(Morgan et al. 1987) receptors that act primarily as transporters for transferin, .... of Sally. Raguet,. Joseph zachwieja and. Rejean. Melan#{231}on. We also thank.
BIOLOGY
OF
REPRODUCTION
40,
27-3
Purification, PAUL
A. KELLY,2’3
MARIKO
2 (1989)
Cloning,
JEAN-MARIE
SHIROTA,3
and Expression BOUTIN,3
MARC
EDERY,4
CHRISTINE ISABELLE
Laboratory
of the Prolactin
Receptor’
JOLICOEUR,3
HIROAKI
DUSANTER-FOURT,4
of Molecular
and
OKAMURA,3 JEAN
DJIANE4
Endocrinology3
McGill University Royal Victoria Hospital Montreal,
Quebec
Canada
H3A and d ‘Endocrinologie
Unite Institut
National
1A1 Mol#{233}culaire4
de la Recherche
CRJJ,
78350
Agronomique
Jouy-en-Josas
France ABSTRACT The
rat
liver
prolactin
receptor
has
been
purified
to
homogeneity,
and
partial
amino
aicd
sequences
have
been obtained. The structure of the receptor has been deduced from a single complementary DNA clone. The mature protein of 291 amino acids has a relatively long extracellular region, a single transmembrane segment, and a short (57 amino acids) cytoplasmic domain. With the rat cDNA used as a probe, the prolactin receptor in rabbit mammary gland and human hepatoma cells has also been isolated. These tissues contain a second, longer form of the receptor (592 receptor show regions of suggesting
that
and 598 amino strong sequence
the prolactin
and growth
acids, respectively). identity with the
hormone
receptors
INTRODUCTION The
anterior
primarily
pituitary
hormone
responsible
mammary number
for
the
gland and lactation. of other reproductive
in nonhuman
species.
In the
prolactin
(PRL)
development However, functions,
of
gland,
monoclonal receptor
a
and itself
the
prolac-
addition
to
the
with
specific,
classical
effects
in
as well as other by the interaction high-affinity
the
mammary
located
plasma membrane and widely distributed of tissues (Posner et al., 1974a, Djiane Different biochemical approaches immunoprecipitation, or immunoblot
on
2
Royal 1A1,
Victoria Canada.
Hospital,
687
Pine
West,
al.,
1984,
in different
tissues.
Prolactin
the
in a number et al., 1977). (cross-linking, analysis with
Ave.
et
ancestor.
have shown that molecular weight
1985,
1987;
levels In rat
pregnancy plays
-
(Kelly by
one
et
al.,
role
1984;
regulated
of the
tissues
with
receptor levels vary et al., 1975), increase
et al.,
estrogens
a major
Kelly
bonds to al., 1983;
are differentially liver,
prolactin binding, estrous cycle (Kelly
stimulated
the prolactin of 40,000
not linked by disulfide subunits (Haeuptle et
1984). receptor
edly
1974), (Posner
in
the
own receptor, inducing both updepending on the concentration
and
and et
are a!.,
mark1974b).
regulation
of its
down-regulation, and duration
of
exposure to prolactin (Posner et al., 1975; Manni a!., 1978; Djiane et al., 1979). As is true for growth hormone (GH), no means signal transduction has been There are no clear effects adenosine 3’, 5’-monophosphate guanosine 5’ -monophosphate pholipids, phosphorylation,
work was supported in part by grants from the Medical ReCouncil of Canada and the National Cancer Institute of Canada. Reprint requests: Dr. Paul A. Kelly, Laboratory of Molecular En-
docrinology, Quebec H3A
apparently to other
Sakai et al., Prolactin
during
‘This search
or
a common
antibodies) a relative
has
the highest during the
effects of prolacof the hormone
receptors
from
is
Katoh
tin stimulates the expression of milk protein genes by increasing both gene transcription and mRNA halflife (Guyette et a!., 1979; Houdebine et al., 1985). In gland, reproductive tin are initiated
originate
is
it also has especially
mammary
Both the short and long forms of the prolactin human and rabbit growth hormone receptors,
Montreal,
27
identified for prolactin. of prolactin on cyclic (cAMP), cyclic (cGMP), inositol phosCa2, or ion channels
et of
28
KELLY
(Matusik bine
and
et
a!.,
ceptor
Rosen, 1985).
appears
1980;
Kelly
Neither to
be
et a!.,
the a
GH
tyrosine
1984;
nor
pletely
Houde-
the
PRL
kinase.
