(Received for publication, June 23, 1994; and in revised form, November 28, 1994)
From the
Chronic administration of thyroid hormone (T)
increases apolipoprotein (apo) A-I gene expression in rat liver. That
transcriptional activity of the apoA-I gene is reduced to 50% of
control, whereas abundance levels of nuclear and total cellular apoA-I
mRNA are increased 3-fold, implies more effective apoA-I mRNA
maturation. To study hormonal effects on apoA-I RNA processing, we
quantified mRNA precursors in control and T
-treated rats
(50 µg/100 g body weight for 7 days). Northern blotting,
amplification of reverse-transcribed RNA, and ribonuclease protection
assays showed that the splicing pathway is branched, in that either
intron 1 or intron 2 is removed first from the primary transcript,
whereas intron 3 is removed last. In T
-treated rats,
abundance levels of the primary transcript, the intron 1-containing
precursor devoid of intron 2, the intron 2-containing precursor devoid
of intron 1, the intron 3-containing precursor lacking both introns 1
and 2, and nuclear mRNA were 65, 183, 78, 195, and 268% of controls.
Compared with control rats, the half-life of the intron 1-containing
precursor, measured after injection of actinomycin D, was increased
2-fold in T
-treated rats. In contrast, half-lives of the
primary transcript and the intron 2-containing precursor were similar
in control and T
-treated rats. Ribonuclease protection
assays revealed an RNA species extending from the transcription start
site close to the 3` end of intron 1. The abundance of this RNA
fragment, probably representing a degradation product, was 2.5-fold
higher in control than in T
-treated animals (p < 0.001). Sequences of apoA-I mRNA precursors were identical in
control and T
-treated rats which excluded hormonal effects
on splice-site selection or post-transcriptional editing of apoA-I
transcripts. Compartmental modeling of apoA-I mRNA processing suggested
that chronic thyroid hormone administration enhances apoA-I mRNA
maturation more than 7-fold by protecting the intron 1-containing
precursor devoid of intron 2 from degradation and by facilitating the
splicing of intron 1 from this precursor.
Plasma concentrations of both high-density lipoprotein
cholesterol and apoA-I, ()high density lipoprotein's
main structural apolipoprotein, are inversely associated with the
incidence of coronary artery disease(1, 2) . Animal
experiments support the notion that increasing apoA-I levels in plasma
may be beneficial. Infusion of high density lipoprotein into rabbits
fed an atherogenic diet induced regression of atherosclerotic lesions (3) , and overexpression of human apoA-I in transgenic mice
decreased the severity of diet-induced atherosclerosis(4) . The
mechanisms whereby apoA-I might protect against atherosclerosis are not
fully understood, but participation by the apolipoprotein in reverse
cholesterol transport may be the key. ApoA-I promotes the translocation
of cholesterol from intracellular pools to the cell
membranes(5) , facilitates the transfer of cholesterol from
cell membranes to nascent high density lipoprotein(6) , and
enhances lecithin:cholesterol acyltransferase-mediated esterification
of cholesterol(7) .
The apoA-I gene is expressed in the
liver and intestine(8, 9, 10) , and a number
of genetic and metabolic factors are known to regulate its
tissue-specific expression
levels(11, 12, 13) . Elucidating the
molecular mechanisms of enhanced expression may allow the development
of approaches for preventing atherogenesis. We and others have shown
that administration of thyroid hormone increases plasma concentrations
of apoA-I in the
rat(14, 15, 16, 17) . Whereas
T does not alter apoA-I mRNA levels in the
intestine(16, 17) , it increases, by complex
mechanisms, apoA-I mRNA levels in the liver. With a single dose of
T
, the transcriptional activity of the apoA-I gene in the
liver as well as the abundance of its mRNA rapidly
rise(14, 18) . With chronic administration, the
synthesis of apoA-I mRNA is reduced by half but abundance levels of
nuclear and total cellular apoA-I mRNA remain elevated, which implies
enhanced apoA-I mRNA maturation(14) .
To gain insight into
the mechanism(s) whereby the hormone affects nuclear apoA-I RNA
processing, we quantified the abundance of apoA-I mRNA precursors. We
describe the splicing pathway of the apoA-I primary transcript in rat
liver and report that chronic administration of thyroid hormone
protects the apoA-I mRNA precursor that contains intron 1 but is devoid
of intron 2 from degradation and may facilitate the splicing of intron
1 from this precursor. ()
Adult male Sprague-Dawley rats (Texas Animal Specialties,
Humble, TX) each weighing about 250 g were housed in a room with a 12-h
light cycle (7-19 h). Animals were fed standard rat chow ad
libitum. Rats were rendered hyperthyroid by daily subcutaneous
injections of T (50 µg/100 g body weight) for 7 days.
This treatment has been shown to lead to plasma T
levels
that result in saturation of more than 80% of hepatic T
receptors(20) . Food was removed at 9 a. m., and rats
were anesthetized with pentobarbital (5 mg/100 g body weight) 3-4
h later. Before liver excision, livers were flushed with ice-cold
saline through the portal vein. Hyperthyroidism was confirmed by
determinations of plasma T
levels using a radioimmunoassay
(Quantimune
T
RIA, Bio-Rad). To block
transcription in vivo, control and T
-treated rats
were injected while anesthetized with actinomycin D (0.5 mg/250 g body
weight) through the portal vein. Liver pieces were excised prior to,
and at various time points after, the injection of actinomycin D and
were used for preparation of nuclear RNA.
Rat liver nuclei were prepared according to the method of Northemann et al.(21) as described previously(22) . Nuclear RNA was extracted from isolated nuclei by the method of Lamers et al.(23) as described (14) and stored at -70 °C. Polyadenylated RNA was isolated from nuclear RNA by chromatography on oligo(dT)-cellulose(24) .
