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J Biol Chem, Vol. 275, Issue 15, 11507-11513, April 14, 2000
From the Thyroid hormone (T3)
coordinates growth, differentiation, and metabolism by binding to
nuclear thyroid hormone receptors (TRs). The TR Thyroid hormone receptors
(TRs)1 mediate the diverse
physiological effects of thyroid hormone (T3) (1-3). TRs
are encoded by two closely related genes, erbA Expression of the two TR Interestingly, the alternative post-transcriptional processing of the
TR One possible explanation for these observations is that the
complementary 3' ends of the RevErb and TR In this study, we examine the requirements for alternative processing
of TR Plasmids--
TR
The minigenes with TR
For further analysis of the splicing enhancer, plasmid pCMV-erbAm was
modified by deleting sequences extending from a BstEII site
260 nt to a XbaI site in the vector. A Bsu36I
site within exon 10 was eliminated by end-filling to produce pErbA,
which contains a unique Bsu36I site immediately downstream
of the TR Cell Transfection and RNA Isolation--
COS-1 and 293 cells
were grown in minimal essential medium supplemented with fetal bovine
serum. Cells were replated at a density of 5 × 105
cells/plate 24 h prior to transfection. Transfection of cells and
isolation of RNA were carried out as described previously (20).
Skeletal muscle RNA was prepared as a control from adult Harlan
Sprague-Dawley rats (26).
RNA Assays--
Northern analysis and RNase protection assays
were carried out as described previously (20). The TR Expression of Accurately Processed TR Site-specific Substitutions within the TR Identification of a Splicing Enhancer within the TR
We hypothesized that intronic sequences may regulate TR
The loss of splicing observed with
The TR Mapping of a Highly Conserved Intronic Splicing Enhancer--
As a
first step toward more precisely mapping sequences required for
enhancer activity, a phylogenetic comparison of sequences from this
region of the TR
In contrast to the conservation of coding sequence among all
vertebrates, the non-coding sequences in non-mammalian vertebrates have
essentially no similarity to the mammalian genes: only 29-39% identity in gapped alignments of 80-nt segments (Fig. 4A).
The very high conservation observed among mammalian sequence within the
first 80 nt of the 3'-untranslated region of TR
To precisely map SE RevErb Gene Expression Regulates the Processing of TR
TR
We also considered the possibility that the shift in ratio of
TR We have identified several features of the TR We have identified a sequence-specific TR The location of SE The evolution of the TR Both TR The present study provides a direct demonstration that RevErb
expression alters the balance between TR RevErb expression could increase the TR The selective decrease in TR We thank Lori Vigue, Leslie Snell, Richard
Baker, Catherine Wickland, and Heather Shoven for skillful assistance;
Kent Wilcox and Mark McNally for providing recombinant
plasmids; and Mark McNally for helpful comments on this manuscript.
*
This work was supported in part by National Institutes of
Health Grants DK 48034 and GM 55922 (to S. H. M.) and DK45586
(to M. A. L.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by a predoctoral fellowship from the Arthur J. Schmitt
Foundation. Current address: Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY 11724.
2
M. L. Hastings and S. H. Munroe,
unpublished results.
The abbreviations used are:
TR, thyroid hormone
receptor;
T3, thyroid hormone;
RevErb, RevErbA
Post-transcriptional Regulation of Thyroid Hormone Receptor
Expression by cis-Acting Sequences and a Naturally
Occurring Antisense RNA*
§,,
Department of Biology, Marquette
University, Milwaukee, Wisconsin 53201 and the ¶ Division of
Endocrinology, Diabetes, and Metabolism, Departments of Medicine and
Genetics, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gene encodes
T3-activated TR1 (NR1A1a) as well as an antagonistic,
non-T3-binding alternatively spliced product, TR2 (NR1A1b). Thus, the TR1/TR2 ratio is a critical determinant of
T3 action. However, the mechanisms underlying this
post-transcriptional regulation are unknown. We have identified a
non-consensus, TR2-specific 5' splice site and conserved intronic
sequences as key determinants of TR mRNA processing. In addition
to these cis-acting elements, a novel regulatory feature is
the orphan receptor RevErbA (NR1D1) gene, which is transcribed from
the opposite direction at the same locus and overlaps the TR2 coding
region. RevErbA gene expression correlates with a high TR1/TR2
ratio in a number of tissues. Here we demonstrate that coexpression of
RevErbA and TR regulates the TR1/TR2 ratio in intact cells.
