(Received for publication, July 20, 1994; and in revised form, October 10, 1994)
From the
We have characterized the 5` and 3` ends of the rat
-adrenergic receptor transcript using RNase protection
assays and have used transient transfection analysis to identify
regions of the
-adrenergic gene 5`-flanking sequences
which are important for expression. The transcript has multiple start
sites, occurring primarily in two clusters at bases -250 and
-280, relative to the first base of the initiation codon. Two
potential polyadenylation signals at +2450 and +2732 are both
functional, although the site at +2732 is preferred both in C6
glioma cells and in heart tissue. Characterization of the gene by
transient transfection analysis has identified a region between bases
-389 and -325 which is necessary for expression. The
specific deletion of a potentially functional inverted CCAAT sequence
within this region does not significantly alter activity. In addition
to the region from -389 and -325, deletion of the bases
between -1 and -159 and between -186 and -211
significantly alters expression. Both of these regions are downstream
from the
-adrenergic receptor gene start sites and may
function either through regulation of transcription or through
alteration of the transcript structure.
Physiological response to the catecholamines epinephrine and
norepinephrine is initiated by agonist interaction with the
-adrenergic receptors (
-ARs)(
)(1) . Two
major
-AR subtypes, the
- and
-adrenergic receptors (
- and
-ARs, respectively), account for most of the mammalian
-AR complement. These two receptors are the products of separate
genes (2) and can be distinguished by their relative affinities
for their endogenous ligands epinephrine and
norepinephrine(3) . In addition to these two subtypes, the gene
for a third subtype, the
-AR, has been isolated
recently from several species (4, 5, 6) .
Northern analysis and in situ hybridization have determined
that the
-AR is the primary or exclusive
-AR in
the cerebral cortex(7, 8) , the pineal
gland(7) , the ventral thalamic nuclear complex(8) ,
and the heart (7) and that
-AR is the primary
or exclusive
-AR in the prostate (7) , the cerebellar
cortex(8) , and the centrolateral and centromedial thalamic
nuclei(8) . By contrast, the
-AR mRNA is
detected exclusively in brown and white adipose tissue, where it is the
predominant
-AR(6) .
Multiple transcriptional start
sites for the -AR mRNA have been identified by S1
nuclease digestion and primer extension analysis(9) . Based
upon these data, it has been suggested that the
-AR
gene contains a promoter that utilizes an inverted CCAAT box upstream
from the multiple start sites(9) . The
-AR
gene has not been assigned a promoter, although potential elements have
been suggested. These elements include an inverted AGCCAAT sequence at
-353 (
)to -359 in the rat gene (10) and
at the corresponding location in the rhesus macaque gene (10) and an inverted CCAAT box at -1001 in the human
-AR gene(11) . Start sites for the mouse brain
-AR transcript occur at the equivalent of rat
-AR gene bases -416, -421, and
-492(12) . Using ribonuclease (RNase) protection assays,
we have identified multiple start sites for the rat
-AR mRNA, occurring primarily in two clusters
surrounding bases -250 and -280. Using the same protocol,
we have identified two polyadenylation sites, both of which are used in
C6 cells and in cardiac tissue. Using transient transfection assays we
have identified the region between bases -389 and -325 as
necessary for expression. We have also determined that the bases
between -1 and -156 and between bases -186 and
-211 contain repressor and enhancer activities, respectively,
which affect the expression of the rat
-AR gene.
Luciferase expression vectors pXP1 and pT109luc (13) were used with the kind permission of Dr. Steven Nordeen,
University of Colorado. Luciferase and -galactosidase activities
were measured using an EG & G Berthold AutoLumat LB 953
luminometer. RNA annealing incubations were performed using an MJ
Research PTC-100 programmable thermal controller. Rat C6 glioma cells
were from Dr. Kim Neve, Veterans Administration Hospital, Portland, OR.
Rat L6 myoblast cells were from ATCC.