A
ET AL.
re-
When
better
disappears
carried is
out
a partially
run
on
understanding involved in
of prolactin receptor structure, regions hormone binding, signal transduction,
Western
and tors
homology shed some
antibody, (Fig. 1B).
possible should
which In
prolactin the present
induces report,
with light
other hormone on the mechanism
its various actions. we describe the
zation, purification, cloning, and expression of the prolactin its structure with PRL receptors lated from hepatoma that
have
(Leung
et al.,
been
characteri-
amino acid sequence receptor and compare we have recently iso-
the rabbit mammary as well as with rabbit
ceptors
recepby
recently
gland and and human cloned
and
human GH re-
sequenced
1987).
CHARACTERIZATION
AND
PURIFICATION
The
PRL
receptor
weight (Mr4O,OOO) the characterization [‘25]I-oPRL to specific band with the hormone-receptor
in rat
liver
is a small
protein. Figure of the receptor hepatic membrane
an Mr of 61,000 corresponding complex is seen, which
A
to corn-
analyzed
using
by
a monoclonal
of the receptor is 41,000 amounts of PRL receptor were
solubilized, estrogen-treated preparations (Katoh et 6 mg of partially purified
and was
to homogeneity after reduction
the protein electroeluted.
band
homogeneous
PRL
analysis.
identified. From three oligonucleotide
B
female rat al., 1987). receptor
in
the
by polyacrylaand alkyla38,000-43,000
receptor
was
reduced
and
Ten the
readable
sequences
first few sequences probes were prepared
were obtained, (Boutin
1988).
Initial library
10
receptor
M
CLONING
MrX
and
1).
(-j
sequence
et al.,
A
of PRL
gel
analysis
is
(Fig.
alkylated and digested exhaustively with trypsin. The resulting peptides were purified on reverse-phase high-performance liquid chromatography (HPLC). Amino acid sequences were obtained by gas-phase
molecular
1 demonstrates by cross-linking receptors.
polyacrylamide
the apparent Preparative
reaction oPRL
preparation
receptor were purified mide gel electrophoresis
The PRL RECEPTOR
purified
binding
of excess
prepared by immunoaffinity-chromatography E2 1, a monoclonal antibody specific to the rat receptor. Preparative amounts 100 i.tg) of
tion, region
OF THE
a
the
presence
immunoblot
purified from liver membrane Approximately were using PRL
when
in the
RECEPTOR
screening with
of
a
cDNA
polysome-enriched
oligonucleotide
Xgtll
PRL-2A
12 5 ‘GT -3
MrXIO
3
detected
TGAT
positive
TCCCA
66-41
31-
bp) to
TC-3’
phages,
contained the peptide probe, plus
clones
El
the
entire used to the regions
corresponding to two flanking tryptic fragments. Insert E2 was shorter and was included within the sequence of El. A longer cDNA clone (F3) was subsequently isolated from a library prepared using size-selected clone El
mRNA as an RNA
screened). A restriction Figure 2. The hepatic FIG. 1. Prolactin receptor characterization: sodium dodecylsulfatepolyacrylamide gel electrophoresis. A) Cross-linked E’2511-ovine prolactin (oPRL) to bound membrane (microsomal) PRL receptor from the rat liver in the absence (Lane 1) and presence (Lane 2) of an excess of unlabelled 0PRL. The PRL-receptor complex migrates with an Mr of 61,000. B) Partially purified receptor revealed by Western immunoblot analysis using a monoclonal antibody (E21) against the rat liver PRL receptor (Lane 3) (Boutin et al., 1988).
TC
recombinant
and E2. Insert El (93 sequence corresponding prepare the oligonucleotide
97-
43-
two
AA
messenger 1635 bp
from probe
estrogen-treated (2.5 X l0
map of clone PRL receptor
-2.2 kb in length are included in the
(see insert
liver with recombinants F3 is shown in is encoded by a
Fig. 4), of which F3, containing a
single open reading frame that corresponds to a protein of 310 amino acids (Boutin et al., 1988). The initial methionine is followed by a 19hydrophobic amino acid stretch indicative of a signal
THE
PROLACTIN
TT
.1 II
I.
