Alternative splicing generates multiple SMRT transcripts encoding cons
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《核酸研究医学期刊》
Institute of Biomedical and Biomolecular Science, School of Biological Science, University of Portsmouth, King Henry I St, Portsmouth PO1 2DY, UK
* To whom correspondence should be addressed. Tel: +44 23 92 84 2062; Fax: +44 23 92 84 2053; Email: colin.sharpe@port.ac.uk
ABSTRACT
Silencing mediator for retinoid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor protein (NCoR) are corepressors that interact with a range of transcription factors. They both consist of N-terminal repressor domains that associate with histone deacetylases and C-terminal interaction domains (IDs) that contain CoRNR box motifs. These motifs mediate the interaction between corepressors and nuclear receptors (NRs), such as the retinoid and thyroid hormone receptors. However, whilst NCoR produces a single transcript during Xenopus development, xSMRT is subject to alternative splicing at four sites in the 3' part of the transcript, the region encoding the C-terminal IDs. Although this provides the potential to produce 16 different transcripts, only five isoforms are found in early embryos. The sites of alternative splicing predict that the resultant isoforms will differ in their ability to interact with NRs, as one site varies the number of CoRNR boxes, the second site changes the sequence flanking CoRNR box-1 and the other sites delete amino acid residues between CoRNR boxes 1 and 2 and so alter the critical spacing between these motifs. SMRT and NCoR therefore represent paralogues in which one form, SMRT, has evolved the ability to generate multiple isoforms whereas the other, NCoR, is invariant in Xenopus development.
INTRODUCTION
Silencing mediator for retinoid and thyroid hormone receptor (SMRT) and nuclear corepressor protein (NCoR) are large proteins of >2300 amino acid residues sharing 37% sequence identity and a conserved gene structure. They act as transcriptional corepressors and this is reflected in their protein architecture (Figure 1), which consists of N-terminal repressor domains (RD1–3) and C-terminal interaction domains (ID-N and ID-C) (2). The RDs recruit histone deacetylases (HDACs) to the transcription complex (3,4), and two adjacent SANT domains target the HDAC activity to the extended tails of nearby histones and this renders the chromatin transcriptionally inactive (5,6). The C-terminal region interacts with a range of transcription factors including Su(H), SRF, NFkB and AP1 (2). However, probably the best-characterized interactions are with the nuclear receptors (NRs) retinoic acid receptor (RAR) and thyroid hormone receptor (THR).
Figure 1. The structure of the Xenopus SMRT cDNA. SMRT is a large corepressor of >2300 amino acids, which in the human genome is encoded by a gene consisting of 49 exons. The protein can be divided into an N-terminal region, which encodes three repressor domains (RD1–3) and two SANT domains between RD1 and RD2. Interaction domains ID-N and ID-C in the C-terminal part of the protein each contain a CoRNR box motif and mediate interaction with nuclear receptors. A third CoRNR box (diagonal bars) has been identified in SMRT located in ID-N at a similar position to CoRNR box-3 of NCoR (see text). The variable exons have been highlighted.
Nuclear receptors act as ligand-dependent regulators of transcription and this depends on their ability to interact with coregulator proteins (1). In the presence of the ligand, they recruit coactivators, such as SRC-1, SRC-3 and CBP/p300 (7). These in turn assemble a multi-component complex that stimulates transcription both through direct interaction with the core transcription machinery (via the DRIP–TRAP and Mediator complexes) and through the acetylation of histone tails that make the adjacent chromatin transcriptionally competent (7). In the absence of the ligand, corepressors such as SMRT and NCoR, are recruited to the DNA-bound NRs to nucleate the assembly of the repressor complex (7). The NRs associate with IDs in SMRT that contain the CoRNR box motif (L/V.x.x.I/V.I) (8–10). Peptide sequences adjacent to the motif contribute to the specificity of interaction between the corepressor and the various NRs suggesting that each box preferentially interacts with a specific NR (9,11). Until recently, a major difference between NCoR and SMRT was the presence of three CoRNR box motifs in NCoR (12) compared to just two in SMRT. However, here we show that the SMRT gene has the capacity to encode a third CoRNR box motif in an additional sequence that arises from the read-through of the exon 37 splice donor to a site within the ensuing intron.
The importance of active repression by corepressors in NR signalling has been demonstrated in Xenopus by the use of dominant negative forms of SMRT, which disrupt normal head development and can be rescued by a dominant negative retinoid receptor or retinoid antagonist (13). However, SMRT and NCoR do not have entirely redundant functions, as knockout mice lacking the NCoR gene are embryonic lethal and show a number of specific defects including impaired neural development (2). The ability of SMRT to interact with NRs is also an important factor in a number of diseases. The PML–RAR gene fusion that contributes to acute promyelocytic leukaemia produces a protein with an increased affinity for SMRT, which accounts for many of the observed molecular defects (14). Resistance to the thyroid hormone can also be due to mutations in THR that increase its affinity to SMRT and which then require elevated levels of thyroid hormone to activate target gene expression (15).