Northern
transfer of RNA was performed as outlined by Thomas(25) .
Nuclear RNA was denatured with 1 M glyoxal and 50%
MeSO and separated by electrophoresis in 1.2% agarose gels.
RNA was transferred by diffusion blotting to S& Nytran membranes
(Schleicher & Schuell Inc., Keene, NH). Membranes were hybridized
with
P-labeled apoA-I cDNA (14) or with
P-labeled DNA consisting of intron sequences only. Probes
for introns 1, 2, and 3 of the rat apoA-I gene were obtained by
amplification of genomic DNA using the polymerase chain
reaction(26) . Primers were synthesized by using a Cyclone Plus
synthesizer and reagents from Milligen Biosearch Division (Burlington,
MA). The following oligonucleotides were used: 5`-TAAGTGCTGCTACCTACC-3`
(+23, +40) and 5`-CCTGGAGAAGAAGAAGAA-3` (+221,
+204) for intron 1-specific amplification;
5`-TGGGTGCCTCTTGGTCTCCAT-3` (+284, +304) and
5`-CCTGAGGATGGAGAGGAGACCCATG-3` (+435, +411) for intron
2-specific amplification; 5`-GCCTTGCAACTGGCACCAAC-3` (+899,
+918) and 5`-CTAGAGGGGAAGAGAGCAGCTGAGAGATGA-3` (+1140,
+1111) for intron 3-specific amplification; the numbers in
parentheses designate the location of the 5` and 3` ends relative to
the major transcription start site(27) . PCR assays contained 1
µg of genomic DNA isolated from rat liver(24) , 0.2
µM of each upstream and downstream primer, 200 µM of each dNTP, 10 mM Tris-HCl, pH 8.3, 50 mM KCl,
2.5 mM MgCl
, 2.5 units of Amplitaq (Perkin-Elmer
Corp.) in a 100-µl reaction volume that was overlaid with mineral
oil. Samples were processed through initial denaturation for 5 min at
94 °C; 30 cycles of amplification each consisting of 1 min at 60
°C (annealing), 1 min at 72 °C (extension), and 1 min at 94
°C (denaturation); and a final extension at 72 °C for 10 min.
PCR products were separated by electrophoresis in 1.2% agarose,
purified using Qiaex (Qiagen Inc., Chatsworth, CA), and labeled with
[
P]dCTP by nick translation(28) .
For
reverse transcription of apoA-I nuclear RNA and quantitative
amplification of cDNA, the following primers were used: exon 1,
5`-GACTGTTGGAGAGCTCCG-3` (-3, +15); exon 3,
5`-CTCATCTTGCTGCCAGAACT-3` (+469, +450); exon 4,
5`-TAGCCCAGAACTCCTGAGTC-3` (+1232, +1213); intron 1,
5`-CCACTCCAGATAGTTCTTCTA-3` (+124, +144); and
5`-GTGCTGCCATGGAGAGGTGCCACACACATTC-3` (+182, +152); intron 2,
5`-CCTGAGGATGGAGAGGAGACCCATG-3` (+435, +411); and
5`-AAGTCTGACATGTCAGCT-3` (+367, +384); intron 3,
5`-TCATTCTTACAGGGTTCCTCTGCCC-3` (+669, +645). The 5` ends of
primers for -actin, used as an internal amplification standard,
were located 14 and 171 base pairs downstream of the intron 4-exon 5
junction (29) and were: 5`-TGAGCCCAAGTACTCTGT-3` and
5`-GTAAAACGCAGCTCAGTAACAGTCC-3`.
Nuclear RNA (1-5 µg) was
reverse-transcribed using 200 units of Moloney murine leukemia virus
reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD), 10
mM Tris-HCl, pH 8.3, 50 mM KCl, 5 mM MgCl, 1 µM downstream primer, or 2.5
µM random hexamers, 1 mM dNTP, and 20 units of
RNasin (Promega Corp., Madison, WI) in a volume of 20 µl. cDNA was
amplified after addition of 0.2 µM upstream primer and 4
µCi of [
-
P]dCTP (3000 Ci/mmol, ICN,
Costa Mesa, CA) using the conditions described, but the number of
amplification cycles was limited to 18. In experiments in which the
downstream primer used for reverse transcription and that used for
amplification of cDNA differed, template nuclear RNA was digested with
RNase A (100 µg/ml) and T1 (1000 units/ml) (Pharmacia, Uppsala,
Sweden) at 37 °C for 20 min to avoid amplification of RNA by Taq
polymerase owing to its reverse transcriptase activity. cDNA was then
purified from primers and nucleotides by using TE 100 Spin Columns
(Clontech, Palo Alto, CA) and amplified as described above. Aliquots of
the PCR solution were applied to polyacrylamide-urea sequencing gels.
Polynucleotide standards were end-labeled with polynucleotide kinase
(New England Biolabs, Inc., Beverly, MA) and
[
-
P]ATP (4500 Ci/mmol, ICN). After removal
of urea, gels were dried and exposed to x-ray film. Intensity levels of
the bands were quantified by scanning autoradiographs with an UltraScan
XL Laser Densitometer equipped with Gelscan XL 2.1 software (Pharmacia)
using the linear range of films.
The quantification of nuclear
apoA-I mRNA precursors was validated in several experiments. All
nuclear RNA samples either poly(A) selected or unfractionated were
treated with RNase A and T1 prior to reverse transcription and
amplification. No PCR products were detected in these experiments
indicating the absence of genomic DNA that could have confounded RNA
measurements by serving as templates. In most assays, coamplification
of -actin RNA was used for internal assay standardization, and
normalization for
-actin PCR products had no significant effect on
the relative abundance levels and proportions of apoA-I RNA species.
