is comparable to that in lane 14. In lane 14,
however, only one-fourth of the amount of the original reac-
tion was loaded on the gel. Other sera precipitated signifi-
cantly lower amounts of Alu RNA (lunes 3, 4, and 7; lane 7
appears to be negative but a weak Alu band could be clearly
seen on the original autoradiograph).
Some of the sera precipitated a labeled band with mobility
slower than that of the yG-globin run-off transcript suggest-
ing a reactivity with high molecular weight nucleic acid (lanes
3 and 9). The origin of this band is currently under investi-
gation. It is possible that ribosomal RNA which was present
in the whole cell extract was labeled during in vitro transcrip-
tion and precipitated with anti-ribosomal RNA antibodies
Immunoprecipitation of in Vitro Transcribed Adenovirus
VA I RNA-We have tested the reactivity of our sera with
the in vitro synthesized adenovirus VA I RNA that was
previously shown to be precipitated by anti-La sera (11, 13)
to determine if Alu and VA RNPs share common antigenic
We have tested four sera in this assay. Three of these sera
contained anti-La antibodies and the fourth serum (AL) had
high levels of anti-Alu antibody (Fig. 2, lane 2). Anti-La sera
precipitated in vitro transcribed VA I RNA (Fig. 3, lanes 2,3,
and 5), in agreement with the observation of Francoeur and
Mathews (13)) but did not react with the A36 Alu RNA (Fig.
3, lanes 7,8, and 10). In contrast, serum AL, which contained
anti-RNP activity (see Figs. 5 and 6), did not precipitate VA
I RNA (lane 4) but did precipitate Alu RNA (lane 9). Normal
human serum was negative in this assay (lanes 1 and 6).
Phenol-extracted intact Alu RNA was not precipitated by AL
serum in the presence or absence of the RNase inhibitor
RNasin (Promega Biotec), suggesting that a protein antigen
is required for recognition by the antibody (results not shown).
The above experiments indicate that Alu RNA binds a
protein which is not common to all RNA polymerase I11
transcripts and as can be seen from the following experiment
this protein binds to RNAs transcribed from other Alu genes.
Precipitation of Other Alu Transcripts-In addition to Alu
and VA transcripts described above we have tested two other
Alu RNAs in our in vitro assay. One Alu sequence is located
downstream from the human a-globin gene (22, plasmid RBa-
l), and the other plasmid (U6-13) carries a truncated copy of
an Alu gene from an undetermined region of the human
genome (23).' The results of the immunoprecipitations are
R. Kole and S. Weissman, unpublished data.
Immunoprecipitation of in Vitro Transcribed Alu RNA
RM SS AL EC N
RM SS AL EC
7 8 9 1 0
FIG. 3. Immunoprecipitation of VA I and Alu RNA by anti-
La and anti-Alu sera. Immunoprecipitation and RNA analysis as
in Fig. 2. Lunes 1-5, immunoprecipitates of VA I RNA. Lanes 6-10,
immunoprecipitates of Alu RNA.
RM AL T
RM AL T
RM AL T
123 456 789
FIG. 4. Immunoprecipitation of A h RNA transcribed from
different DNA templates. Immunoprecipitation and RNA analysis
as in Fig. 2. Lunes I, 4, and 7, immunoprecipitation by serum RM,
an anti-La-containing serum. Lanes 2,5, and 8, immunoprecipitation
by serum AL, an anti-Alu serum. Lanes 3, 6, and 9, RNA transcribed
from plasmids indicated at the top of figure, loaded directly on a gel.
presented in Fig. 4 . All three Alu transcripts were precipitated
by serum AL, an anti-Alu serum (lanes 2, 5, and 8). As has
been shown in Fig. 3, lane 4, serum AL did not precipitate the
VA RNA. In addition two other anti-Alu sera were negative
versus VA I RNA (not shown). Since three Alu transcripts
were precipitated by anti-Alu sera and a non-Alu transcript,
VA I, was not precipitated by anti-Alu sera these
suggest that the antigen recognized by these sera may be Alu
specific. As expected from previous experiments (Fig. 3, lane
7) the Alu transcript derived from plasmid A36 was
precipitated by serum RM, an anti-La containing serum (lane
4). However, weak bands of the Alu RNAs derived from RBa-
1 and U6-13 could be detected in immunoprecipitations with
this serum (lanes 1 and 7), suggesting a possibility that these
transcripts bind, albeit weakly, the La antigen in addition to
the Alu antigen. It is likely that the differences in the se-
quences around the transcription termination sites are re-
sponsible for this difference in binding of the La antigen by
Alu transcripts (see “Discussion”).