RNase protection assays were based on the
method of Gilman(14) . Template-containing plasmids were
linearized and then stored in 1-µg aliquots in 2 µl of water at
-20 °C. Antisense transcripts were generated from 1 µg of
template in a total of 20 µl of reaction mix (40 mM TrisHCl, pH 7.9, 6 mM MgCl
, 2 mM spermidine, 10 mM dithiothreitol, 0.5 mM rNTPs
(rATP, rGTP, rUTP), 12 µM rCTP, 20 units of RNasin, 20
units of T7 or SP6 RNA polymerase, and 50 µCi of 800 Ci/mmol
[
-
P]rCTP). The reaction was incubated at 37
°C for 1 h. 25 µg of tRNA was added, and the RNA was
precipitated with ethanol. The pellet was resuspended in 5 µl of
50% formamide with xylene cyanol and bromphenol blue and applied to a
6% acrylamide, 8.3 M urea gel to separate transcript from
template and unincorporated label. The transcript was visualized by
autoradiography, excised, and then eluted into 140 µl of 10 mM Tris
HCl, pH 7.5, 400 mM NaCl, and 1 mM EDTA for 1-3 h.
Antisense transcript (10 cpm)
was combined with either 20 or 100 µg of total RNA and recovered by
ethanol precipitation. The RNA was resuspended in 30 µl of
hybridization solution (40 mM PIPES, pH 6.8, 400 mM NaCl, 1 mM EDTA, 80% formamide) and then denatured by
incubating the mix at 95 °C for 30 min. Annealing conditions were
transcript-dependent and are described in the figure legends. Following
the annealing step, 29 µl of the hybridization mix was added to an
RNase mixture (final volume 400 µl; final composition: 10 mM Tris
HCl, pH 7.5, 300 mM NaCl, 1 mM EDTA,
5.4% formamide, with RNases A and T
as indicated in the
figure legends). Incubation conditions are indicated in the figure
legends. The remaining 1 µl of the hybridization solution was used
as an undigested control. RNase digestion was terminated by the
addition of 10 µl of 20% sodium dodecyl sulfate and 25 µl of 2
mg/ml proteinase K. The mix was incubated first at room temperature for
10 min and then at 37 °C for 15 min. The solution was extracted
with phenol/chloroform and ethanol precipitated. The recovered sample
was resuspended in formamide/dye solution, and the products were
separated by electrophoresis on a 6% acrylamide, 8.3 M urea
gel. A DNA sequencing ladder using pGEM3Zf(+) as the template was
prepared to size the protected fragments. To correct for any
differences that might occur between the migration of RNA and DNA, RNA
standards of known size were compared with the DNA ladder.
The
-AR gene PstI fragment from base -484
to +270 was subcloned into the PstI site of
pGEM3Zf(+). Bases +1 to +270 were then removed from this
fragment by exonuclease III deletion(15) . A HindIII
linker was inserted immediately 3` of base -1, and the
5`-flanking sequence of the
-AR gene was reconstructed
using this fragment as the 3` end. Deletion mutants of the rat
-AR 5`-flanking sequences were then generated by one
of two methods. For the first method, deletion mutants were generated
by using endogenous restriction sites and available sites in the vector
multicloning sites. For the second method, when no conveniently located
restriction sites were available, unidirectional overlapping deletions
were generated using exonuclease III(15) . Deletion mutants
were first screened by size and then characterized by sequencing.
Selected deletion fragments generated by either protocol were subcloned
into the multicloning site of pXP1 or pd09. All constructs were
verified by dideoxy sequencing and restriction digests. The
nomenclature of the
-AR gene constructs is [5`
end, 3` end] to indicate the 5` and 3` limits of a segment of DNA.
Figure 1:
Transcription
start sites of the rat -AR gene. Panel A,
probes used to determine the start site of
-AR gene
transcription by RNase protection assay. The open bar represents the
-AR transcript. The shaded
bars represent the RNase protection assay probes. Panel
B, RNase protection assay products of antisense RNA probe
[-299, -81] hybridized to total RNA. Lane
1, RNA standards; lanes 2-5, pGEM3Zf(+)
sequence using the reverse primer; left to right, the lanes are A, C, G, T; lane 6, rat C6 glioma total
RNA; lane 7, rat L6 myoblast total RNA. The sizes of the RNA
standards are indicated by the numbers on the left.