Typically, quantification was at the end of 18 cycles; to exclude
plateau effects, aliquots of PCR mixtures were removed after 12, 15,
20, and 24 cycles and analyzed as described above. Over this full
range, the distribution of cDNAs representing the various apoA-I mRNA
precursors remained constant. In addition, varying the input of
template nuclear RNA over a range of 0.2 to 5 µg did not alter the
distribution among amplification products of apoA-I transcripts.
To determine the nucleotide sequence of mRNA precursors, PCR products were subjected to denaturing polyacrylamide gel electrophoresis. Gel pieces containing amplification products of distinct RNA species were excised, and DNA was eluted in 100 mM Tris-HCl, pH 8.3, 500 mM NaCl, 1 mM EDTA. After precipitation in ethanol, reamplification, and purification in 2% agarose gels, DNA was cloned into pGEM-3Zf+ (Promega Corp., Madison, WI) as described(30) . Individual clones were sequenced by the dideoxy chain termination method (31) using the Sequenase 2.0 kit (U. S. Biochemical Corp., Cleveland, OH). Genomic sequencing was performed by ligation mediated PCR(32) .
RNase protection assays were
performed as an additional method of quantification of apoA-I mRNA
precursors. Using exon 1 and exon 3 primers described above, a
466-nucleotide segment of genomic rat apoA-I DNA was amplified and
cloned into pGEM-3Zf+. After linearization of DNA, labeled
antisense RNA was transcribed from the SP6 promoter using Maxiscript
(Ambion Inc., Austin, TX), mG(5`)ppp(5`)G (Ambion), and
[
-
P]UTP (specific activity 3000 Ci,
mM, DuPont NEN) according to the instructions of the
manufacturer. Newly synthesized
P-labeled RNA was gel
purified, and aliquots of 0.4 ng corresponding to about 5
10
cpm were coprecipitated with nuclear RNA (1-10
µg). Pellets were dissolved in hybridization buffer (RPA II Kit,
Ambion), heated at 95 °C for 5 min, and incubated at 45 °C
overnight. The following day, samples were incubated with 0.5 unit of
RNase A and 20 units of RNase T1 for 30 min at 37 °C, in a total
volume of 220 µl, to digest unhybridized RNA. RNase
inactivation/precipitation mixture, supplied by the manufacturer, was
added to precipitate
P-labeled RNA-RNA hybrids.
Precipitates were washed with 70% ethanol and subjected to 10%
polyacrylamide-urea sequencing gels. Protected fragments were
quantified by scanning of autoradiographs. For assay standardization,
sense RNA was transcribed, in the presence of
[
-
H]UTP (specific activity 35.8 Ci,
mM, DuPont, Boston, MA) and m
G(5`)ppp(5`)G, from
the T7 promoter of pGEM-3Zf+ clones containing inserts
complementary to the primary transcript and to nuclear RNA lacking
intron 1 or intron 2 or both. A standard curve was constructed using
the gel-purified [
H]RNA to estimate the content
of individual apoA-I mRNA precursors in total nuclear RNA. For all four
standard [
H]RNA species a linear relationship (r < 0.998) between signal intensity (integrated area of
the protected fragment on autoradiographs) and dose of
[
H]RNA was found over the range tested
(1.5-40 pg). After correction for specific activity of the
[
P]RNA protected, slopes between RNA input and
signal were identical for the three larger RNA species, but the signal
intensity of the smallest RNA was reduced to 50% of the larger RNA
species. To validate the quantification of distinct apoA-I mRNA
precursors in nuclear RNA, mixtures of the four standard
[
H]RNA species containing up to 16-fold
differences in the mass of individual RNAs were used in protection
assays. In these RNA mixtures, the relation between the dose of an
individual RNA and the discriminatory signal, i.e. the
intensity of the band specific for the RNA, was identical to that of
assays containing the respective RNA only. In other control
experiments, in which yeast RNA was used instead of rat hepatocyte
nuclear RNA, protected RNA fragments were not detected. When digestion
with ribonucleases was omitted, the
P-labeled antisense
probe was quantitatively recovered and exhibited the electrophoretic
mobility expected.
Compartmental analysis of apoA-I mRNA processing
was performed using the CONSAM V31.0 computer program (33, 34) which was kindly supplied by Dr. Loren A.
Zech from the Laboratory of Mathematical Biology, National Institutes
of Health, Bethesda, MD. Fractional transfer rate constants (k) and mass flux (R
) are reported here using the k
nomenclature(33, 34) .
The processing of nuclear mRNA was modeled in two stages. In the first stage, the decline in mass of the various apoA-I transcripts after administration of actinomycin D, was modeled as a linear, time-invariant, nonsteady-state compartmental model. The criterion of parsimony was used to select the simplest model in which each transcript consisted of a single compartment. During development of this minimal model, it became apparent that several similar structures would fit the data. The steady-state mass of each mRNA transcript before actinomycin D treatment defined the initial mass of each compartment. Because the mass of compartments containing the intron 2 containing precursor lacking intron 1 and the transcripts devoid of intron 1 and intron 2 did not change following actinomycin D, the information content in these experiments was insufficient to permit independent estimates of the fractional transfer rates. Consequently, dependence equations were used to compute values for k based on the steady-state mass (M) of the linked compartments z, y, and x by the following formula,
The adjustment of the fractional transfer rate parameters (k) by the program was complete when no
further improvement in the sums of squares of the residual values could
be achieved, and the fractional error of the fitted parameters was not
improved by further iteration of the system. In the second stage, the
computed fractional transfer rates and the initial steady-state masses
were used to compute the flux of material through the system in a
pseudo-steady-state model. This model is valid if actinomycin D does
not alter the activity of the enzymes involved in processing the
primary mRNA transcript over the period of observation.