500
0
1000
3’
1500
FIG. box tion
(PRL)
2. Prolactin
indicates sites are
receptor
F3
El cDNA
restriction
the predicted coding region of clone indicated, as well as the localization
map.
The
F3. Unique of El (Boutin
blot analysis glycosylation,
purified receptor trifluoromethanesulfonic an Mr fragments
______________________ -
29
Western for by
-
5’
RECEPTOR
hatched restricet al.,
1988).
of
sequence
attempts
to
(Gascuel obtain
unsuccessful, which 20 may be present receptor
thus
the
and
Danchin,
N-terminal
suggests that as a pyro-Glu
corresponds
protein
with
a theoretical
between and the
the predicted Mr Mr of the hepatic
to Mr
sequence
Two were
the GIn at position residue. The mature a
of
1986).
291-
33,368.
amino
acid
Differences
of the F3-encoded protein PRL receptor detected by
34,000 previously
were
located
from
F3
to
the
strongly (230-25
to the separating region
plasmic
region,
and
the
ASN
an
tissue tissues
RNA
234
-r--
acids region
region of the extracellular short
C-terminal
to probe
BLOT
ANALYSIS
derived
from
of mRNA This figure
PRL PRL cyto-
El
were
Large amounts used to clearly
of Poly demonstrate
(A)’
of receptor mRNA relative abundance
the
cDNA
El,
was analyzed in 17 shows that mRNAs present
that previously had been shown receptors and were absent in all other
only
in tissues
to contain PRL tissues examined.
mRNA this fact
(100 zg) and confirm
were the
in negative tissues. of the mRNAs correlated
well with specific binding (Posner et al., 1974a; Kelly
of PRL in these tissues et a!., 1984). Indeed, the
strongest
in
signal
was
found
the
liver,
and a weak signal expressing the PRL
followed
by
was present receptor,
in in-
cluding the mammary gland. Moreover, the level of expression was hormone-dependent in liver, since the signal was weak or absent in hypophysectomized and normal males, higher in normal females, and markedly (Posner ple
increased et al., 1974b,
As is the case for forms of mRNAs
only one found species in the FIG. 3. Schematic representation of the rat liver prolactin (PRL) receptor. The localization of the three Asn-glycosylation sites and the five Cys residues in the extracellular domain are indicated.
other
amino this
it probably is involved in signal 3 schematically demonstrates rat liver prolactin PRL receptor.
probe
distribution (Fig. 4).
ovary, and prostate, all other tissues
211
In addione
illustrated.
hybridizing
(203)
sequence
of the five Cys residues (amino acid 31, and 203) and the three potential Asn sites (amino acids 54, 99, and 127) are
absence The
-CYS
tryptic sequence
The location 41, 70, 81, glycosylation
(41)
(127)-
peptide,
with with
Figure of the
(31)
ASN ( 99)-
signal
very
All
protein
mismatches.
transmembrane the N-terminal,
from
F or a band
transduction. the structure
the rat
(70) (81)
the any
hydrophobic region of 24 3) was observed. Presumably
With
-CYS -CYS
in
accounted of highly
shown). by protein
within
without
hydrophobic
binding
1
ASN ( 54)-
be
endoclycosidase acid results
NORTHERN
-CYS -CYS
might treatment
(data not identified
deduced
corresponds receptor, peptide
with
analysis tion
(41,000) since
band form
was was
seen the
in estrogen-treated 1975; Djiane et a!., other exist.
in the prostate
hormone The 2.2
liver and
female 1979).
receptors, kb band
rats
multiwas the
and was clearly a major ovary. A slightly larger
in the adrenal. Another major mRNA 4 kb band, which is the predominant
KELLY
30
ET AL.
.2 o
52
M
-
...,
)
)_
-
.2
.
N
52
.4
5252
...‘
0
53
*1:
A
#{149}t.’.
-
‘
V.
-
-2.8
.
-.
-2.1 -1.7
.
-1.3
-0.6
FIG. 4. Northern blot analysis of prolactin receptor mRNA expression. Northern blot of 17 rat mentary to clone El (93 bp). Poly (Aye mRNA (100 big) was used in each lane. The film was exposed for 36 h with an intensifying screen to detect low-abundance mRNA5 in testis, kidney, adrenal, and son, all lanes have been mounted on the same figure (Boutin et al., 1988).
form
in the
were testis,
also the
kidney seen signal
and
mammary
were seen at 2.2 and mRNA was detected et a!.,
gland.