The truncated Alu transcript derived from the plasmid U6-
13 contains 139 base pairs from the 5‘-end of the Alu sequence
and includes the polymerase I11 promoter. When transcribed
in vitro, this plasmid gave rise to a transcript about 200
nucleotides long. The size of the transcript as
blot experiments (not shown) indicated that the transcript
initiates at the 5’-end of the Alu gene and terminates within
the vector sequences, about 60 nucleotides downstream from
the 3’ end of the Alu fragment. The fact that the truncated
Alu was precipitated by the anti-Alu serum AL suggests that
the Alu antigen binds to the 5’ half of the Alu transcript.
However, the possibility that this antigen binds to the down-
stream vector sequences cannot be excluded.
Precipitation of RNA and Proteins in the in Vivo Labeled
Extracts-We have used [35S]methionine and [3H]uridine la-
beled extracts (25) from HeLa cells to study the immunopre-
cipitation of the in vivo labeled ribonucleoprotein particles
and have tested five anti-Alu sera in these assays. Fig. 5 shows
immunoprecipitates of a 35S-labeled extract. All five sera
precipitated a common potein band with an apparent molec-
ular mass of about 68 kDa (lanes 5-8, and 10, band indicated
by dots). This band was not observed in the immunoprecipi-
tates obtained with Alu negative sera (lanes 2-4, 9, 11, and
12). Immunoprecipitation of [3H]uridine-labeled extracts by
the same five anti-Alu sera is shown in Fig. 6. We did
detect any common RNA band precipitated by anti-Alu sera;
hence the role and association of Alu antigen in vivo remains
to be established.
Analysis of the gels in Figs. 5 and 6 indicates that the anti-
Alu sera, in addition to anti-Alu specificity, exhibit anti-RNP
(Figs. 5 and 6, lanes 5 and 8), anti-Ro (lanes 6, 7, and 10 in
Fig. 5 and lanes 6, 7, and 9 in Fig. 6) and anti-U2/Ul
specificities (lane 7 in both figures). Control Alu-negative sera
exhibit specificities versus Sm (Fig. 5, lanes 9 and 11 and Fig.
6, lanes 10 and 12), RNP (Fig. 6, lane 13), La and Ro (Fig. 5,
lanes 3,9, and 11 and Fig. 6, lanes 3 and lo), To (Figs. 5 and
6, lane 4), and U2-U1 (Fig. 5, lane 12 and Fig. 6, lane 11)
antigens. These results support the conclusions based on the
in vitro experiments that anti-Alu specificity is not associated
with and is distinct from any known lupus antibody.
We were unable to detect the 68-kDa band on immunoblots
(28) using either nuclear (19) or whole cell extracts (18) as a
source of the antigen. In the same experiments Sm and La
proteins could be easily detected (results not shown). Since
the 68-kDa band was immunoprecipitated from 35S-labeled
extracts in much smaller quantities than La and Sm proteins
(compare bands below 50 kDa in Fig. 5 with the 68-kDa band)
well as Southern
Immunoprecipitation of in Vitro Transcribed Alu RNA
S N La To AL EJ RMc CF RM JM RM U2/!
I 2 3 4 5 6 7 8 9 1011 12
FIG. 5. Immunoprecipitation of ["S]methionine in vivo la-
beled HeLa cell extracts. Immunoprecipitated proteins were ana-
lyzed by electrophoresis on a 10% sodium dodecyl sulfate-polyacryl-
amide gel (26). Lane I, molecular mass markers. Molecular mass is
indicated in kilodaltons. Lanes 2-4,9,1 I and 12, immunoprecipitation
by Alu-negative sera. Specificities of the control sera are indicated at
top of the figure. N, serum from a healthy individual. Lanes 5-8 and
10, immunoprecipitation by anti-Alu sera. A putative Alu antigen
common to all anti-Alu sera is indicated by dots. Patient initials are
at the top.