Thus, both cis- and trans-regulatory mechanisms
contribute to cell-specific post-transcriptional regulation of TR gene
expression and T3 action.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and erbA
, in all
vertebrates (4). Additional receptor diversity is generated by
alternative processing of the TR and TR pre-mRNAs. The
alternatively spliced isoforms of TR, TR1 (NR1A1a) (5) and TR2
(NR1A1b), are of particular interest. Although both are widely
expressed, they are functionally antagonistic. Due to its variant C
terminus, TR2, unlike TR1, does not bind T3 and lacks
the major activation function present in other TRs (6, 7). The dominant
negative activity of TR2 appears to reflect both competition for
binding to TR target genes as well as altered protein-protein
interactions (8-11).
isoforms is highly regulated, with each
mRNA expressed in a tissue-specific and developmentally regulated
fashion. In some tissues TR2 represents a relatively minor fraction
of the TR-related isoforms, while in other tissues such as brain,
kidney, and testes, TR2 is the most abundant isoform (12, 13). A
developmental increase in the T3 responsiveness of some
cells correlates with a decrease in the relative expression of TR2
mRNA, suggesting that TR2 expression modulates T3
action (13). Thus, TR2 may act as a tissue-specific antagonist of T3-responsive gene activation.
transcript that gives rise to TR2 mRNA occurs exclusively in mammals (14-17). The mammalian TR gene is also remarkable in that it partially overlaps the gene for another nuclear receptor that
is encoded on the opposite DNA strand. This receptor gene, RevErbA
(RevErb, NR1D1), is convergently transcribed with respect to the TR
gene such that its 3' end overlaps sequences coding for TR2, but not
TR1 (18, 19). The unusual organization of these two genes, which
code for structurally related transcription factors, may have important
implications for regulation. In many cells and tissues, high levels of
RevErb mRNA correlate with low levels of TR2 mRNA relative
to TR1 (18, 20). Furthermore, conditions that alter RevErb
expression levels often result in a reciprocal change in the ratio of
TR2 to TR1 (21, 22).
2 transcripts form duplexes that negatively regulate TR2 mRNA. In this case
specific base pairing interactions between RevErb and TR2 RNA may
provide a transcript-specific mechanism for regulating the level of
TR1 and TR2 mRNA levels. Although antisense RNA provides a
well established mechanism for regulation of gene expression in
prokaryotic systems, it is poorly characterized in eukaryotes (23).
Thus, the molecular mechanisms regulating alternative processing of the
TR mRNAs are of interest from both a physiological and
mechanistic perspective.
1 and TR2 mRNA using a model erbA minigene. Our
results demonstrate that the activity of the TR2-specific 5' splice
site (ss) is dependent on sequence elements located within the
TR2-specific intron. We also find that RevErb expression results in
a decrease in TR2 mRNA relative to TR1, consistent with a
role for RevErb mRNA in blocking TR2 expression. These findings
have important implications for the regulation and evolution of the
thyroid hormone response in mammals.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and RevErb transcripts were expressed from a
9.1-kb MscI/BamHI fragment that spans exons 7-10
of the rat TR gene and exons 2-8 of the overlapping RevErb gene
(18). This fragment was cloned into the HindIII and
NotI sites of the pRcCMV vector (InVitrogen) to create
pCMV-erbAm. A plasmid expressing TR and RevErb was constructed by
reversing the orientation of this insert relative to the CMV promoter
and inserting the SV40 early promoter adjacent to the TR gene. In
this plasmid, pSVerbA/Rev(CMV), the 9.1-kb insert was cloned between
the NotI and ApaI sites of pRcCMV and a 344-bp
PvuII/HindII SV40 fragment replaced downstream
vector sequences extending through another SV40 promoter to a
BclI site. A control plasmid, pSVerbA(
CMV), lacking the
CMV promoter, was made by deleting the NruI/NotI fragment
upstream of the insert in pSVerbA/Rev(SV). The pSVerbA/Rev plasmids,
with and without the CMV promoter, were modified by insertion of the
complete green fluorescent protein (GFP) coding sequence as a 0.75-kb
KpnI/NotI fragment upstream of RevErb to produce
pSVerbA/GFP-Rev(CMV) and pSVerbA/GFP-Rev(CMV). pTKerbA/Rev(RSV) was
constructed by replacing the CMV promoter in pSVerbA/Rev(CMV) with the
RSV promoter and the SV40 promoter with the HSV1 thymidine kinase
(TK) promoter. The RSV and TK promoters in this construct were obtained
as a NdeI/HindIII fragment of pRSV2 (the gift of
M. T. McNally, Medical College of Wisconsin) (24), and a
BglII/HindIII fragment from pXK70R (the gift of
K. W. Wilcox, Medical College of Wisconsin) (25), respectively. A
control plasmid, pTKerbA(RSV), was made by omitting the RSV promoter.