The alternative splicing of primary transcripts generates different but related proteins from the same gene and may account for much of the additional diversity in the vertebrate proteome that compensates for the relatively low number of genes. It has been estimated that up to 60% of human genes may undergo some form of alternative splicing (16,17) and in some cases the isoforms produced have opposing functions (18–20), whilst in others alternative splicing generates vast numbers of isoforms that provide exceptional levels of protein diversity (21,22). However, it is also likely that closely related isoforms fine-tune the efficiency of cellular processes by producing related proteins with subtly different abilities.
As SMRT and NCoR have a similar gene organization and protein architecture, we decided to examine the extent of alternative splicing in each gene. In SMRT, we identified one difference in the 5' part of the transcript that encodes the RDs, but this is not due to alternative splicing. However, there is one site of alternative splicing in this region of the mouse gene that is not found in the frog gene. In contrast, we found extensive alternative splicing in the SMRT C-terminal domain in both frog and mouse. However, we did not detect alternative splicing in the same region of NCoR. Consequently, the introduced variability from alternative splicing is confined to just one paralogue. If alternative splicing in Xenopus SMRT was not regulated, the four sites would produce 16 distinct isoforms. However, examination of the SMRT transcript profile identifies just five significant alternatively spliced isoforms in the early embryo.
MATERIALS AND METHODS
Animals
Xenopus embryos were grown in 0.1x MBS (23) and collected at specific stages (24). Embryos were manually dissected in 1x MBS.
Cloning of SMRT
A Xenopus neurula stage cDNA library in lambda phage (25) was screened with an xNCoR cDNA clone derived from IMAGE clone 347360 that encodes the N-terminal RDs. One clone was isolated and subsequently identified as a SMRT cDNA clone. The full sequence of xSMRT was derived from 5' and 3' RACE reactions (First Choice RLM, Ambion).
Analysis of alternative splicing by RT–PCR
Total nucleic acid was extracted from embryos and the DNA was removed by RNAse-free DNase. RNA was converted to cDNA using Superscript II (Invitrogen) and stored at –20°C. cDNA from the equivalent of 0.1 embryos was used for each semiquantitative PCR using specific primers and the products run on 2% agarose gels. The standard programme involved 32 cycles, which was within the logarithmic phase of amplification for SMRT, and an annealing temperature of 53°C. For RT–PCR transcript profile analysis, cDNA from 0.1 embryos was amplified with primers adjacent to the alternatively spliced sites and 5 μCi of dATP in a 25 μl reaction and the products resolved on a denaturing 6% polyacrylamide gel. The amplified DNA was visualized on a Fujifilm FLA 5000 phosphorimager.
Cloning and characterization of the transcript profile products
An unlabelled RT–PCR using RNA from the equivalent of 0.1 embryos was run on an agarose gel and the bands from 900–1400 bp eluted and ligated directly into a TA vector system (Invitrogen). Recombinants were isolated and characterized by automated sequencing (MWG Corp.). Clones corresponding to the radiolabelled bands on the transcript profile were identified by PCR using the same primers and comparison by electrophoresis on polyacrylamide gels. Each clone corresponded to an element of the profile pattern and the abundance of each clone corresponded to the approximate intensity of its band in the RT–PCR transcript profile. Although this approach is semiquantitative, it provides an indication of the relative abundance of the different transcripts. This is supported by the observation that the same pattern with the same relative intensities was seen in both Xenopus tropicalis and Xenopus laevis and that the pattern and intensities vary consistently during development (C. Sharpe and S. Short, unpublished data).
RESULTS
A neurula stage cDNA library was screened with a probe for the corepressor gene, xNCoR. Of the clones identified, one encoded the closely related protein, xSMRT. The full sequence of xSMRT was determined from this cDNA clone and from clones encoding the 5' and 3' end of the transcript obtained by RACE reactions. These sequences show that xSMRT has extensive similarity to human and mouse SMRT with >70% conservation of amino acid sequence in the N-terminal RD1. In contrast, xSMRT differs significantly from xNCoR (with which it shares 37% similarity) (26) indicating that the frog does encode both corepressor genes.
As the SMRT corepressor is thought to interact with many different transcription factors (2) and has been shown to contain at least one site of alternative splicing (27), we looked for evidence of additional isoforms across the transcript using three approaches. First, we compared the xSMRT sequence to that found in other species, aligned according to the annotated exon boundaries of the human gene. Second, we looked for differences amongst the 5' and 3' RACE clones that again mapped to exon boundaries and finally we evaluated candidate sites by RT–PCR using primers from adjacent exons. This strategy identified two sites in the N-terminal repressor region and four around the C-terminal IDs.