Northern blots of total nuclear RNA showed several apoA-I RNA
species (Fig. 1). The most abundant nuclear apoA-I RNA displayed
an electrophoretic mobility similar to mature cytoplasmic RNA (about
1050 nucleotides). Additional RNA species, not well separated from each
other but clearly separated from the smallest nuclear apoA-I mRNA, were
identified on blots probed with apoA-I cDNA. These larger species
hybridized with sequences specific for intron 3, intron 2, and intron 1 (Fig. 1B) and were thus apoA-I mRNA precursors. The
band(s) above the mature apoA-I mRNA consisted of about 1600
nucleotides as estimated from their electrophoretic mobility. Since
introns 1, 2, and 3 contain 199, 152, and 552 base pairs(26) ,
respectively, the 1.6-kilobase RNA was likely to contain intron 3.
Nuclear apoA-I RNA species were also analyzed after reverse
transcription and PCR with primers complementary to exons 1 and 4. All
PCR products that could theoretically be expected were identified in
sequencing gels. In both control and T-treated rats, the
abundance of PCR products corresponding to precursors containing intron
1, intron 2, or both, but devoid of intron 3 was less than 5% of the
abundance of intron 3-containing precursors (not shown). These findings
were thus in agreement with Northern blots showing only RNA species
with 1050 and 1600 or more nucleotides even after hybridization with
intron 1- and intron 2-specific probes. Hence, the removal of intron 3
must have occurred after the removal of introns 1 and 2 or at a slower
rate. In hepatocyte nuclei of chronically hyperthyroid rats, the
abundance levels of mature apoA-I mRNA forms increased to 305 ±
81% of control (mean ± S.D., five pools per group, two rats per
pool, p < 0.01, Mann-Whitney U test), while the
abundance level of precursor forms was not significantly different from
controls (133 ± 35% of control). Furthermore, the difference in
hybridization signals between control and experimental animals appeared
to be greater for the intron 3-specific probe than for intron 1- or
intron 2-specific probes (Fig. 1B).
Figure 1:
Influence of chronic
hyperthyroidism on the abundance of apoA-I RNA in rat hepatocyte
nuclei. A, Northern blots of (a) 5 µg and (b) 10 µg of nuclear RNA of control rats (Co) and
rats injected daily with 50 µg of T/100 g body weight
for 7 days (T
). Membranes were hybridized with
P-labeled apoA-I cDNA. Arrows indicate positions
of the mature nuclear RNA (about 1050 nucleotides) and the smallest
precursor visible (about 1600 nucleotides). B, Northern blots
of 10 µg of nuclear RNA hybridized with
P-labeled
apoA-I cDNA (c). Blots were stripped and subsequently
hybridized with
P-labeled probes specific for intron 3 (3), intron 2 (2), and intron 1 (1). Control versus T
-treated rats. Mobilities of nucleotide
standards are shown on the right.
To overcome the
limitations of Northern blotting in separating intron 3-containing mRNA
precursors, a PCR-based assay was used. Using primers complementary to
exons 1 and 3 (Fig. 2A) and nuclear poly(A) RNA
prepared from six control and six T-injected rats, four PCR
products were visualized in denaturing polyacrylamide gels (Fig. 2B). In chronically hyperthyroid rats, the
intensity of the largest band representing the amplification product of
the primary transcript was decreased to 52% of control rats. This was a
consistent finding in three independent assays using the same poly(A)
RNA preparations. A similar result was obtained when primary
transcripts were quantified in unfractionated nuclear RNA (Fig. 2C); in hyperthyroid rats, the abundance of the
primary transcript was reduced to 59 ± 5% of its abundance in
control rats (mean ± S.D., triplicate determinations in three
pools per group, two rats per pool, p < 0.002, t test). Abundance levels of
-actin mRNA were similar in
control and T
-treated rats.
Figure 2:
Analysis of apoA-I mRNA precursors by
reverse transcription and PCR amplification in control rats and rats
with chronic hyperthyroidism. A, diagram of the portion of the
apoA-I gene encompassed by the primers used. A, B, and C refer to the first three exons of the apoA-I gene; I and II refer to intron 1 and intron 2, respectively. Arrows depict the locations of primers used for PCR, and numbers in parentheses indicate the 5` ends of primers relative
to the major transcription start site(26) . B,
autoradiograph of gel-separated PCR products. Nuclear poly(A)-selected
RNA (1 µg) isolated from six control (Co) and six
T-injected (T3) rats was reverse-transcribed using
random hexamers prior to amplification. Products were analyzed by
denaturing electrophoresis in 10% polyacrylamide gels. Lane M shows end-labeled standards containing the number of nucleotides
indicated. C, autoradiograph of gel-separated PCR products
obtained from unfractionated nuclear RNA of control (a, c, and e) and T
-treated rats (b, d, and f); amplification of apoA-I (a and b) or
-actin transcripts (e and f), coamplification of
apoA-I and
-actin transcripts (c and d); D, identity of bands visualized in B and C.
The exact number of nucleotides was determined by sequencing. Because
of deletions of two and three nucleotides in introns 1 and 2,
respectively, the nucleotide numbers shown differ from those expected
from the apoA-I sequence described by Haddad et
al.(27) .
In contrast to the decreased abundance of the primary transcript, the intensity of the smallest PCR product (Fig. 2, B and C) was increased in chronic hyperthyroidism. Since this band comprised amplification products of mature mRNA and intron 3-containing RNA devoid of introns 1 and 2, these results were in agreement with the Northern blots shown in Fig. 1. The two PCR products of intermediate size were identified by intron-specific primer extensions of unlabeled PCR products with end-labeled primers. As expected, extension with an intron 1-primer created two products of about 340 and 190 nucleotides, while extension with an intron 2-primer resulted in two products of 430 and 240 nucleotides (not shown). The larger extension product in both assays matched the size expected for the primary transcript; the smaller extension products showed mobilities calculated for precursors devoid of either intron 2 or intron 1. Thus, the two middle bands in Fig. 2B were precursors of apoA-I mRNA that contained either intron 1 or intron 2. Hence, either intron 1 or intron 2 can be spliced first in vivo to give rise to precursors schematized in Fig. 2D.