2-specific 5'ss mutations were constructed
using polymerase chain reaction. In pCMV-erbAm(5'ss), the wild-type
sequence (AG/GUGACU) at the 5'ss (/) is replaced with the sequence
CC/CACACA. pCMV-erbAm(5'ss+5G) and pCMV-erbAm (5'ss+6G) contain a
single G residue substitution at positions 5 and 6 of the
TR2-specific 5'ss, respectively. pCMV-erbAm(BS) and
pCMV-erbAm(XS) were made by deleting a 3.5-kb
Bsu36I/SspI and a 3.2-kb
XbaI/SspI from the TR2 intron, respectively.
pCMV-erbAm(BS+344H) was constructed by inserting a 344-bp
SphI/PstI fragment of 
phage into the deletion in pCMV-erbAm(BS).
1 stop codon. Expression and processing of the TR
mRNA from pErbA is comparable to that of pCMV-erbAm. pErbA(BS)
and pErbA(XS) were derived from pErbA and include the same deletions
as pCMV-erbAm(BS) and pCMV-erbAm(XS), respectively. pErbA(SE30)
and pErbA(SE80) were constructed by polymerase chain reaction
amplification of the pErbA minigene using primers for sequences
extending exactly 160 and 210 nt downstream, respectively, of the
TR2 5'ss. Sequences of plasmids and primers are available upon request.
2 poly(A) site
probe was prepared from a cloned PstI/DdeI
fragment yielding an RNA probe that overlaps the final 323 nt of
TR2. The TR1 poly(A) site probe was made from a cloned
StyI/HincII fragment, which overlapped the final
140 nt of TR1. The TR2 cDNA probe was prepared from a cloned
135-bp StuI/FseI fragment of TR2 cDNA
plasmid p2C-Sac/stop (27) that produced a probe that overlapped
80 nt of TR1 and 134 nt of TR2 mRNA. The exon 9A probe was
prepared from a cloned DdeI/EcoO109I fragment
that overlapped 97 nt of intron 8 and 154 nt of exon 9. RevErb poly(A)
site probes were prepared from a cloned 516-bp
StyI/EcoRI fragment. All other probes have been described (20). Detailed information on each probe is available upon
request. RNA assays were quantitated as by direct radioanalytic scanning of 32P-labeled blots or gels, following
appropriate corrections for background and probe length and composition
as described previously (20).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Minigene
Transcripts--
Formation of TR1 and TR2 reflects competition
between two mutually exclusive alternative processing reactions:
polyadenylation at the proximal TR1-specific poly(A) site or
splicing of the TR2-specific 5'ss to exon 10 (Fig.
1A). To facilitate analysis of
the requirements for alternative processing of TR mRNAs, we constructed a minigene expression plasmid that includes the
alternatively spliced exons and the entire region of overlap between
TR2 and RevErb in addition to other TR and RevErb sequences (Fig.
1A). When this minigene was transiently expressed in COS-1
cells, the resulting transcripts were alternatively spliced and
polyadenylated at exactly the same sites used in the endogenous
transcripts. RNase protection assays (Fig. 1B) using probes
complementary to the TR2-specific 5'ss (lane
2), spliced TR2 mRNA (lane 6),
and to both polyadenylation sites (lanes 8-10)
confirmed that the minigene transcripts were accurately processed. The
sizes of the protected fragments are identical to those observed for
endogenous mRNAs isolated from skeletal muscle (Fig. 1B,
lanes 1, 5, and 11).
Similarly, Northern blotting revealed two transcripts (3.9 and 1.4 kb;
Fig. 2B, lane
1) corresponding exactly to the expected sizes of the
alternatively processed minigene transcripts. Only very low levels of
endogenous transcripts were present in these cells, which were seen
upon a 10-fold overexposure of lanes containing RNA from untransfected
COS-1 cells (cf. Fig. 1B (lanes
3 and 7) and Fig. 2B (lane
5)). These results validate the use of the minigene as a
model system for alternative processing of the TR mRNAs.
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Fig. 1.
Structure and expression of the endogenous
TR gene locus and TR
minigenes. A, diagram of the TR/RevErb genes
(top) and the pCMV-erbAm minigene (bottom).
Boxes represent exons; lines, introns;
hatched boxes, untranslated regions;
block arrows, direction of transcription and
poly(A) sites (pA); horizontal arrow,
hCMV promoter; shaded box, TR1-specific
sequence; stippled box, TR2-specific sequence;
angled line, TR2 splicing. Dotted
lines indicate the RevErb and TR2 overlap. Probes used in
RNase protection assays are labeled and presented as bars
beneath protected regions. Sizes of protected fragments are noted for
each probe. B, RNase protection assays of endogenous and
minigene TR RNA. Assays were carried out with total RNA from COS
cells transfected with pCMV-erbAm (T, lanes
2, 6, and 8-10), rat skeletal muscle
(S, lanes 1, 5, and
11), untransfected cells (C, lanes
3 and 7) or with tRNA alone (t,
lanes 4 and 12). Marker lanes
(M) are MspI-cut pBR322. Probes used to assay
erbA RNA processing were the TR2-specific 5'ss (lanes
1-4), a TR2 cDNA probe spanning the TR2-specific
5'ss/3'ss junction (lanes 5-7) or the TR1 and
TR2 poly(A) probes separate or in a mixture (lanes
8, 9, and 10-12, respectively).