Variation in the N-terminal domains of SMRT
Compared to human and mouse, the Xenopus SMRT cDNA clone lacks exon 19 that encodes a short, 17 amino acid peptide. However, we found no evidence for the alternative splicing of this exon (data not shown). Similarly, comparison of human, mouse and Xenopus cDNA sequences identified the lack of exon 25 in the published mouse cDNA sequence (28) (Figure 2A). However, hypothetical translation of the intron sequence between the adjacent exons in the mouse genome identifies a sequence with extensive similarity to exon 25, bounded by splice acceptor and donor sequences, which has so far escaped annotation (Figure 2B). The alternative splicing of exon 25 in the mouse SMRT transcript has been confirmed by RT–PCR and additionally shows that the exon 25(+) isoform is predominant in all tissues examined (Figure 2C). However, RT–PCR analysis using Xenopus mRNA fails to detect exon 25(–) transcripts during early development (Figure 2D). This exon encodes part of RD3, though the specific contribution of this peptide to HDAC recruitment has not been thoroughly evaluated (29).
Figure 2. Exon 25 is alternatively spliced in mouse but not in Xenopus. Exon 25 is not currently annotated in the mouse genome, but can be identified and is subject to alternative splicing. (A) Alignment of exon 25 showing extensive conservation of the predicted mouse sequence with the identified sequences in Xenopus and humans. (B) Mouse genome sequence from exons 24 to 26. The intron was translated and sequence corresponding to exon 25 identified and shown to be flanked by splice acceptor (AG) and donor (GT) sequences (in upper case) (C) RT–PCR analysis of RNA from dissected mouse tissues (B = brain, M = muscle, T = testes, K = kidney, H = heart, L = liver) identifies two bands corresponding to SMRT exon 25(+) and 25(–) transcripts. (D) Just one band, corresponding to xSMRT exon 25(+) transcripts, is detected during Xenopus development (numbers represent stages of development).
Variations in the C-terminal domains of xSMRT
Exon 37
The characterization of SMRT cDNA clones from 3' RACE reactions identified two variants that differed by the inclusion or exclusion of an additional 38 amino acids at the boundary between sequences predicted to be encoded by exons 37 and 38 (Figure 3A). Comparing the additional sequence to the hypothetical translation of the intron between these two exons in the mouse genome identified a region of extensive similarity that arises from read-through of the conventional exon 37 splice donor (Figure 3B). We have called this additional sequence exon 37b. The same organization is seen in the human genome (data not shown). RT–PCR with primers from exons 37 and 38 generated two bands using both Xenopus and mouse RNA of sizes predicted for exon 37b(+) and 37b(–) transcripts (Figure 3C) and this was confirmed by sequence analysis of the cloned fragments. The amino acid sequence encoded by exon 37b shows extensive similarity to the equivalent region in xNCoR (Figure 3D). This region in NCoR contains CoRNR box-3, IDVII, which shows enhanced affinity for THR (12). In xSMRT this region is IDAII and this sequence is conserved from frog to man. The sequence I.x.x.I.I conforms to the consensus sequence for corepressor NR interaction motifs (12). This region is not subject to alternative splicing in Xenopus NCoR as the internal splice donor is changed from GT to a non-functional GG. Hence, in response to alternative splicing xSMRT can encode either a 2-box or a 3-box form of corepressor.
Figure 3. Alternative splicing at exon 37 generates a third SMRT CoRNR box. Read-through of the standard splice donor in exon 37 generates exon 37b which encodes a sequence with extensive similarity to the region encoding NCoR CoRNR box-3 and includes a novel third SMRT CoRNR box. (A) Additional 38 amino acids identified in 3' RACE reactions that lie between exons 37 and 38. (B) Translation of the mouse genomic sequence beyond the exon 37 splice donor generates sequence with extensive similarity to the additional sequence seen identified in Xenopus cDNA clones and is followed by a new splice donor. (C) RT–PCR analysis of RNA from Xenopus blastula and mouse brain using primers from exons 37 and 38 generates two bands corresponding to 37b(+) and 37b(–) transcripts. (D) Alignment of sequences around CoRNR box-3 in SMRT and NCoR from human, mouse and Xenopus show extensive similarity. The CoRNR boxes are shown in boldface.
Exons 41, 42 and 43
Analysis of cDNA clones from 3' RACE reactions of the xSMRT cDNA clone identified transcripts in Xenopus that lack exon 41 indicating alternative splicing at this site (Figure 4A). This was confirmed by the amplification of two bands of the correct predicted sizes in an RT–PCR of Xenopus RNA using primers adjacent to the splice sites (Figure 4B) and the cloning and sequencing of the PCR products. In contrast, the same approach, using mouse RNA, generated a single band and this corresponded to the exon 41(+) transcript (Figure 4B) suggesting that alternative splicing at this site is not prevalent. Sequence analysis of exon 41 identified no known domains or motifs.
Figure 4. Exon 41 is alternatively spliced during Xenopus development. (A) Alignment of human, mouse and Xenopus SMRT sequences identifies the absence of exon 41 from some xSMRT transcripts. (B) RT–PCR using Xenopus and mouse RNA and specific primers that flank exon 41 identify two bands of the sizes that indicate alternative splicing in Xenopus but not in mouse. (C) Isoform specific primers (arrows) for exon 41(+) (top panel) and 41(–) (middle panel) transcripts used in RT–PCR reactions show that both transcript types are expressed throughout early development (numbers refer to Nieuwkoop and Faber stages). The additional band in the 41(–) panel (open arrow) represents alternatively spliced transcripts from exon 42b (see Figure 5). ODC primers (lower panel) were used as a control for the RNA levels.