The approximate locations of the protected start site clusters are
indicated by the numbers on the right. Probe was
hybridized to 100 µg of total RNA. The RNA/probe mix was denatured
at 95 °C for 30 min and then cooled to 85 °C. After 2 h at 85
°C, the incubation temperature was reduced in 5 °C increments
with a 2-h incubation at each increment. The final incubation was at 55
°C. Hybridized RNAs were digested with 140 µg/ml RNase A and 7
µg/ml of RNase T
at room temperature for 2 h. After the
RNases were inactivated, the RNA was recovered and the products
separated on a 6% acrylamide, 8.3 M urea gel. Variation
between RNA standard and DNA migration is less than 2%. Panel
C, RNase protection assay products of antisense RNA probe
[-479, -159] hybridized to total RNA. Panels
B and C were prepared from the same autoradiogram. Lane 1, rat L6 myoblast total RNA; lane 2, rat C6
glioma total RNA; lane 3, RNA standard; lanes
4-7, pGEM3Zf(+) sequence using the reverse primer; left to right, the lanes are A, C, G, T. The
approximate locations of the start site clusters are indicated by the numbers on the left. In addition to the start site
clusters at -250 and -280, lane 2 has additional
bands that correspond to start sites at -295, -304, and
-309. The length of the RNA standard is indicated by the number on the right. Panel D, the sequence
of the rat
-AR gene indicating the start sites
(
) identified by RNase protection assay. The sequence is from
Searles et al.(10) .
RNase protection assays using probe [-216, -1]
annealed to 20 µg of total RNA showed only a single band about 40
bases shorter than intact probe (data not shown). The 40-base
difference is equivalent to the pGEM3Zf(+)-derived sequence in the
probe and indicates that the entire length of the -AR
antisense probe was protected. RNase digestion of probe
[-299, -81] annealed to 20 µg of total C6 RNA
produced three faint bands. Two of these bands were diffuse and
somewhat smaller than the full-length probe; the third appeared to be
equivalent to probe minus vector sequence. To increase the signal from
these multiple start sites, we increased the amount of total C6 RNA
5-fold to 100 µg and increased the amount of RNase A and T
4-fold to 140 µg/ml and 7 µg/ml, respectively, to
accommodate the increased amount of RNA. The increased amounts of RNase
for this assay did not change the pattern of the digestion, but the
increased signal allowed us to identify two clusters of start sites at
approximately -250 and -280 (Fig. 1B, lane 6). In addition, the third band represented RNA initiated
close to or upstream of -299, indicating that all
-AR start sites are not within the -250 and
-280 clusters. Control experiments using 100 µg of C6 cell
RNA and probe [-299, -81] digested at 15 °C
for 2 h with 3.5 µg/ml RNase T
verified that the
clusters are not the result of RNA ``breathing'' (data not
shown).
To identify the start sites with greater resolution and to
identify additional start sites upstream of base -299, RNase
protection assays were performed with probe [-484,
-159]. This probe identified the same clusters of start
sites as probe [-299, -81] (Fig. 1C, lane 2). In addition to the
-280 and -250 clusters, digests using probe
[-484, -159] contained several additional bands (Fig. 1C, lane 2). These are derived from
transcriptional start sites at -295, -304, and -309.
These additional sites contribute to the band at -299 for probe
[-299, -81] in lane 6 of Fig. 1B. In total, 18 sites are identified by probe
[-484, -159] (Fig. 1D). The
darkest band in the -280 cluster (Fig. 1C, lane 2) corresponds to base -281. The probe has a
cytosine at the base complementary to base -281. This cytosine is
3` to 2 consecutive adenosines. Since neither RNase A nor T digests 3` of adenosines, the signal at -281 may represent
the signal from start sites at -279, -280, and -281.
Alternatively, base -281 may be the primary transcription start
site. The digest of probe [-484, -159] also has a
product which indicates intact probe minus vector sequence (data not
shown). This suggests that there are more start sites upstream of base
-484, which is consistent with the transfection analysis of the
-AR 5`-flanking sequences (below). The control
experiments with each of these probes using L6 RNA did not produce any
protected fragments.
Figure 2:
Polyadenylation sites of the rat
-AR gene. Panel A, RNase protection assay of
-AR transcript using probe [+2084,
+2901] with total RNA from rat C6 glioma and L6 myoblast
cells. Lane 1, undigested probe; lane 2, rat L6
myoblast total RNA; lane 3, rat C6 glioma total RNA; lane
4, RNA standards. The location of the polyadenylation site
corresponding to the product is indicated by the numbers on
the left. 2901 corresponds to the product derived
from unprocessed transcript. The sizes of the RNA standards, which
include the undigested probe, are indicated by the numbers on
the right. The probe and RNA were denatured at 95 °C for
30 min and then cooled to 45 °C for an overnight incubation. The
annealed RNA/probe mix was digested with 3.5 µg/ml RNase T
for 2 h at 15 °C. After the RNase was inactivated, the RNA
was recovered, and the products were separated on a 6% acrylamide, 8.3 M urea gel. Panel B, RNase protection assay of
-AR transcript using probe [+2084,
+2901] with C6 cell and cardiac tissue total RNA. Lane
1, undigested probe; lane 2, cardiac tissue total RNA; lane 3, C6 glioma total RNA; lane 4, RNA standards.