Protected fragments corresponding to TR1 mRNA (1)
and TR2 mRNA (2) and RNAs reading through the
poly(A) sites (open arrows) are indicated.
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Fig. 2.
TR 2 5' splice site
sequence is critical for balance between TR1
and TR2 mRNA processing.
A, metazoan consensus 5'ss (28), the TR1 coding sequence
for mammals and non-mammals, and three separate 5'ss mutations made in
pCMV-erbAm (+5G, +6G, ss) are
shown. Mutations are in lowercase, and non-consensus
nucleotides are underlined. TR consensus sequences
correspond to the following data base files (GenBankTM
accession nos.): human (X55068), rat (M18028), mouse (X51983), sheep
(Z68308), pig (AJ005797), frog (Xenopus laevis) (M35343,
M35344), chicken (Y00987), penguin (AJ002363), flounder (D16461,
D16462), and zebrafish (U54796). B, Northern blot analysis
of 2 µg of poly(A)+ RNA from COS cells transfected with
indicated plasmids. RNA from untransfected cells was included as a
control (C, lane 5). Blots were
hybridized with a probe from the region common to TR1 and
TR2.
2 5'ss Dramatically
Affect Alternative Processing--
The sequence of the TR2 5'ss
differs from the metazoan 5'ss consensus at the highly conserved +5
position (Fig. 2A) (28). This sequence is also conserved in
non-mammalian TR genes, except for the presence of a purine at the
position corresponding to +6 of the TR2 5'ss (Fig. 2A).
This difference may account for the lack of splicing in the
non-mammalian TR gene. To test this hypothesis, we constructed three
5'ss mutations in the context of the rat TR minigene (Fig.
2A). The first mutation (+5G) changes the +5C residue to G,
thereby creating a 5'ss that matches the consensus. A second mutation
(+6G) converts position +6 of the 5'ss from a U to a G as found in
non-mammalian TR1 genes. The third mutation (ss) eliminates this
5'ss by changing six residues of the consensus sequence. Northern blot
analysis of poly(A) mRNA from the different constructs demonstrates
that the wild-type TR transcripts were alternatively processed with
a TR1/TR2 ratio of 3.3 (Fig. 2B, lane
1). Strikingly, the 5'ss consensus mutation (+5G) results in
a complete switch to TR2 splicing, with no detectable use of the
TR1 polyadenylation site (Fig. 2B, lane
2). The apparent increased expression of TR2 (and some endogenously expressed RNAs) in lane 2 resulted
from sample loading variation and is not reproducible. However, this
overloading serves to demonstrate the complete absence of the TR1
minigene transcript in the +5G mutation. The single-nucleotide
difference between the mammalian and non-mammalian sequences is also
important for splice site activity, as was apparent from the lack of
splicing of the +6G mutant, which increased the ratio of TR1/TR2
mRNA about 7-fold (Fig. 2B, lane
3). Remarkably, this single base change reduced splicing
nearly to the level of the ss mutant, which has a completely
crippled splice site and expresses only TR1 (Fig. 2B,
lane 4). The switch to TR1 processing and
absence of cryptic splicing with the +6G and ss mutations, and the
switch to TR2 expression with the +5G mutation, demonstrate that the suboptimal TR2 5'ss allows both TR1 and TR2 to be derived from the primary transcript. Taken together, these results demonstrate that
the strength of the TR2 5'ss is critical for determining the balance
between TR1 and TR2 mRNA processing.
2
Intron--
The suboptimal 5'ss in TR2 mRNA presumably allows
other factors to influence the balance between TR2 alternative
splicing and TR1 polyadenylation. We considered that the activity of
the TR1 poly(A) site may be important in determining TR
alternative processing. However, deletion of the TR1 poly(A) site
produced only a modest enhancement of TR2 mRNA splicing (data
not shown). In addition, decreasing the length of the TR2-specific
intron by removing more than 60% of the central portion of this intron had little effect on TR alternative processing (data not shown).
2 mRNA
processing. To test this, a deletion beginning 130 nt downstream of the
TR2 5'ss and ending 60 nt upstream of the TR2 3'ss was constructed. The resulting minigene has a drastically shortened TR2
intron of about 200 nt (BS, Fig.