Figure 5. Exon 42 is alternatively spliced from an internal exon donor. (A) The alternative splicing of exon 42 depends on an exonic GT (underlined and in boldface) donor that is part of codon 2216, but which is not found in the equivalent sequence (lower case) in NCoR. The shaded portion of exon 42 represent exon 42b. (B) Alignment of exon 42 sequences shows that the alternatively spliced sequence of exon 42b is extensively conserved from frog to man. (C) RT–PCR analysis demonstrates alternative splicing in Xenopus (left-hand panel), but not in mouse brain (right-hand panel) . (D) Developmental series of Xenopus RNA used in an RT–PCR reaction to identify alternative splicing of exon 42 shows that both isoforms are present during early development. The decreased levels at stages 11 and 13 are consistent with the overall lower levels of SMRT in gastrula and early neurula stages. ODC is presented as a loading control.
Exon 41(+) and 41(–) transcripts are expressed throughout the early development (Figure 4C) and in all Xenopus tissues examined (data not shown). The specific primers used to detect exon 41(–) transcripts (Figure 4C, lower panel) generated two bands in RT–PCR reactions from Xenopus RNA, which suggests the alternative splicing of either exon 42 or exon 43. The size of the smaller band corresponds to the use of an internal splice donor in exon 42 and the use of this site was confirmed by cloning and sequence analysis (Figure 5A). This splicing event cannot occur in NCoR where the equivalent sequence is a CT, which is not active as a splice donor (Figure 5A).
Exon 42 can therefore be considered as a composite of exons 42 and 42b (Figure 5A). A BLAST search using exon 42b, or the alternative exons 42 and 43, sequence failed to identify sequences other than SMRT or NCoR and there is no apparent sequence motif within this region (Figure 5B). Alternative splicing in Xenopus was demonstrated by RT–PCR (Figure 5C), but the identification of a single band using equivalent primers and mouse brain RNA suggests that the alternative splicing of exon 42 is not prevalent in this tissue (Figure 5C) and this observation was extended to a range of other mouse tissues (data not shown). The ratio of exon 42b(+) and 42b(–) transcripts remains approximately constant during early Xenopus development (Figure 5D).
We did not identify alternatively spliced forms of exon 43 in Xenopus, but the screen of EST (expressed sequence tag) databases identified an exon 43(–) human SMRT clone (Image clone 1341723). Analysis of mouse RNA by RT–PCR confirmed alternative splicing at this site in each of several tissues (data not shown). Although the sequence of exon 43 is highly conserved between frog, mouse and human, there is no evidence that it encodes a functional motif.
Exon 44
The alternative splicing of exon 44 has been reported briefly in (27). An exon-splice donor within exon 44 can be recognized and will generate either a short exon 44 or a longer form, which we term exon 44b to maintain nomenclature with exons 37 and 42 (Figure 6A). Although the sequences of SMRT and NCoR are extensively conserved, the critical exon donor GT is changed to a GC in NCoR. The site of the internal exon 44 splice donor is adjacent to the 3' end of CoRNR box-1 in ID-C, and alternative splicing therefore changes the flanking sequence next to this motif (Figure 6B). The alternative splicing of exon 44 is found in both frog and mouse (Figure 6C) and is therefore a conserved event between two divergent species.
Figure 6. Alternative splicing of exon 44 in xSMRT. (A) Alternative splicing of xSMRT at exon 44 depends on the use of an exonic splice donor (GT) within codon 2294 (underlined and in boldface) that is not conserved in the equivalent sequence in NCoR (lower case). CoRNR box-1 is shown in diagonal stripes and exon 44b is shaded. (B) Alignment of sequences around the alternative splice site in exon 44 for 44b(+) and 44b(–) transcripts. The CoRNR box motif (LEAII) is shown in boldface. The flanking sequence in both transcripts shows conservation from frog to man. (C) RT–PCR using Xenopus and mouse cDNA with primers flanking exon 44b generate two bands of sizes corresponding to the 44b(+) (black arrow) and 44b(–) isoforms (open arrow). In Xenopus, the additional band (light shaded arrow) may indicate the production of further isoforms.
The SMRT paralogue, NCoR, does not undergo alternative splicing during early Xenopus development
NCoR-specific oligonucleotide primers located adjacent to exons that in xSMRT are alternatively spliced and were used in RT–PCR with Xenopus embryonic RNA (Figure 7A). In each case, the combination of primers gave a single band, indicating a lack of alternative splicing (Figure 7B). This confirms the prediction from sequences for exons 37, 42 and 44 where the critical splice donor GT in SMRT is altered in NCoR. During the development of the embryo, the xSMRT primary transcript is subject to extensive alternative splicing, however, this source of diversity is apparently unavailable to the NCoR gene.