To determine the exact size of mRNA precursors and to exclude differences in the splicing pathway between control and experimental rats that could have remained undetected in our assay, the four PCR products were cloned and sequenced (Fig. 3). These sequence analyses showed complete homology to the apoA-I gene sequence reported by Haddad et al.(27) with the exception of two and three nucleotide deletions in introns 1 and 2, respectively. The splice sites for introns 1 and 2 were the same in control and hyperthyroid rats and were identical to those reported by Haddad et al.(27) .
Figure 3:
Comparison of the apoA-I gene sequence
extending from the transcription start site into exon 3, reported by
Haddad et al.(26) , with sequences of genomic DNA and
various mRNA precursors of control and T-treated rats. The
major transcription start site is indicated by an asterisk.
Exon nucleotides are underlined. Solid lines below the published sequence (26) refer to: A, genomic
DNA isolated from livers of rats used for mRNA maturation studies; B, primary transcript; C, mRNA precursors containing
intron 1 but devoid of intron 2; D, mRNA precursors containing
intron 2 but devoid of intron 1; E, mRNA precursors devoid of
introns 1 and 2. B-E represent sequences from pGEM-3ZF+
clones containing cDNA inserts corresponding to the respective nuclear
RNA species of three pools of two animals per group. Deletions at
position 50, 53, 330, 331, and 388 were present in all sequences
containing the respective intron. In the primary transcript, three
nucleotide changes were noted in 3 (indicated by superscripts a, b, and c) of 16 T
clones and 1 (superscript d) of 8 control clones. In the intron 1-containing precursor,
two changes in 2 (superscript e and f) of 8 T
clones were found, whereas no changes were found in 4 control
clones. In the intron 2-containing precursor, four sequence changes
were found in 3 (superscripts g, h, and i) of 15
T
clones, whereas no changes were found in 4 control
clones. In the precursor devoid of introns 1 and 2 no sequence changes
were observed in 3 control and 3 T
clones.
Since the PCR-based assay with primers for exon 1 and exon 3 did not
distinguish between nuclear mRNA and intron 3-containing precursors
devoid of introns 1 and 2, the abundance of intron 3-containing
precursors was quantified by using a primer complementary to intron 3
for reverse transcription. To generate bands that were comparable in
size to the previous studies, primers for exon 1 and exon 3 were used
for amplification of cDNA. Autoradiographs of these studies, a
representative experiment performed in the presence of -actin
primers is shown in the inset of Fig. 4, revealed a
substantial portion of nuclear apoA-I RNA devoid of intron 1 and intron
2 but containing intron 3. In hyperthyroid rats, the abundance level of
this precursor was increased to 195 ± 51% of control rats (mean
± S.D., six pools per group, two rats per pool, p <
0.001). In T
-treated rats, the abundance level of the
primary transcript was reduced to 55 ± 9% of control rats (p < 0.001), a decrease nearly identical to that measured under
different assay conditions (Fig. 2).
Figure 4:
Effect of chronic hyperthyroidism on
abundance levels of apoA-I primary transcript and mRNA precursors
containing intron 3, but devoid of intron 1 and 2. Compared are
abundance levels between T-treated rats (full
columns) and control rats (open columns). Columns and bars represent mean values and S.D., respectively, of
six pools per group (two rats per pool). Asterisks indicate
significant differences between T
-treated and control rats (p < 0.01). Nuclear RNA was reverse-transcribed using an
intron 3-specific primer; cDNA was amplified with primers for exons 1
and 3. The inset shows gel-separated PCR products from two
pools of control (lanes 1 and 2) or
T
-treated rats (lanes 3 and 4).
Coamplification of
-actin transcripts was used for assay
standardization. Lanes 5 and 6 show amplification
products of
-actin RNA in control and T
-treated rats,
respectively. Band 1, PCR product of the primary transcript of
the apoA-I gene; band 2, PCR product of the
-actin
transcript; band 3, PCR product of the apoA-I mRNA precursors
containing intron 3 but lacking introns 1 and
2.
The PCR-based assays in
poly(A) RNA preparations suggested differences in the proportion of
intron 1- to intron 2-containing RNA between control and hyperthyroid
rats (Fig. 2B). Relative abundance levels of these
precursors were determined in unfractionated nuclear RNA of several
animals (Fig. 5). Molar abundance ratios of the precursors
containing intron 1 but devoid of intron 2 to precursors containing
intron 2 but devoid of intron 1 were 0.201 ± 0.044 and 0.492
± 0.030 in control and T-treated rats, respectively
(mean ± S.D., duplicate determinations in three pools of two
rats per group, p < 0.001, t test). A similar
difference was observed in a second set of rats exhibiting molar
abundance ratios of 0.187 ± 0.022 and 0.448 ± 0.068 in
control and T
-treated rats, respectively (mean ±
S.D., four rats per group, p < 0.001). In contrast, no
difference between control and experimental rats was found in the
abundance ratio of precursors containing intron 2 but lacking intron 1
to primary transcripts (Table 1).
Figure 5:
Effect of
chronic hyperthyroidism on abundance levels of apoA-I mRNA
intermediates. Nuclear RNA (2 µg) of control (Co) or
T-injected (T3) rats was reverse transcribed with
random hexamers and amplified with primers for exon 1 and exon 3. Only
the two RNA intermediates, identified on the right, are shown.