Larger
in the ovary and the adrenal. was very weak, although faint 4 kb. only
In addition, in this tissue
forms
in
In the bands
These curves ity confirming
CHO
cells
tissues analyzed with an RNA probe complefor 16 h. The same blot was autoradiographed mammary gland. For convenience of compari-
stably
transfected
demonstrate
with
classical
that
clone
pKCR2/F3.
hormone
F3
encoded
specificthe
PRL
a 0.6 kb (Boutin
1988).
120 A
EXPRESSION
A
To the
determine
whether
synthesis
of
heterologous
and vector
clone
functional
cell
ent vectors Bluescript
the
system,
F3
F3
would
rat
PRL
was
inserted
direct
receptor
expressed in various with its transcription
in
into
0
a
differ-
cell types. promoter
0
The T3
0
was used to synthesize F3 mRNA, which was microinjected into Xenopus oocytes. The expression of the PRL receptor at the cell surface was shown by significant
specific
of
[‘25I]-PRL
(data
the
transiently
and
PRL
stably
containing and Transient
receptor
was
expressed
in mammalian
the promoter polyadenylation transfactions
cells
by
conferring
G4l8.
Similar
shows
the
and competition
stable curve
resistance results
were
transfections. of
[‘25I]-oPRL
to
the
rPRL
.-u
hPL oGH
0
region, origin of signal of the SV4O were performed in
neo
pSV2 analog
#{149}-. hGH a’c
INSULIN
using
plasmid
transient
a-c oPRL
z
A-a
both
kidney cells using the pECE/F3 stable CHO cell lines were established of the pKCR/F3 construct with the
both
60
not
COS-7 monkey construct, and by cotransfection mycin
80
40
shown). In addition, vectors replication, genome.
binding
100
0 .01
HORMONE
neo-
observed
for
Figure binding
20
5
.1
1
10
IN INCUBATION
100
1000
(ng/tube)
FIG. 5. Expression of rat liver prolactin (PRL) receptor/F3 CHO cells stably transfected with pKCR2/F3. Values are a percentage of specific binding calculated in the absence beled hormone (Boutin et al., 1988).
.:DNA in expressed as of any unla-
THE
receptor.
Transformation
Scatchard
analysis
extracts with an liver
of both affinity
PRL
of the
PRL
binding
demonstrates COS similar
receptor
that
data
by
membrane
7 and CHO cells to that reported
(Posner
PROLACTIN
bound PRL for the rat
et al. (1974b;
Kelly
et a!.
1983). REGULATION
OF PRL
RECEPTOR
EXPRESSION
GENE
Sex steroids are major regulators expression in rat liver. Using a probe
of PRL encoding
receptor the rat
PRL
receptor
mRNA
receptor
levels
in
(F3), female
response
to
we rat
studied liver
estrogen
PRL during
and
androgen
ontogeny
and
treatment.
Steady
PRL mRNA
analysis. receptor levels
stimulated
From
prepuberty
numbers increased
PRL
to
increased 8-fold, 3-fold. Estrogen
receptor
numbers
6-fold,
levels were only increased 3-fold. androgen administration, the number tors in rats previously 3-fold, but mRNA These
results
pression or
with only
estrogen declined
that
PRL
receptor
liver
is regulated
post-transcriptional
tional
level
level
(unpublished
at the as well
form of the rat mammary
which
multiple
single ever,
decreased 1.5-fold.
(Fig. 4). mechanism GH
receptors
moto
et
1987)
receptors
al.
transferin, respectively. milk,
cerebral
clone
F3
used
as a prove,
splicing is the the different
thus
of
a part
receptors. Howkinase activity nor
of
the
structural
IGF-II/M6P primarily
and prolactin fluid,
short
PRL
arrangement
et a!. 1984),
act
of the
LDL
(Yama-
(Morgan
et
as transporters
mannose 6-phosphate, has been detected and
semen.