Figure 7. NCoR transcripts are not alternatively spliced across the 3' regions during early Xenopus development. (A) NCoR and SMRT are paralogues that share a related exon organization and CoRNR box (black boxes) distribution. Although alternative splicing from the equivalent of exons 37, 42 and 44 (cross-hatched in SMRT) is unlikely as the important donor GT sequences are mutated in NCoR, we checked for alternative splicing across the 3' region of the coding sequence using a series of primers (F1-F4 and R). (B) Each set of primers gave a single band of expected size by RT–PCR indicating the presence of a single transcript in Xenopus embryos.
Transcript profiles for the region encoding the C-terminal isoforms
If alternative splicing occurred independently at each identified site in the 3' part of the gene, it would generate 16 different SMRT transcripts. However, it is possible that not all isoforms are produced or that some may be more abundant than others, indicating either that some sites are inherently more prone to alternative splicing or that the process is regulated. To determine the xSMRT transcript profile, primers were used that flank all the variable 3' exons in the X.tropicalis gene and the RT–PCR products resolved on acrylamide gels (Figure 8A). At the blastula stage, there are five expressed SMRT isoforms. The same profile was seen using X.laevis embryos (data not shown) and when an independent upstream primer was used. This demonstrates that some SMRT isoforms are preferentially expressed in the early embryo. The same approach using NCoR-specific primers detected just one isoform corresponding to the entire 3' coding region, confirming the lack of alternative splicing across this region of NCoR (Figure 8B). The predominant SMRT isoforms were cloned, sequenced and compared to the original RT–PCR to identify the bands (Figure 8A). Five isoforms are consistently found, of which four (SMRTc, e, j and m) are prevalent and one (SMRTa) is less abundant in the blastula embryo.
Figure 8. Only a subset of xSMRT transcript isoforms are expressed in the blastula stage embryo. (A) Xenopus RNA was subject to RT–PCR using primers that span the 3' region that harbours the four alternatively spliced exons. This generates a series of bands corresponding to the isoforms expressed at the blastula stage of development. The bands were isolated and cloned and the clones analysed by PCR using the same primer set. These clones were aligned with the bands in the RT–PCR reaction to identify the expressed transcript isoforms. On denaturing acrylamide gels, the DNA strands dissociate and run as doublets. The 16 possible isoforms are named in the order of decreasing sizes from SMRT-a to SMRT-p and the expressed isoforms and their composition are indicated. (B) Primers that span the equivalent 3' region of NCoR generate a single isoform (seen as a strand-separated doublet) confirming the lack of alternative splicing in NCoR.
DISCUSSSION
SMRT and NCoR are paralogues that act as corepressors for a range of transcription factors including a number of NRs (2). In the absence of ligand, they repress transcriptional activation by recruiting a multicomponent complex that includes HDAC proteins (4). Although SMRT and NCoR have similar protein architecture, we show that they differ in the number of protein isoforms each produces. xSMRT has four sites of alternative splicing that increase diversity in the C-terminal ID region. This could potentially generate 16 related proteins. In contrast, xNCoR is not alternatively spliced during early development and so produces a single isoform.
Variation within the N-terminal repressor domain
Two sites of variation corresponding to exons 19 and 25 (all numbering from the human genome) were identified. Exon 19 is apparently absent in Xenopus but is present in human and mouse genomes, but is not subject to alternative splicing (data not shown). The NCoR gene also lacks the equivalent exon in frog, mouse and human, suggesting that exon 19 was added after the duplication of the SMRT/NCoR precursor and after the divergence of amphibians from the line leading to mammals. Exon 19 is located between repressor domains RD1 and RD2, 51 residues C-terminal to the second SANT domain, but does not encode a peptide with a known functional motif. Exon 19 may represent an example of exon shuffling during evolution (30).
In contrast, exon 25 was identified in frog cDNA and in human genome and cDNA sequences, but not in published mouse cDNA sequences or in its annotated genome. However, a BLAST search of sequence from the appropriate mouse intron identified an open reading frame bounded by splice acceptor and donor sequences that shows extensive similarity to human exon 25. This exon is alternatively spliced in the mouse transcriptome, but there is no evidence for this in Xenopus. Although the exon 25-encoded peptide is in RD3, it maps to a region not directly involved in HDAC binding (29) and it would be interesting to compare the repressive abilities of SMRT isoforms that either lack or contain this sequence.
Variation in the C-terminal interaction domains
Previous analysis of the CoRNR boxes of SMRT and NCoR has proposed three mechanisms by which they achieve differential affinity for specific NRs (Figure 9). These are the use of multiple CoRNR boxes (12), the contribution of regions flanking the CoRNR boxes (9,11,31) and the spacing between CoRNR boxes (11). In this paper, we show that alternative splicing in SMRT affects each of these parameters and could therefore have a significant impact on the activity of SMRT as a corepressor.