Intervening sequence-1 (IVS) and -2 refer to intron 1 and 2; A, B, and C refer to exons 1, 2, and 3, respectively.
Each lane corresponds to a separate pool of nuclear RNA obtained from
two rats.
RNase protection assays were
performed to validate the PCR-based quantification of nuclear apoA-I
RNA species (Fig. 6). In T-treated rats, the
abundance level of the primary transcript was reduced to 65% of control
rats. In contrast, levels of intron 1-containing precursors devoid of
intron 2 and the nuclear apoA-I RNA species lacking introns 1 and 2
were increased to 183 and 253%, respectively (Table 1). No
significant difference was observed in the abundance of the intron
2-containing mRNA precursors lacking intron 1. Abundance levels of
nuclear apoA-I RNA species relative to abundance levels of primary
transcripts are also displayed in Table 1for comparison with the
PCR-based quantification. The two methods showed reasonable agreement
in the quantification of the early mRNA precursors. Compared with the
PCR-based method, the RNase protection method revealed lower abundance
levels of the smaller precursors devoid of introns 1 and 2. This
difference may be attributed to the propensity of PCR reactions to
amplify smaller templates to a greater extent(35) .
Nevertheless, increased abundance levels of the smallest nuclear RNA
species in T
-treated rats were a consistent finding with
both techniques. To estimate the abundance of intron 3-containing
precursors devoid of introns 1 and 2, a similar degree of
overestimation of smaller templates was assumed in assays shown in Fig. 4. This assumption appeared justified, since the primers
used for amplification of cDNA were identical to those shown in Fig. 2and Fig. 5. Using the abundance level of primary
transcripts (Table 1) and the corrected signal intensity ratio
obtained in PCR based assays (Fig. 4), abundance levels of the
intron 3-containing precursor devoid of introns 1 and 2 in control and
experimental rats were 20 ± 2.8 and 39 ± 10 pmol/g of
nuclear RNA, respectively. Thus, mean abundance levels of mature
nuclear apoA-I mRNA were 75 and 201 pmol/g of nuclear RNA in control
and T
-injected rats. This result is consistent with the
data from the Northern blots showing a 3-fold higher abundance of mRNA
in nuclei of T
-treated rats.
Figure 6:
Characterization of nuclear apoA-I RNA in
control and T-treated rats by ribonuclease protection
assays. A, structural relationship among apoA-I gene,
antisense [
P]RNA (probe), primary
transcript (1), intron 1-containing precursor (2),
intron 2-containing precursor (3), and precursor devoid of
introns 1 and 2 (4). Boxes refer to RNA fragments
protected, and shaded boxes represent the RNA fragments
discriminatory for each of the nuclear RNA species. B, the
[
P]RNA probe (nucleotides 1-468) was
hybridized with 5 µg of nuclear RNA of two pools (two rats per
pool) of control rats (C1, C2) or T
-treated (T1, T2) rats. R1, R2, R3, and R4 refer to protection assays, in
which the [
P]RNA probe was hybridized with
[
H]RNA transcribed from clones containing cDNA
inserts corresponding to the RNA precursors shown in A. Assay
controls containing probe and yeast RNA subjected to RNase digestion or
not are shown in lanes Ca and Cb, respectively. M refers to end-labeled standards containing the number of
nucleotides indicated. Numbers on the left (1-4) refer to nuclear RNA species identified in A. x and y refer to RNA fragments, which did
not match the size of fragments whose protection was expected from the
structure of apoA-I mRNA precursors.
In addition to RNA
fragments, whose protection was expected from the intron-exon
composition of individual mRNA precursors determined previously (Fig. 2, 4, 5), additional bands, labeled x and y in Fig. 6B, were visualized in sequencing gels.
The abundance of band x was 2.5-fold greater in control than
in T-treated rats (Table 1). The abundance of band y relative to the primary transcript did not differ between
control and experimental animals. Furthermore, band y was also
present in [
H]RNA species serving for assay
standardization (Fig. 6B, lanes R1, R2, and R3). Hence, this band probably represented an artifact of the
assay.
To identify the apoA-I RNA sequence protected in band x, the sense and antisense RNA fragments contained in band x and the primary transcript band, used as control, were
eluted from gel slices, reverse-transcribed, and amplified in the
presence of [P]UTP. With use of exon 1 and
intron 2 primers for reverse transcription and amplification of cDNA,
the intensities of PCR products of band x were 56% of those of
the primary transcript. In contrast, use of intron 2 and exon 3 primers
resulted in negligible quantities of PCR products for band x when compared with those for the primary transcript (not shown).
Since band x contained about 210 nucleotides (Fig. 6B), this RNA species must have extended from the
transcription start site close to the 3` end of intron 1.
To study
the mechanism of the hormone-induced increase in abundance of intron
1-containing precursors, apoA-I RNA intermediates were quantified in
liver nuclei of control and T-treated rats after injection
of actinomycin D (Fig. 7). The disappearance rates of primary
transcript and intron 2-containing precursors did not differ between
the two groups of rats over the time period studied; however, the
apoA-I RNA species containing intron 1 but devoid of intron 2 exhibited
a slower rate of disappearance in the hyperthyroid rats (p = 0.004, analysis of covariance), and its half-life was
increased from 5 min in control to 12 min in experimental rats.
Figure 7:
Comparison of disappearance rates of
primary transcript (), intron-1 containing mRNA precursors
devoid of intron 2 (
), and intron 2-containing mRNA precursors
devoid of intron 1 (&cjs0801;) in control (Panel A) and
T
-treated (Panel B) rats after injection of
actinomycin D into the portal vein. Liver pieces were removed prior to
and for up to 7 min after injection for isolation of nuclear RNA.