La To AL EJ RMcCF JM RMU211 Sm RNP
9 1 0 1 1 1 ' 2 1 3
FIG. 6. Immunoprecipitation of ['Hluridine-labeled HeLa
cell extracts. Immunoprecipitation and RNA analysis
2. Lane 1, RNA isolated directly from the extracts and used as size
markers. Lanes 2-4 and 10-13, precipitation by Alu-negative control
sera. Sera specificity is indicated at the top. Lanes 5-9, precipitation
by anti-Alu sera. Patients' initials are indicated at the top of the
was as in Fig.
Prevalence of antidlu in sera containing other antinuclear antigens
Known extractable nuclear antigen specificities were determined
bv immunodiffusion and comDarison to standard reference antisera.
it is possible that the 68-kDa protein band was lost in a
somewhat higher background of immunoblots. Alternatively
it is likely that the antigen was not recognized by the antibody
after denaturation and separation on SDS-containing gels
and subsequent blotting.
Screening of the Sera for antidlu Specificity-Using the in
vitro transcription of the A36 plasmid in our assay we have
screened 114 sera from selected patients with other antinu-
clear antibodies for the ability to precipitate A h RNA. The
results of the screening, summarized in Table I, again support
the observation that anti-Ah antibody does not correspond
to any known antibody to established nuclear antigens. There
was no correlation of anti-Ah with the presence of any of the
other specificities examined.
As seen in Table I1 patients with the anti-Ah antibody
specificity suffered from scleroderma or from systemic lupus
(two patients had discoid lupus and two mixed connective
tissue disease). No obvious clinical association with anti-Alu
antibody was observed. The antibody was not a marker for
scleroderma, as it was present in only 6 of 19 scleroderma
sera. Similarly, the antibody was present in a minority of
systemic lupus erythematosus sera.
In results presented here 12% of the sera from selected
patients with autoimmune diseases contained antibodies
Number of Alu
which precipitated RNA transcribed in vitro from the A h
family gene located upstream from the human yc-globin gene.
Two other A h transcripts were also precipitated by anti-Ah
sera. Because the Alu transcripts are not uniformly precipi-
tated by sera known to contain any of the major classes of
lupus autoantibodies, i.e. anti-RNP, -Sm, -La, and anti-Ro,
anti-Alu most likely represents a distinct and novel specific-
ity. In addition, anti-Alu sera precipitate a 68-kDa protein
band which is not precipitated by the A h negative sera further
suggesting that anti-Ah is a novel specificity recognizing a
previously unidentified lupus antigen.
Alu RNA is transcribed and terminated by RNA polymerase
I11 in uitro, and it might be expected that this RNA should
bind La antigen and be precipitable by anti-La antibodies as
are other polymerase I11 transcripts. However, it should be
noted that the sequence around the termination site of the
A h gene in plasmid A36 is 5' CTTATTATT (30); hence the
transcript derived from this gene will have a UU dimer at the
3'-end. Since it has been shown that La antigen binds to the
Immunoprecipitation of in Vitro Transcribed Alu RNA
Clinical characteristics ofpatients with anti-Alu
Known nuclear antigen specificities were determined as in Table I. Abbreviations: Ab, antibody; SCL, sclero-
derma; SLE, systemic lupus erythematosus; DLE, discoid lupus; MCTD, mixed connective tissue disease; N,
nucleolar antinuclear antigen (ANA); S, speckled ANA; H, homogeneous ANA; X, unknown precipitin line. RNP,
Ro, DNA, La, and Sm denote appropriate antibody specificity.
initials Age Sex Diagnosis
Calcinosis; pulmonary fibrosis
Myositis; pulmonary fibrosis
RNP CNS lupus
Sm Autoimmune hemolytic anemia
Myositis; pituitary adenoma
oligo U sequence at the 3'-end of the polymerase I11 tran-
scripts (16) and that the binding to longer U oligomers is
much stronger than binding to dimers (31) it is likely that the
Alu RNA sequence was not recognized efficiently by the La
antigen. In contrast, VA I RNA terminates at a sequence 5'
CUUUU (32) and, as expected, was efficiently precipitated by
anti-La sera. The fact that an anti-La serum weakly precipi-
tated Alu transcripts derived from plasmids RBa-1 and U6-
13 seems to suggest that these RNAs bind La antigen
addition to the Alu antigen. Since the termination sites of
these transcripts are not well mapped it is difficult to correlate
this result with the sequences that might be responsible for
the weak binding of the La antigen.