Figure 9. The alternative splicing of xSMRT may determine its interactions with NRs by altering three CoRNR box parameters. Between exons 37 and 45 in xSMRT, there are four sites of alternative splicing. At exon 37, this results in the presence or absence of CoRNR box-3 and the formation of SMRT-3-box and SMRT-2-box isoforms. In NCoR, CoRNR box-3 has been shown to have a preference for specific NRs and it is likely that the complement of CoRNR boxes present in the protein will influence its interaction capability (12). The alternative splicing at exon 42 alters the spacing between CoRNR boxes 1 and 2, at least in the primary sequence, and it has been speculated that this parameter can modulate interactions with specific NRs (11). Finally, the alternative splicing of exon 44 results in the combination of CoRNR box-1 with different flanking regions, which may determine the specificity of interaction with NRs (9,11).
The first mechanism is the inclusion or exclusion of a CoRNR box. Alternative splicing at exon 37 involves the read-through of the normal exon donor to a second donor 114 bp downstream. The additional sequence (exon 37b) encodes a third CoRNR box, which is located within ID-N such that SMRT exists in both 2-box and 3-box isoforms where the 3-box isoforms are similar in architecture to NCoR (12). The CoRNR box-3 in NCoR is important for interactions with THR (12), and the sequence around this motif is conserved in SMRT and NCoR.
The second mechanism depends on alternative splicing at exon 44 (27), which generates isoforms that differ in amino acid sequence flanking CoRNR box-1. Mutational analyses of CoRNR box flanking sequences have demonstrated their importance for the specificity of interaction with NRs (9,11). The different flanking sequences provided by exon 44b and by exon 45 are each conserved between frog, mouse and human and this may reflect their function.
The final mechanism depends on alternative splicing of Xenopus exons 41 and 42. There are no identified functional domains in either exon and the alternatively spliced isoforms of exons 41 and 42 do not directly affect the CoRNR motifs or their flanking sequences. However, they do affect the spacing between the boxes and it has been suggested that this parameter may influence the heterodimeric NR complexes with which SMRT may bind (11). We have been unable to identify exon 41(–) or 42b(–) transcripts in mice. However, exon 43 is alternatively spliced in humans where 43(+) and 43(–) transcripts are represented in the EST databases though we have not been able to detect this transcript in Xenopus. It is likely though that there are other sites of alternative splicing in this region of the mouse transcript (C. Sharpe and S. Short, unpublished data).
SMRT and NCoR are paralogues of which one, SMRT, is subject to alternative splicing whilst the other is not. It is possible that this difference represents an evolutionary strategy that allows diversification without compromising the fundamental activity of the protein. This would allow the rapid evolution of diverse isoforms with a range of related functions. A similar relationship is seen in the p53 family. p53 itself is not alternatively spliced but the closely related genes, p63 and p73, generate multiple isoforms (32). Till date, the functional analysis of SMRT in Xenopus (13) has used a mouse dominant negative with an exon composition equivalent to SMRT-c: 37b(–)/41(+)/42b(+)/43(+)/44b(+). Although this is one of the prevalent transcripts in the early embryo, it may not act as a dominant negative for all SMRT–transcription factor interactions. The analysis of dominant negative transcripts of the other isoforms may be informative.
A common and highly successful approach to examine the response of a cell to a particular stimulus, or to identify tissue-specific gene expression, is to examine changes in the transcriptome using microarrays based on sequences from the 3' end of transcripts. However, this approach would fail to appreciate the diversity of SMRT protein isoforms. Given that some estimates suggest that up to 60% of human genes may show alternative splicing, to ignore this phenomenon may be to overlook a vital aspect of complexity within the vertebrate genome.
ACKNOWLEDGEMENTS
Mouse tissue samples were a kind gift from Chun Fu and Darek Gorecki, University of Portsmouth. We would like to thank members of the IBBS Genes and Development division for helpful discussion and Matt Guille and Sarah Brickwood for comments on the paper. M.M. was supported by a University of Portsmouth IBBS Graduate Bursary, S.S. by a BBSRC studentship and C.S. by a BBSRC Project Grant.
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* To whom correspondence should be addressed. Tel: +44 23 92 84 2062; Fax: +44 23 92 84 2053; Email: colin.sharpe@port.ac.uk
ABSTRACT
Silencing mediator for retinoid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor protein (NCoR) are corepressors that interact with a range of transcription factors. They both consist of N-terminal repressor domains that associate with histone deacetylases and C-terminal interaction domains (IDs) that contain CoRNR box motifs. These motifs mediate the interaction between corepressors and nuclear receptors (NRs), such as the retinoid and thyroid hormone receptors. However, whilst NCoR produces a single transcript during Xenopus development, xSMRT is subject to alternative splicing at four sites in the 3' part of the transcript, the region encoding the C-terminal IDs. Although this provides the potential to produce 16 different transcripts, only five isoforms are found in early embryos. The sites of alternative splicing predict that the resultant isoforms will differ in their ability to interact with NRs, as one site varies the number of CoRNR boxes, the second site changes the sequence flanking CoRNR box-1 and the other sites delete amino acid residues between CoRNR boxes 1 and 2 and so alter the critical spacing between these motifs. SMRT and NCoR therefore represent paralogues in which one form, SMRT, has evolved the ability to generate multiple isoforms whereas the other, NCoR, is invariant in Xenopus development.