Abundance of primary transcript and intron-containing precursors was
quantified by reverse transcription of nuclear RNA and PCR
amplification of cDNA.
Using the data from the actinomycin D experiment (Fig. 7) and
the steady-state levels of nuclear apoA-I transcripts in control and
experimental rats ( Fig. 4and Fig. 6and Table 1),
a compartmental model was developed (Fig. 8). The limited
information content in this system required a manually determined rate
constant for the catabolism of the precursor containing intron 1 but
devoid of intron 2 to its putative degradation product consisting of
exon 1 and a large portion of intron 1. A value was selected that
simultaneously gave good estimates for the mass in all other
compartments. In order to minimize the number of degrees of freedom,
the mass of the degradation product was not modeled here because it
was on a terminal catabolic path. Because the mass of the intron
2-containing precursor devoid of intron 1 was largely unresponsive to
acinomycin D treatment in both control and T groups, we
assumed that it had a relatively slow rate constant, and consequently a
small overall flux of mRNA through it. Since the mass ratio of the
primary transcript to this precursor was not affected by T
treatment (Table 1), the fractional transfer constants
between these compartments were set identical in the T
group and the control group. Using these assumptions, the
percentage deviation of computed and observed pool sizes ranged from 0
to 20% ( Table 1and Fig. 8). This model suggested that the
primary effect of thyroid hormone is to alter the processing of the
intron-1 containing precursor devoid of intron 2 by retarding its
degradation and increasing its conversion to the intron 3-containing
precursor devoid of introns 1 and 2. As a result, mRNA maturation was
increased 7-fold in chronically hyperthyroid rats.
Figure 8: The compartmental model of apolipoprotein A-I mRNA processing in hepatocyte nuclei of control and chronically-hyperthyroid rats. Boxes refer to compartments containing the apoA-I mRNA precursors specified. Numbers within boxes refer to computed RNA concentrations within the respective compartment (pmol/g nuclear RNA). A-I-B-II-C-III-D, primary transcript; A-I-B-C-III-D, mRNA precursor containing introns 1 and 3 devoid of intron 2; A-B-II-C-III-D, mRNA precursor containing introns 2 and 3 devoid of intron 1; A-B-C-III-D, mRNA precursor containing intron 3 devoid of introns 1 and 2; A-I, RNA species consisting of exon 1 and the majority of intron 1; and A-B-C-D, mature mRNA. Compartment fluxes (pmol/g nuclear RNA/min) are above and beside the arrows noting the transfer direction.
In rat liver,
T enhances post-transcriptional editing of apoB mRNA,
leading to increased synthesis of apoB-48 at the expense of
apoB-100(36) . To determine whether such a mechanism
contributed to the enhanced apoA-I mRNA maturation, nuclear mRNA
precursors were sequenced. Since editing in rat liver is not complete
and sequences obtained from PCR products may be ambiguous, we sequenced
numerous clones with cDNA inserts corresponding to the various nuclear
apoA-I RNA species (Fig. 3). Deletions of two and three
nucleotides found in intron 1 and intron 2, respectively, were observed
in all cDNA inserts including those obtained by PCR amplification of
genomic DNA isolated from livers of the same rats. The G-deletions in
intron 2 were verified by genomic sequencing (data not shown) and are
therefore not PCR artifacts. A number of sequence changes were detected
in individual clones, but were not consistent across separate clones.
Unlike the G-deletions, these base substitutions may therefore be
attributed to infidelity of Taq polymerase(37) , and
post-transcriptional editing of apoA-I transcripts is not likely to
account for the difference in apoA-I mRNA maturation between control
and T
-treated rats.
The present studies provide insight into
T-induced stabilization or more effective processing of
apoA-I mRNA precursors that have been postulated on the basis of mRNA
synthesis and abundance data(14) . In livers of euthyroid and
chronically-hyperthyroid rats, the apoA-I mRNA pathway is branched, in
that intron 1 or intron 2 is removed first from the primary transcript,
whereas intron 3 is removed last (Fig. 1, Fig. 2, and Fig. 5). Removal of the smaller introns 1 and 2 may be a
prerequisite for a successful attack on intron 3 by splicing factors.
Alternatively, intron 3 removal may simply be slower than that of
introns 1 and 2.
The most striking difference between control and
experimental rats was the 2.7-fold greater abundance level of mature
nuclear mRNA in T-treated rats. Since cytoplasmic apoA-I
mRNA abundance also increased 3-fold in T
-treated
rats(14) , enhanced formation rather than reduced nuclear
export must have accounted for the nuclear accumulation of mature
apoA-I mRNA. That an increase in mRNA maturation results in increased
abundance of mature nuclear apoA-I mRNA implies a faster rate constant
for intron removal of pre-mRNA than for nuclear export of mRNA. Such a
conclusion is consistent with the observation that mRNA is the
predominant nuclear RNA species of other apolipoproteins we have
studied(14, 30, 38) .
The abundance of
primary transcripts fell to 65% of control, a finding consistent with
reduced apoA-I gene transcription quantified previously by run-on
assays(14) . Since the reduction in primary transcript
abundance was similar in poly(A) selected and total nuclear RNA
preparations, chronic hormone administration had little influence on
polyadenylation of apoA-I primary transcripts. The impact of chronic
hyperthyroidism on apoA-I mRNA processing becomes even more evident
after normalization for primary transcript abundance or mRNA synthesis.
Relative abundance levels of precursors containing intron 1 but not 2,
precursors containing only intron 3, and mature nuclear RNA increased
2.6-, 3.0-, and 4.1-fold, respectively. In contrast, the abundance
level of intron 2-containing precursors relative to primary transcript
was similar in control and T-treated rats. Thus, an
increased abundance level of the precursor containing intron 1 but
lacking intron 2 must have reflected the earliest step in the
processing cascade of apoA-I mRNA that showed sensitivity to T
administration.