This
al. for in sug-
transcriptional
as at the
PRL RECEPTOR
transla-
RABBiT
Gli RECEPTOR RUMAI4
HUMAN/RABBIT
uvu
MAIPATOMA
STRUCTURE 1_
With
been
site in the cytoplasmic growth factor receptors.
segment
spinal
have
ex-
RAT
RECEPTOR
are
of the
1984),and cholesterol In fact,
RNA for
class tyrosine
(Schneider that
both The
appear.
cytoplasmic
results).
OF PRL
that
is reminiscent
of
in the
in which identified.
mRNAs
Alternative responsible
receptor
transferin
species been
of receptor
a potential phosphorylation domain, as is true for many short
form
identified
is probably also present and in other tissues in
and
PRL
second
been
first have
membrane-spanning they do not possess
uvu COMPARISON
has
receptor gland,
forms
of the
this
(PRL-R2)
longer in the
Both
GH receptors in this of both the rabbit and
domain
making it the the receptor
identified probable
and
PRL
Interestingly,
receptor
rat ovary, forms of
of the
mRNA
gene
receptors. PRL
receptor
whereas treatment but
the
The
response to of PRL recep-
treated levels
suggest
in rat
In
between the cytoplasmic
human
in
adulthood,
31
identity extended
forms
state mRNA levels were determined by Northern blot analysis and densitometric analysis. Messenger RNA levels were compared to receptor numbers assessed by Scatchard
RECEPTOR
cDNAs
1_
1
encoding
the prolactin receptor from rabbit mammary gland and human hepatoma have just been isolated. In addition, cDNAs encoding the PRL receptor in rat mammary gland, ovary, fied recently. A second appears rabbit
to be the predominant and human (Fig. 4). The
a 592-amino acid prolactin receptor ever, a much longer PRL receptor (598 structure second size to
and kidney form of
have been identithe PRL receptor
species in both rabbit PRL receptor
the is
211
#{149}..
211
291
protein highly similar to the rat (Edery et al., 1989), with, howcytoplasmic domain. The human amino acids) has a similar overall
to the rabbit mammary receptor. The form of the PRL receptor is much closer in the rabbit and human GH receptor, which has
recently been cloned and sequenced (Leung et a!., 1987). In addition to the highly conserved sequence just past the transmembrane segment (Boutin et a!., 1988),
211
there
are 3 additional
regions
of strong
sequence
592 FIG. 6. Schematic comparison rat liver, rabbit mammary gland, rabbit or human growth hormone
820 of the prolactin (PRL) receptor and human hepatoma compared (GH) receptor.
from to the
KELLY
32 gests
that
and
testis-where
in
the
localized-that their normal
mammary
these function
transport proteins, one compartment investigate
The the
localized
GH
mains Whether
await
than
regions
and
PRL
suggests
form
where
membrane transfer
more
in the
important alone
been
may, in addition transduction, act
of sequence
regions,
mutagenesis
one
plexus, have
the hormone It will be interesting
receptors some
these
some other ble for the
receptors of signal
in tissues
choriod
receptors
translocating to another.
if more
tor is present occurs.
gland,
prolactin
to as
from to
of the
PRL
recep-
than
one
action
identity cytoplasmic
between do-
functional
role.
or in combination
with
of
component the hormonal
studies
in functional
(s)
are responsimessage must systems.
ACKNOWLEDGMENTS These studies were supported in part by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada. We gratefully acknowledge the technical support of Sally Raguet, Joseph zachwieja and Rejean Melan#{231}on. We also thank Nirmala Brunel for typing the manuscript.