INTRODUCTION
Silencing mediator for retinoid and thyroid hormone receptor (SMRT) and nuclear corepressor protein (NCoR) are large proteins of >2300 amino acid residues sharing 37% sequence identity and a conserved gene structure. They act as transcriptional corepressors and this is reflected in their protein architecture (Figure 1), which consists of N-terminal repressor domains (RD1–3) and C-terminal interaction domains (ID-N and ID-C) (2). The RDs recruit histone deacetylases (HDACs) to the transcription complex (3,4), and two adjacent SANT domains target the HDAC activity to the extended tails of nearby histones and this renders the chromatin transcriptionally inactive (5,6). The C-terminal region interacts with a range of transcription factors including Su(H), SRF, NFkB and AP1 (2). However, probably the best-characterized interactions are with the nuclear receptors (NRs) retinoic acid receptor (RAR) and thyroid hormone receptor (THR).
Figure 1. The structure of the Xenopus SMRT cDNA. SMRT is a large corepressor of >2300 amino acids, which in the human genome is encoded by a gene consisting of 49 exons. The protein can be divided into an N-terminal region, which encodes three repressor domains (RD1–3) and two SANT domains between RD1 and RD2. Interaction domains ID-N and ID-C in the C-terminal part of the protein each contain a CoRNR box motif and mediate interaction with nuclear receptors. A third CoRNR box (diagonal bars) has been identified in SMRT located in ID-N at a similar position to CoRNR box-3 of NCoR (see text). The variable exons have been highlighted.
Nuclear receptors act as ligand-dependent regulators of transcription and this depends on their ability to interact with coregulator proteins (1). In the presence of the ligand, they recruit coactivators, such as SRC-1, SRC-3 and CBP/p300 (7). These in turn assemble a multi-component complex that stimulates transcription both through direct interaction with the core transcription machinery (via the DRIP–TRAP and Mediator complexes) and through the acetylation of histone tails that make the adjacent chromatin transcriptionally competent (7). In the absence of the ligand, corepressors such as SMRT and NCoR, are recruited to the DNA-bound NRs to nucleate the assembly of the repressor complex (7). The NRs associate with IDs in SMRT that contain the CoRNR box motif (L/V.x.x.I/V.I) (8–10). Peptide sequences adjacent to the motif contribute to the specificity of interaction between the corepressor and the various NRs suggesting that each box preferentially interacts with a specific NR (9,11). Until recently, a major difference between NCoR and SMRT was the presence of three CoRNR box motifs in NCoR (12) compared to just two in SMRT. However, here we show that the SMRT gene has the capacity to encode a third CoRNR box motif in an additional sequence that arises from the read-through of the exon 37 splice donor to a site within the ensuing intron.
The importance of active repression by corepressors in NR signalling has been demonstrated in Xenopus by the use of dominant negative forms of SMRT, which disrupt normal head development and can be rescued by a dominant negative retinoid receptor or retinoid antagonist (13). However, SMRT and NCoR do not have entirely redundant functions, as knockout mice lacking the NCoR gene are embryonic lethal and show a number of specific defects including impaired neural development (2). The ability of SMRT to interact with NRs is also an important factor in a number of diseases. The PML–RAR gene fusion that contributes to acute promyelocytic leukaemia produces a protein with an increased affinity for SMRT, which accounts for many of the observed molecular defects (14). Resistance to the thyroid hormone can also be due to mutations in THR that increase its affinity to SMRT and which then require elevated levels of thyroid hormone to activate target gene expression (15).
The alternative splicing of primary transcripts generates different but related proteins from the same gene and may account for much of the additional diversity in the vertebrate proteome that compensates for the relatively low number of genes. It has been estimated that up to 60% of human genes may undergo some form of alternative splicing (16,17) and in some cases the isoforms produced have opposing functions (18–20), whilst in others alternative splicing generates vast numbers of isoforms that provide exceptional levels of protein diversity (21,22). However, it is also likely that closely related isoforms fine-tune the efficiency of cellular processes by producing related proteins with subtly different abilities.
As SMRT and NCoR have a similar gene organization and protein architecture, we decided to examine the extent of alternative splicing in each gene. In SMRT, we identified one difference in the 5' part of the transcript that encodes the RDs, but this is not due to alternative splicing. However, there is one site of alternative splicing in this region of the mouse gene that is not found in the frog gene. In contrast, we found extensive alternative splicing in the SMRT C-terminal domain in both frog and mouse. However, we did not detect alternative splicing in the same region of NCoR. Consequently, the introduced variability from alternative splicing is confined to just one paralogue. If alternative splicing in Xenopus SMRT was not regulated, the four sites would produce 16 distinct isoforms. However, examination of the SMRT transcript profile identifies just five significant alternatively spliced isoforms in the early embryo.
MATERIALS AND METHODS
Animals
Xenopus embryos were grown in 0.1x MBS (23) and collected at specific stages (24). Embryos were manually dissected in 1x MBS.