Thyroid hormone status may affect splice-site
selection. In hypothyroidism, the replacement of juvenile tau variants
by the adult form is delayed(39) . Our sequencing studies
showed, however, that the splice sites for introns 1 and 2 were not
altered by the hormone (Fig. 3). Additional studies in control
and T-treated rats excluded alternative splicing of intron
3 (data not shown). Other mechanisms which may control the efficiency
of mRNA maturation relate to splicing and stabilization of mRNA
precursor forms. Several examples supporting on/off regulation of gene
expression at the splicing level have been reported(40) , and
intronic sequences that prevent splicing in mammalian cells have been
identified(41) . Differences in the stability of nuclear RNA
may explain the different expression levels between osteoblast-derived
cells and HepG2 cells in the liver/bone/kidney alkaline phosphatase
gene, since transcriptional activity is similar between these cell
lines(42) . Changes in mRNA maturation have also been
implicated in the increased apoA-IV gene expression in livers of obese
Zucker fatty rats(43) . Furthermore, the adenovirus E1B-55K
protein enhances the accumulation of late viral message
post-transcriptionally by stabilizing mRNA prior to its translocation
across the nuclear envelope(44, 45) .
The kinetic
studies after actinomycin D injection suggested that stabilization of
the intron 1-containing precursor was a likely mechanism, whereby
T would increase the maturation of apoA-I mRNA. Compared
with control rats, T
-treated rats exhibited a 2.5-fold
increase in the abundance of an RNA species extending from the
transcription start site into intron 1 (Fig. 6). Such an RNA
species could have resulted from degradation of primary transcripts or
intron 1-containing precursors. The latter possibility is more
plausible, since control and experimental rats were identical in the
abundance ratio of intron 2-containing precursors to primary transcript (Table 1) as well as in the disappearance rate of primary
transcript (Fig. 7). Thus, steady-state levels of and kinetic
data for mRNA precursors and abundance levels of an RNA degradation
product are all consistent with stabilization of intron 1-containing
precursors as a result of thyroid hormone administration (Fig. 8).
Compartmental analysis of the processing pathway supports this conclusion. In addition, our model also suggested that the hormone enhances the splicing of intron 1 from this precursor as the fractional transfer rate constant between the respective compartments was increased 10-fold in comparison to control. This compartmental model embodied two major assumptions; (i) an unidirectional flux of mass from the primary transcript leading either to catabolism via the intron 1-containing precursor devoid of intron 2 to a degradation product consisting of exon 1 and the majority of intron 1, or to formation of mature mRNA via the intron 1-containing precursor and the intron 3-containing precusor devoid of introns 1 and 2; and (ii) production of mature mRNA via both the intron 1-containing precursor devoid of intron 2 and the intron 2-containing precursor devoid of intron 1. Assumption 1 is probably valid, because reinsertion of introns and repair of degradation products is unlikely to occur. The validity of assumption 2 cannot be ascertained by our experimental data. Notwithstanding the constraints of the model due to experimental limitation, the mechanisms implied by the model may be physiologically plausible. The size of the putative degradation product is consistent with cleavage close to the intron 1-exon 2 border. It is thus possible that stabilization of the RNA intermediate influences the rate of splicing. Alternatively, a splicing block may lead to degradation of mRNA precursors by permitting the attack of introns by nucleolytic enzymes. Such a mechanism may regulate the expression of the gene coding for the L1 ribosomal protein in Xenopus laevis(46) .
To definitely validate our assumptions, a true steady-state experiment would be required in which an isotopic precursor is administered and the specific activity of each mRNA transcript is measured as a function of time. Alternatively, transfection experiments using hormone-sensitive cells and apoA-I gene constructs devoid of introns 1 or 2 or both may eliminate the constraints of our model.
The enhanced stability/processing of the
intron 1-containing apoA-I mRNA precursors could have resulted from
post-transcriptional editing. Such a mechanism appeared to be
plausible, since thyroid hormone enhances editing of apoB transcripts
in rat liver(36) . However, neither in control nor
T-treated rats was a consistent nucleotide substitution
found in any of the apoA-I mRNA precursors (Fig. 3). In
addition, the sequences of the exon 3-intron 3 border were identical in
control and T
-treated rats (data not shown). These studies
argue against a role of apoA-I transcript editing in its processing,
but do not exclude hormonal effects on editing of other transcripts
which may perhaps control the processing of several mRNA including that
of apoA-I. Thyroid hormone status also regulates the hepatic expression
of the apoC-III gene which is closely linked to the apoA-I gene (27) at the level of mRNA maturation. In hypothyroid rats,
apoC-III gene transcription is increased in comparison with euthyroid
rats, but RNA processing is reduced. Conversely, reduced apoC-III gene
transcription in chronically hyperthyroid rats is, at least in part,
compensated for by enhanced mRNA maturation(38) . Similar
mechanisms may therefore be involved in the hormonal control of
maturation of apoC-III and apoA-I mRNA.
Because of the inverse relation between nuclear mRNA abundance and transcription rate in euthyroid and chronically-hyperthyroid rats, feedback inhibition of apoA-I gene transcription by more efficient mRNA maturation has been postulated(14) . Indeed, apoA-I gene transcription in rats with chronic hyperthyroidism is suppressed by a transcription elongation block that is competitively relieved by RNA fragments(47) . An inverse association also exists between apoC-III transcription and abundance of nuclear apoC-III RNA, across hypothyroidism, euthyroidism, and chronic hyperthyroidism(38) . Hence, the mechanisms regulating the rate of mRNA maturation of the apoA-I and apoC-III genes may not only be similar, but may provide novel information about regulation of transcription in eukaryotes.