3A). By shortening the intron
and removing the competing TR1 polyadenylation site, this mutation
was expected to enhance TR2 splicing efficiency. Surprisingly, the
deletion caused a striking decrease in splicing (Fig. 3B, cf. lanes 2 and 4).
Northern blot analysis and RNase protection assays confirm that the
major product of this deletion is a transcript that is unspliced at the
TR2-specific 5'ss and extending to the TR2 poly(A) site (results
not shown).
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Fig. 3.
Sequences within the
TR 2 intron enhance splicing.
A, diagram of minigenes with intron deletions.
Dotted lines indicate deletions starting at
XbaI (X), Bsu36I (B), and
SspI (S) sites. Numbers show position
of these sites relative to the 2 5'ss. Black
box represents Bsu36I/XbaI fragment.
Hatched box is heterologous sequence. Other
symbols are as detailed in Fig. 1. B, RNase protection
assays of total RNA from transfected COS cells using the 5'ss probe.
Fragments corresponding to mRNAs unspliced and spliced at the
TR2-specific 5'ss are labeled u and s,
respectively, since deleted minigenes lack the TR1 poly(A) site.
C, quantitation of RNase protection assays is shown as the
percentage of the total TR RNA that is spliced. D, RNase
protection assays with the 2 5'ss probe of RNA from COS cells
transfected with minigenes containing the indicated deletions
(lanes 1-4) and the 5'ss consensus mutation +5G
(Fig. 2) alone (lanes 5 and 6) or in
combination with BS (lanes 7 and
8).
BS indicates either that specific
sequences within the deleted intron are required for TR2 splicing or
that the intron is shorter than a nonspecific minimal length. To test
these possibilities, 344 nt of heterologous sequence was inserted into
the BS deletion (BS+344H, Fig. 3A). This nonspecific
lengthening of the intron did not improve the efficiency of splicing
(Fig. 3B, lanes 7 and 8).
In contrast, adding back 250 nt of the TR2 intron to the 5' end of
the BS deletion in the XS deletion (Fig. 3B,
lanes 5 and 6) restored splicing
efficiency to wild-type levels as shown quantitatively in Fig.
3C. These results provide evidence for a splicing enhancer sequence within a 250-nt region located 130-380 nt downstream of the
TR2 5'ss.
2 splicing enhancer sequence was required for splicing at the
inherently weak TR2-specific 5'ss. However, the enhancer was not
required when the TR2 5'ss was mutated to the consensus (+5G) 5'ss
sequence (Fig. 3D, cf. lanes
4 and 8). This suggests that the function of the
TR2 intronic splicing enhancer is to regulate the suboptimal
splicing characteristic of the wild-type TR2 5'ss.
2 gene in mammals and non-mammalian vertebrates was
carried out. The region analyzed includes coding and non-coding
sequences from the first 500 nt of exon 9. The coding regions common to
TR1 and TR2 and specific to TR1 are very highly conserved
among mammals with 96-98% identity (Fig. 4A). These sequences also are
highly conserved among other vertebrates, with 80-88% identity
between rat and frog. The conservation of these coding sequences
reflects the highly conserved structure of the C-terminal portion of
TR1. It is noteworthy that a short, 80-nt segment (dark
shading, Fig. 4A) immediately downstream of the
TR1 stop codon, and within the 250-nt
Bsu36I/XbaI splicing enhancer fragment, is also
conserved among mammals at a level (95%) comparable to that of the
coding sequences immediately upstream. This high level of similarity
drops off sharply (70-72% identity) further downstream of the stop
codon, as seen in a comparison of three additional segments of
identical length. Thus, the first 80 nt of the TR1 3'-untranslated
region are conserved at a level similar to the region coding for the
highly conserved C-terminal portion of TR1.
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Fig. 4.
Mapping of the intronic splicing enhancer for
TR 2 mRNA. A,
representation of sequences within the 5' end of exon 9. The TR2
5'ss and TR1 stop codon, UAA, are noted. The region of exon 9 common
to TR1 and TR2 along with downstream sequences specific to the
TR1 exon and TR2 intron were compared in human, rat, sheep, and
frog. Sequences were compared using GCG programs PILEUP and GAP within
segments of indicated sizes. A highly conserved 80-nt region is
darkly shaded, TR1-specific exonic sequences are
small boxes, and untranslated regions are
hatched. The percentage identity of each segment is shown in
bold beneath the region compared. GenBankTM
accession nos. are as follows: human, X55068; rat, M18028; sheep,
Z68308; frog (X. laevis) X17385. B, schematic
representation of TR minigenes derived from pErbA. Sequences downstream of the
Bsu36I site that were added back to the BS intron
deletion are indicated by +80 and +30. Positions
of sequences relative to the 2-specific 5'ss are noted.