REFERENCES JM, Jolicoeur C, Okamura H, Gagnon J, Edery M, Shirota M, Banville D, Dusanter-Fourt I, Djiane J, Kelly PA, 1988. Cloning and expression of the rat prolactin receptor, a member of the growth hormone/prolactin receptor gene family. Cell 53:69-77 Djiane J, Clauser H, Kelly PA, 1979. Rapid down-regulation of prolactin receptors in mammary gland and liver. Biochem Biophys Res Commun 90:1371 -78 Djiane J, Durand P. Kelly PA, 1977. Evolution of prolactin receptors in rabbit mammary gland during pregnancy and lactation. Endocrinology 100:1348-56 Edery M, Jolicoeur C, Levi-Meyrueis C, Dusanter-Fourt I, P#{233}tridou B, Boutin
Boutin J-M, Lesueur L, Kelly PA, Djiane J, 1989. Identification and sequence analysis of a second form of prolactin receptor by molecular cloning of complementary DNA from rabbit mainmary gland. Proc Nati Acad Sci (USA): (In press) Gascuel 0, Danchin A, 1986. Protein export in prokaryotes and eukaryotes: indications of a difference in the mechanism of exportation. J Mol Evol 24:130-42 Guyette WA, Matusik RJ, Rosen JM, 1979. Prolactin-mediated transcriptional and post-transcriptional control of casein gene expression. Cell 17:1013-23 Haeuptle MT. Aubert ML, Djiane J, Kraehenbuhl JP, 1983. Binding
ET AL. sites for lactogenic and somatogenic hormones from rabbit mammary gland and liver. J Biol Chem 258:305-14 Houdebine LM, Djiane J, Dusanter-Fourt I, Martel P, Kelly PA, Devinoy E, Servely JL, 1985. Hormonal action controlling mammary activity. J Dairy Sci 68:489-500 Katoh M, Djiane J, Kelly PA, 1985. Prolactin binding components in rabbit mammary gland: characterization by partial purification and affinity labelling. Endocrinology 116:2612-20 Katoh M, Djiane J, Leblanc G, Kelly PA, 1984. Characterization of antisera to a partially purified prolactin receptor: effect on binding in different target tissues. Mol Cell Endocr 34:191 -200 Katoh M, Raguet S. Zachwieja J, Djiane J, Kelly PA, 1987. Hepatic prolactin receptors in the rat: characterization using monoclonal antireceptor antibody. Endocrinology 120:739-49 Kelly PA, Djiane J, Katoh M, Ferland LH, Houdebine LM, Teyssot B, Dusanter-Fourt I, 1984. The interaction of prolactin with its receptors in target tissues and its mechanism of action. Recent Prog Horm Res 40:379-436 Kelly PA, Djiane J, Leblanc G, 1983. Preferential establishment of a slowly dissociable component in plasma membrane compared to intracellular prolactin receptors. Proc Soc Exp Biol Med 172: 219-24 Kelly PA, Posner BI, Friesen HG, 1975. Effects of hypophysectomy, ovariectomy, and cycloheximide on specific binding sites for lactogenic hormones in rat liver. Endocrinology 97:1408-15 Kelly PA, Posner BI, Tsushima T, Friesen HG, 1974. Studies of insulin, growth, hormone, and prolactin binding: onrogenesis, effects of sex and pregnancy. Endocrinology 96:532-39 Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Henzel WJ, Barnard R, Waters Mi, Wood WI, 1987. Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature (Lond) 330:537-43 Manni A, Chambers MJ, Pearson OH, 1978. Prolactin induces its own receptors in rat liver. Endocrinology 103:2168-71 Matusik RJ, Rosen JM, 1980. Prolactin regulation of casein gene expression: possible mediators. Endocrinology 106:252-59 Morgan DO, Edman JC, Standring FN, Fried VA, Smith MC, Roth RA, Rutter Wi, 1987. Insulin-like growth factor II receptor as a multifunctional binding protein. Nature (Lond) 329:301-07 Posner
BI, Kelly PA, Friesen HG, 1974b. Induction of a lactogenic receptor in rat liver: influence of estrogen and the pituitary. Proc NatI Acad Sci USA 71:2407-10 Posner B!, Kelly PA, Friesen HG, 1975. Prolactin receptors in rat liver: possible induction by prolactin. Science 188:57-59 Posner BI, Kelly PA, Shiu RPC, Friesen HG, 1974a. Studies of insulin growth hormone and prolactin binding: tissue distribution, species variation and characterization. Endocrinology 96:521-31 Sakai S. Katoh M, Berthon P, Kelly PA, 1984. Characterization of prolactin receptors in porcine mammary gland. Biochem J 224: 911-22 Schneider C, Owen MJ, Banville D, Williams JG, 1984. Primary structure of human transferrin receptor deduced from the mRNA sequence. Nature (Lond) 311:675-78 Yamamoto T, Davis CG, Brown MS. Schneider WJ, Casey ML, Goldstein JL, Russell DW, 1984. The human LDL receptor: a systeinerich protein with multiple alu sequences in its mRNA. Cell 39: 27-38