The fact that La antigen did not bind to A36 Alu RNA
seems to indicate that binding of La protein to RNA is not
required for correct transcription and termination of at least
certain polymerase I11 transcripts. However, the possibility
that the La protein may act as a transcription or termination
factor by interacting with DNA or other components of the
transcription complex cannot be excluded.
It has been recently suggested that Alu sequences are proc-
essed pseudogenes for 7 S RNA (29). The 7 S RNA complexed
with 6 polypeptides forms the signal recognition particle
(SRP), which participates in the translocation of secretory
proteins across membranes (33). One of the SRP polypeptides
has a molecular mass of 68 kDa which is similar to that of
the common band visible in 35S-labeled anti-Alu immunopre-
cipitates. However, it has been shown that the 68-kDa SRP
protein binds to sequences of the 7 S RNA which lack ho-
mology to Alu sequences (34), making the SRP protein an
unlikely candidate for the Alu antigen. In addition, anti-Alu
sera did not precipitate 7 S RNA from the in vivo labeled
extracts prepared from HeLa cells (see Fig. 6) or baby hamster
kidney cells (not shown).
Another possibility is that Alu antigen is an RNA polym-
erase 111 transcription factor, analogous to TFIIIA (35, 36).
Similar to the AIu antigen, which binds to Alu RNA and does
not bind VA RNA, TFIIIA binds to the promoter region in 5
S RNA but does not bind to VA RNA (35-37). In our prelim-
inary experiments we were unable to inhibit transcription of
Alu RNA by pretreating the extracts
suggesting that the Alu antigen is not a transcription factor.
However, due to the volume limitations and the presence of
RNAse in the sera we were not able to ascertain that all of
with anti-Alu sera,
the antigen had been removed leaving open the possibility
that Alu antigen does play a part in transcription. More
experiments will be required to establish the identity and the
in vivo role of the Alu antigen.
Two recent reports (38,39) indicate that a high percentage
of lupus sera contain antibodies against
RNP. Transcription of the plasmid A36 (21) which has been
used in this study allows for simultaneous transcription of
Alu RNA and yc-globin RNA. Globin RNA forms a hetero-
geneous nuclear RNP-like particle during transcription in
vitro (40),' so it could be expected that this complex will be
precipitated by some sera. However, none of the 114 sera
tested precipitated yc-globin RNA in our assay. A few sera
(Fig. 1, lunes 3 and 9) precipitated a labeled high molecular
weight band, but the origin and identity of this band is not
The significance of the clinical findings regarding the pa-
tients which exhibit anti-Alu antibody is unclear. All of the
patients with this reactivity had either systemic lupus or
scleroderma. There was no apparent common manifestation
of disease nor was there any evidence of association of anti-
Alu with antibodies to other known nuclear antigens. As the
sera screened for the presence of anti-Alu were from a bank
of sera known to have antinuclear antigens, no estimate can
be made of the frequency of this specificity in the general
Acknowledgments-We thank Kathleen Kaiser-Rogers and Sylvia
Craven for excellent technicaI assistance and Dr. Paul Furdon for
helpful discussions and critical reading of the manuscript.
1. Tan, E. M. (1982) Adv. Immunol. 33, 167-240
2. Lerner, M. R., and Steitz, J. A. (1979) Proc. Natl. Acad. Sci.
U. S. A. 76,5495-5499
3. Steitz, J. A., Wolin, S. L., Rinke, d., Pettersson, I., Mount, S. M.,
Lerner, E. A., Hinterberger, M., and Gottlieb, E. (1983) Cold
Spring Harbor Symp. Quant. Biol. 47,893-900
4. Fisher, D. E., Reeves, W. G., Conner, G. E., Blobel, G., and
Kunkel, H. G. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3185-
5. Padgett, R. A., Mount, S. M., Steitz, 3. A., and Sharp, P. A.
(1983) Cell 35,101-107
6. Fradin, A., dove, R., Hemenway, C., Keiser, H. D., Manley, J. L.,
and Prives, C. (1984) Cell, 37,927-936
7. Yang, V. W., Lerner, M. R., Steitz, J. A., and Flint, S. J. (1981)
Proc. Natl. Acad. Sci. U. S. A. 78. 1371-1375