C, RNase protection assay (top) using the 5'ss
probe shown in Fig. 1. Quantitation of splicing as determined by RNase
protection assays is shown graphically (bottom) and
represents the average of four to eight experiments with the exception
of BS+30, which represents a single experiment. D, RNase
protection assay using the exon 9A probe, which extends into intron 8. The top band (unmarked) represents undigested probe.
I8/I9 indicates unspliced transcripts containing both intron
8 and intron 9. Other protected fragments are labeled as in Fig.
3B.
1 mRNA strongly suggests that this region has a specific function, such as splicing, that is conserved in mammals but not in non-mammalian vertebrates. To
test the role of the conserved 80-nt region in splicing, this sequence
was inserted into the BS deletion and assayed for its ability to
promote splicing of TR2 (Fig. 4B). Splicing of this transcript, SE80, was compared with that of identical constructs that
contain the BS and XS sequences previously described. The addition of 80 nt to the 5' end of BS (positions 130-210 in the TR2-specific intron) results in splicing levels slightly higher than
that seen in XS or the wild-type TR minigenes (Fig.
4B, lanes 5 and 6). This
result indicates that SE80 includes all of the sequences specific to
XS that are needed to enhance TR2 splicing. We refer to this
80-nt sequence as the TR2 splicing enhancer, SE2.
2, a 30-nt segment at the 5' end of the conserved
80 nt region was also tested for enhancer activity (Fig. 4B). This transcript, SE30, displays nearly the same reduced
level of splicing seen in BS (Fig. 4C, lanes
7 and 8). These results demonstrate that 50 nt
located between positions 160 and 210 of the TR2-specific intron are
required for efficient splicing. To determine if SE2 affects
splicing of intron 8, as well as the TR2-specific intron 9, splicing
was assayed with a probe, spanning both the 3'ss and 5'ss of exon 9A.
The results show that intron 8 was spliced out of approximately 95% of
the transcripts from BS, XS, and SE80, a level similar to that
observed for the wild-type transcript (Fig. 4D). Thus, the
presence or absence of SE2 downstream of exon 9A has no effect on
the activity of the upstream 3'ss, while dramatically altering the
activity of the 5'ss for exon 9A.
Pre-mRNA--
The structure of the TR locus is remarkable in
that the final exon of TR2 overlaps the 3' end of RevErb transcripts
encoded on the opposite DNA strand (Fig. 1A). We have shown
previously that antisense transcripts derived from RevErb sequences
regulate TR splicing in vitro (29). However, such
regulation has not been shown in vivo. To investigate the
effect that the naturally occurring RevErb antisense RNA has on
alternative processing of TR mRNAs, we expressed RevErb from the
opposite strand of the same insert used to express TR mRNAs.
RevErb minigene mRNA, beginning with exon 2, was expressed at high
levels (Fig. 5, A and
B, lane 4). This mRNA is correctly
spliced and polyadenylated, as indicated by Northern blotting and RNase
protection assays carried out at selected splice junctions (Fig. 5,
B and C, and data not shown). Importantly, the 3'
end of the transcript mapped to a site that precisely matched that of
endogenous RevErb mRNA from skeletal muscle (data not shown). Thus,
the 3' end of the rat RevErb minigene transcript, like the endogenous
RNA, overlaps only TR2 and not TR1 (18, 30).
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Fig. 5.
RevErb expression results in lower level of
TR 2 mRNA relative to
TR1. A, diagram of
TR/RevErb minigene expression system. Filled
arrows indicate convergent promoters, and black
bars show probes for Northern blots. B, Northern
analysis (left) of cytoplasmic poly(A)+ mRNA
from 293 cells transfected with pSVerbA/Rev(CMV) (+R,
lanes 1 and 4) or pSVerbA(CMV)
(
R, lanes 2 and 5), or
untransfected cells (C, lanes 3 and
6) using probes specific for TR (lanes
1-3) or RevErb mRNA (lanes 4-6).
Quantitation of results (right) from two separate
experiments is expressed as the ratio of 1/2 in the presence
(+R) and absence (R) of RevErb. C,
RNase protection assays of total RNA from 293 cells transfected with
pSVerbA/Rev(CMV) (e/R, +R, lanes
1 and 2), pSVerbA(CMV) (e/R,
R, lanes 3 and 4),
pSVerbA/GFP-Rev(CMV) (e/R(G), +R,
lanes 5 and 6),
pSVerbA/GFP-Rev(CMV) (e/R(G), R,
lanes 7 and 8) or untransfected cells
(C, lane 9). TR and RevErb mRNA
was detected with the 2 cDNA probe (upper) and RevErb
probe (lower), respectively. Graphical representation of
data at right represents results of assays of two experiments.
mRNA levels in 293 cells transfected with the minigene
expressing both the TR and RevErb genes (+R) were
compared with those in cells expressing only TR (R,
Fig. 5A). A probe hybridizing to the common regions of
TR1 and TR2 mRNA was used for Northern analysis of
poly(A)+ RNA (Fig. 5, A and B) to
allow the TR1/TR2 ratio to be determined accurately. A comparison
of TR expression with and without RevErb coexpression
showed a striking 2.1-fold increase in the ratio of TR1/TR2 in
the presence of RevErb relative to mRNA levels in its absence (Fig.