The location of the polyadenylation site corresponding to a given
product is indicated by the numbers on the left. 2901 corresponds to the product from unprocessed transcript.
The sizes of the RNA standards, which include the undigested probe, are
indicated by the numbers on the right. The parameters
for the RNase protection assay are described in the legend for panel A.
Figure 3:
Expression analysis of the 5`-flanking
sequences of the rat -AR gene. The left side is a schematic diagram of the rat
-AR 5`-flanking
sequences subcloned into the luciferase expression vector and
transfected into rat C6 glioma cells. The right side represents the expression of the
-AR constructs
relative to the expression of the plasmid pT109luc. The error bars represent ± S.E. for the combined results of two or three
transfections. Each transfection utilized triplicate
samples.
To examine further the region between -484 and -1, two series of overlapping deletions were generated and used to promote luciferase expression. Unlike the deletions performed above, these deletions do not result in a significant change in the size of the overall plasmid, and therefore interpretation of changes in expression is not complicated by the change in molar ratios of the transfected DNAs. The first set of deletions progressed from the 5` side of [-484, -1] (Fig. 4). Overall, there is a 78% difference in activity between [-484, -1] and [-160, -1]. Deletion of the bases between -484 and -389 does not significantly change expression, but when the 18 bases between -389 and -370 are deleted, there is a 35% decline in expression. Expression declines by 61% when the bases between -370 and -299 are deleted. Continued deletion from -299 to -160 does not affect expression. The expression of the last three constructs ([-299, -1], [-244, -1], [-160, -1]) differs significantly from the expression of pXP1 (p = 0.05). This may result from cryptic promoters present in the high GC regions of the remaining DNA.
Figure 4:
Expression analysis of fragment
[-479, -1] using 5` deletions. The left side is a schematic diagram of the rat -AR 5`-flanking
sequences subcloned into the luciferase expression vector and
transfected into rat C6 glioma cells. The right side represents the expression of the
-AR constructs
relative to the expression of the plasmid pT109luc. The error bars represent ± S.E. for the combined results of two or three
transfections. Each transfection utilized triplicate
samples.
A second set of recombinants was prepared by deletion of [-484, -1] from the 3` side. Luciferase activity varied in response to the deletion of three specific regions of the gene (Fig. 5). First, there is a 35% increase in signal when the region between bases -1 and -159 is deleted. Second, deletion of the region between bases -159 and -258 results in a 62% decline in activity. The majority of this decline occurs with the deletion of the region between -186 and -211, removal of which results in a 52% loss of activity. Interestingly, activity remains essentially level when the bases between -211 and -325 are removed, even though all of the start sites are deleted. Deletion of bases between bases -325 and -367 results in a loss of 81% of the remaining activity. Expression from the plasmid [-484, -367] is not significantly different from that of pXP1 (p = 0.05). These data imply that the region between -325 and -367 is necessary and sufficient for activity, even in the absence of the endogenous start sites.
Figure 5:
Expression analysis of fragment
[-479, -1] using 3` deletions. The left side is a schematic diagram of the rat -AR 5`-flanking
sequences subcloned into the luciferase expression vector and
transfected into rat C6 glioma cells. The right side represents the relative expression of the
-AR
constructs relative to the expression of the plasmid pT109luc. The error bars represent ± S.E. for the combined results of
two or three transfections. Each transfection utilized triplicate
samples.
Figure 6:
Expression analysis of the internal
deletion of the AGCCAAT element. Panel A, the
-AR gene fragment [-479, -1] was
reconstructed as described under ``Experimental Procedures''
to replace the sequence from -348 to -359 with an 8-base
linker sequence. This deleted the inverted AGCCAAT sequence at
-353 to -359. The resultant recombinant was subcloned in
the luciferase expression plasmid pXP1 and transfected as described
under ``Experimental Procedures.'' Panel B,
expression from the recombinant expression plasmid(-)CCAAT is
compared with the expression from intact [-479,
-1]. Expression from both plasmids is presented as a
percentage of the expression of the plasmid pT109luc. The error
bars represent ± S.E. for the combined results of two or
three transfections. Each transfection utilized triplicate
samples.