5B). These results were confirmed by RNase protection assays
of total RNA, using the TR2 cDNA probe shown in Fig. 1 (Fig.
5C). Using this probe, coexpression of RevErb resulted in
2.8 ± 0.4-fold increase in the TR1/TR2 ratio (Fig. 5C). The ratio of RevErb/TR2 in these experiments was
34 ± 5 (Fig. 5C), which is similar to ratios observed
in cells expressing high levels of endogenous RevErb mRNA (20). The
results were not due to promoter-related artifacts because similar
results were obtained with a minigene employing the RSV early promoter to express RevErb and the herpes simplex virus TK promoter to drive TR expression (data not shown).
1/TR2 observed upon expression of RevErb reflects direct or
indirect effects of expressing the RevErb receptor protein, rather than
the overlapping RevErb transcript. Although RevErb translation
normally initiates at a start codon in exon 1, which is missing in the
minigene, it is possible that the processed RevErb transcript is
translated from an in-frame start codon in exon 2 (18). To eliminate
possible translation of the RevErb reading frame, cells were
transfected with plasmids that have the complete coding sequence for
GFP terminated by a stop codon placed upstream of the RevErb minigene.
This construct, pSVerbA/GFP-Rev(CMV), produced a GFP-RevErb mRNA
transcript that efficiently expressed GFP (results not shown).
Expression of this plasmid also showed a very similar shift in
TR1/TR2 ratio as compared with the same construct lacking the CMV
promoter (Fig. 5C). Since translation of any portion of the
RevErb coding sequence downstream of the stop codon following GFP is
unlikely (31), transcription but not translation of the RevErb
sequences is required for an increase in the TR1/TR2 ratio. Taken
together, these results demonstrate that overexpression of RevErb
antisense RNA in vivo is sufficient to alter expression of
the alternatively processed TR1 and TR2 mRNAs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gene that
contribute to the tissue-specific and developmentally regulated
alternative mRNA processing required for expression of
functionally antagonistic TR proteins in mammals. These features,
including a suboptimal 5'ss, a cis-acting splicing enhancer,
and a naturally occurring antisense transcript, are absent in
non-mammalian vertebrates, which do not express TR2. The suboptimal
nature of the splice site allows for the regulation of TR1 and
TR2 mRNA expression by both cis and trans
mechanisms. Results of our analysis provide insight into the evolution
of this complex locus as well as requirements for its regulation.
2 intronic splicing
enhancer, SE2, located in a region 130-210 nt from the TR2 5'ss,
and immediately downstream of the TR1 stop codon (Fig. 4). A
sequence of just 80 nt, but not 30 nt, within this region stimulates
TR2 splicing 4-fold. SE2 is also required for efficient splicing
of pre-mRNA in vitro
(29).2 SE2 is unusual in
that it represents an intronic splicing enhancer that influences
differential splicing and polyadenylation. Sequence analysis of SE2
reveals that the element is very purine-rich (73% A+G). In this
respect it resembles purine-rich exonic splicing enhancers (ESEs)
(32-34). ESEs generally function by binding members of the
arginine-serine-rich family of splicing factors (SR proteins) and
activating a weak upstream 3'ss (34). SE2 differs from most other
purine-rich splicing enhancers in that it is located directly
downstream of an alternative 5'ss rather than a 3'ss. In two other
instances where purine-rich splicing regulatory elements have been
identified within introns, the sequences inhibit rather than enhance
splicing (24, 35). These elements appear to act as a decoy by directing
the partial assembly of competing but non-functional spliceosomes.
However, examples of purine-rich intronic splicing enhancers have also
been identified, suggesting that purine-rich sequences within an intron
can regulate splicing in a positive as well as negative manner (36,
37).
2 between two alternative processing sites also
resembles in some respects a purine-rich splicing enhancer located
between two alternative 5'ss of exon 5 in the caldesmon gene (38, 39).