We have identified multiple cap sites for the rat
-AR mRNA by RNase protection assay. The use of
multiple transcription start sites is common among adrenergic receptor
genes(9, 20) . Of particular interest, the rat
-adrenergic receptor gene has been reported to use
five start sites controlled by three different promoters(21) .
In contrast to this, the human
-adrenergic receptor
transcript was shown by RNase protection assay and primer extension
analysis to possess a cluster of start sites about 177-193 bases
upstream from the translation initiation codon(22) . Similarly,
the rat and the mouse
-AR genes both use multiple
start sites, but there are no start sites common to the two genes. This
may represent species variations, or it may represent tissue variation:
the mouse transcript was isolated from brain, and the rat transcript
was isolated from a cultured glioma cell line. Overall, the use of
multiple start sites may reflect the high GC content commonly found in
the genes for the adrenergic receptors, since multiple start sites are
common in genes with high GC
promoters(23, 24, 25) .
In addition to the
heterogeneity of the 5` end of the transcript, our data also identify
two functional polyadenylation sites for the -AR gene.
Multiple sites of polyadenylation for single genes are known, and they
can be part of the complex regulation of transcript
processing(26) . For example, multiple polyadenylation sites
are present in the adenovirus major transcriptional unit, and selection
of specific polyadenylation sites varies over the course of infection (27) . Functionally, the switching of polyadenylation signals
may have an effect on the stability of a transcript by altering the
secondary structure of the 3`-untranslated sequence(28) .
Therefore, regulation of
-AR gene expression may be a
complex function involving promoter/enhancer elements modulating
transcription levels while changes in polyadenylation site alter mRNA
stability. Variation in the use of the polyadenylation sites is not
apparent in the comparison of C6 cell and cardiac tissue. Whether this
is the case for
-AR mRNAs obtained from other tissue
is under investigation. Also, whether there is a shift in the selection
of polyadenylation sites under conditions that have an effect on
receptor number (e.g.in vitro during agonist-induced
down-regulation; (31) ) and in vivo during conditions
such as congestive heart failure, which exhibits a marked decline in
the levels of
-AR expression (29) is also
currently under investigation.
Transfection analyses of specific
upstream flanking sequences of the rat -AR gene
inserted into luciferase expression vectors show that there is no
change in expression when the primary start sites at -280 and
-250 are deleted. However, sequences both upstream and downstream
from the start sites appear to have a significant effect on expression.
First, the combination of 5` and 3` deletion constructs identifies the
region between bases -389 and -325 as important for
expression. There may be multiple elements within this region, since
the decline in activity with progressive 5` deletion has a notably
smooth appearance, suggesting the progressive deletion of additive
elements. A linker mutant removing the consensus AGCCAAT sequence in
this region demonstrates that it is not functional. We are currently
examining this region more closely to characterize specific functional
elements.
Two more important regions of the -AR
gene flanking sequences, -1 to -186 and -186 to
-211, are identified by the 3` deletion constructs. Both exist
downstream of the start sites of transcription, so deletions of these
regions must be considered in the context of both transcriptional
regulation and transcript structure. The increase in activity
accompanying the 3` deletions from -1 to -211 may represent
destabilization of the DNA. Just as we have encountered difficulty in
the in vitro transcription of this gene, the high GC content
may impede transcription of the gene in vivo, and removal of
this portion of the gene may facilitate the progress of the
transcription complex. The dramatic decline in activity with the
deletion of the 25 bases between -186 and -211 may result
from the loss of two overlapping AP-4 (30) sites (bases
-204 to -196 and bases -201 to -193). However,
as indicated above, these variations in expression may also result from
changes in transcript structure.
In conclusion, we have identified
multiple transcription start sites for the rat -AR
gene. These sites occur primarily in two clusters, located at about
bases -280 and -250. Our data indicate that deletion of the
sites does not affect expression. Sequences located between -389
and -325 appear to be critical for expression, presumably through
the presence of a promoter or an enhancer. An inverted AGCCAAT sequence
within this region is not functional. The regions between -1 and
-159 and between -186 and -211 also appear to have a
significant effect upon the levels of expression. Because these regions
are downstream from the transcription start sites, it is not clear
whether their effect is transcriptional or if it results from an
alteration in the structure of the mRNA. We have also identified two
functional polyadenylation signals in the rat
-AR
gene. Further analysis is currently under way to characterize elements
necessary for the expression of the
-AR gene.