However, the caldesmon splicing enhancer appears to function as an ESE,
whereas several results show that SE2 functions as an intronic
element. Unlike ESEs, SE2 does not affect the activity of the
upstream 3'ss of intron 8 (Fig. 4D). SE2 is also active
in vitro in transcripts lacking an upstream 3'ss (results
not shown). Finally, SE2 functions independently of a competing
TR1 polyadenylation signal, thus indicating that its location within
an exon is not a requirement for activity. These results suggest that
SE2 represents an unusual type of purine-rich enhancer, which
promotes splicing from an intronic location. Further analysis of such
elements is needed to determine whether they promote splicing by a
mechanism distinct from that of exonic elements.
2 5'ss within a highly conserved region of
the TR gene provides a unique opportunity to assess the process by
which a novel regulatory function evolves by establishment of an
alternative processing pathway. The co-evolution of the overlapping
TR and RevErb genes defines a unique series of events. Whereas a
RevErb homolog has been identified in insects (40), the expression of
TR1 is restricted to vertebrates, and TR2 to mammals. This
evolutionary evidence suggests that RevErb arose first, followed by
TR (41). Since the C-terminal end of RevErb is homologous to that of
many other nuclear receptor proteins, whereas that of the overlapping
TR2 is unique, it seems clear that the evolution of TR2
alternative splicing was the final step in the evolution of this locus
(41). Our analysis extends the insight into this evolutionary process.
Once the RevErb and the TR genes were juxtaposed, formation of an
alternative 5'ss required only a single base change (+6T) within TR.
Although this change is sufficient for a low level of splicing
(Fig. 2), the activity of this non-consensus site may have been
augmented through the gradual evolution of SE2. The intronic region
of TR2 may provide the only available site for positioning a
splicing enhancer (Fig. 4A), as the flanking coding regions
in exons 9A and 10 are very highly conserved (98-99% identity in
humans and rats).
and RevErb mRNAs encode nuclear receptor proteins of the
NR1 type (5). The similar structure of these closely linked genes
raises the possibility that the genetic organization reflects
requirements for coordinate regulation such as that observed for many
other loci where tight linkage between similar, but functionally distinct genes is highly conserved. Interestingly, both RevErb and
TR2 are inhibitors of gene transcription, RevErb as an active repressor and TR2 primarily as an inhibitor of thyroid
hormone-stimulated transcription (42-44).
1 and TR2 mRNA
processing in vivo. The 2-3-fold effect of RevErb
expression on TR mRNA levels with the minigene system (Fig. 5)
agrees with what we have observed upon increasing endogenous RevErb
expression by pharmacological means (22, 30). The increase in the ratio
of TR1/TR2 mRNAs with increasing levels of RevErb relative to
TR2 mRNA is also similar to that observed in a series of B
lymphocyte-derived cell lines (20) as well as other tissues (18).
However, in the minigene system, the change in TR1 and TR2
mRNA with increasing RevErb seems restricted to a range that is
somewhat narrower than that observed in vivo. The
identification of cis-acting elements that influence TR1
and TR2 processing may indicate that balanced expression is also
determined by regulatory proteins binding to SE2. Regulation of
TR1 and TR2 at multiple levels likely explains why the
relationship between RevErb and TR mRNAs does not hold in all
instances (45).
1/TR2 ratio by a number of
mechanisms, including selective interference with exon 10 transcription, inhibition of TR2 processing, or destabilization of
TR2 mRNA. However, the kinetics of TR transcription and
mRNA turnover upon induction of RevErb expression are consistent
with a block in TR2 processing as opposed to a change in TR2
turnover or transcription (21, 22). Furthermore, RevErb antisense RNAs complementary to exon 10 efficiently and specifically block 2 splicing in vitro (29). The simplest explanation for these
results and those described here is that RevErb interferes selectively with the splicing process that generates TR2.
2 mRNA levels in response to RevErb
expression has implications for regulation of T3 action. Because TR2 inhibits transcriptional activation by TR1 in a dose-dependent manner (15, 16), any change in the ratio of these regulatory proteins is expected to alter T3 action.
The physiological significance of a 2-3-fold change in the expression of these receptors is supported by studies demonstrating that tissues
with a higher TR1/TR2 mRNA ratio are more responsive to
T3 than tissues with a 2-fold lower ratio (46). Such an
effect would be especially important in tissues such as the heart that depend specifically on TR for the T3 response (47).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biology,
Wehr Life Sciences Bldg., Marquette University, P. O. Box 1881, Milwaukee, WI 53201. Tel.: 414-288-1485; Fax: 414-288-7357; E-mail: stephen.munroe@marquette.edu.
ABBREVIATIONS
;
ss, splice site;
nt, nucleotide(s);
ESE, exonic splicing enhancer;
TK, thymidine kinase;
GFP, green fluorescent protein;
bp, base pair(s);
kb, kilobase pair(s);
CMV, cytomegalovirus;
RSV, Rous sarcoma virus.
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