Cloning of SMRT
A Xenopus neurula stage cDNA library in lambda phage (25) was screened with an xNCoR cDNA clone derived from IMAGE clone 347360 that encodes the N-terminal RDs. One clone was isolated and subsequently identified as a SMRT cDNA clone. The full sequence of xSMRT was derived from 5' and 3' RACE reactions (First Choice RLM, Ambion).
Analysis of alternative splicing by RT–PCR
Total nucleic acid was extracted from embryos and the DNA was removed by RNAse-free DNase. RNA was converted to cDNA using Superscript II (Invitrogen) and stored at –20°C. cDNA from the equivalent of 0.1 embryos was used for each semiquantitative PCR using specific primers and the products run on 2% agarose gels. The standard programme involved 32 cycles, which was within the logarithmic phase of amplification for SMRT, and an annealing temperature of 53°C. For RT–PCR transcript profile analysis, cDNA from 0.1 embryos was amplified with primers adjacent to the alternatively spliced sites and 5 μCi of dATP in a 25 μl reaction and the products resolved on a denaturing 6% polyacrylamide gel. The amplified DNA was visualized on a Fujifilm FLA 5000 phosphorimager.
Cloning and characterization of the transcript profile products
An unlabelled RT–PCR using RNA from the equivalent of 0.1 embryos was run on an agarose gel and the bands from 900–1400 bp eluted and ligated directly into a TA vector system (Invitrogen). Recombinants were isolated and characterized by automated sequencing (MWG Corp.). Clones corresponding to the radiolabelled bands on the transcript profile were identified by PCR using the same primers and comparison by electrophoresis on polyacrylamide gels. Each clone corresponded to an element of the profile pattern and the abundance of each clone corresponded to the approximate intensity of its band in the RT–PCR transcript profile. Although this approach is semiquantitative, it provides an indication of the relative abundance of the different transcripts. This is supported by the observation that the same pattern with the same relative intensities was seen in both Xenopus tropicalis and Xenopus laevis and that the pattern and intensities vary consistently during development (C. Sharpe and S. Short, unpublished data).
RESULTS
A neurula stage cDNA library was screened with a probe for the corepressor gene, xNCoR. Of the clones identified, one encoded the closely related protein, xSMRT. The full sequence of xSMRT was determined from this cDNA clone and from clones encoding the 5' and 3' end of the transcript obtained by RACE reactions. These sequences show that xSMRT has extensive similarity to human and mouse SMRT with >70% conservation of amino acid sequence in the N-terminal RD1. In contrast, xSMRT differs significantly from xNCoR (with which it shares 37% similarity) (26) indicating that the frog does encode both corepressor genes.
As the SMRT corepressor is thought to interact with many different transcription factors (2) and has been shown to contain at least one site of alternative splicing (27), we looked for evidence of additional isoforms across the transcript using three approaches. First, we compared the xSMRT sequence to that found in other species, aligned according to the annotated exon boundaries of the human gene. Second, we looked for differences amongst the 5' and 3' RACE clones that again mapped to exon boundaries and finally we evaluated candidate sites by RT–PCR using primers from adjacent exons. This strategy identified two sites in the N-terminal repressor region and four around the C-terminal IDs.
Variation in the N-terminal domains of SMRT
Compared to human and mouse, the Xenopus SMRT cDNA clone lacks exon 19 that encodes a short, 17 amino acid peptide. However, we found no evidence for the alternative splicing of this exon (data not shown). Similarly, comparison of human, mouse and Xenopus cDNA sequences identified the lack of exon 25 in the published mouse cDNA sequence (28) (Figure 2A). However, hypothetical translation of the intron sequence between the adjacent exons in the mouse genome identifies a sequence with extensive similarity to exon 25, bounded by splice acceptor and donor sequences, which has so far escaped annotation (Figure 2B). The alternative splicing of exon 25 in the mouse SMRT transcript has been confirmed by RT–PCR and additionally shows that the exon 25(+) isoform is predominant in all tissues examined (Figure 2C). However, RT–PCR analysis using Xenopus mRNA fails to detect exon 25(–) transcripts during early development (Figure 2D). This exon encodes part of RD3, though the specific contribution of this peptide to HDAC recruitment has not been thoroughly evaluated (29).
Figure 2. Exon 25 is alternatively spliced in mouse but not in Xenopus. Exon 25 is not currently annotated in the mouse genome, but can be identified and is subject to alternative splicing. (A) Alignment of exon 25 showing extensive conservation of the predicted mouse sequence with the identified sequences in Xenopus and humans. (B) Mouse genome sequence from exons 24 to 26. The intron was translated and sequence corresponding to exon 25 identified and shown to be flanked by splice acceptor (AG) and donor (GT) sequences (in upper case) (C) RT–PCR analysis of RNA from dissected mouse tissues (B = brain, M = muscle, T = testes, K = kidney, H = heart, L = liver) identifies two bands corresponding to SMRT exon 25(+) and 25(–) transcripts. (D) Just one band, corresponding to xSMRT exon 25(+) transcripts, is detected during Xenopus development (numbers represent stages of development).
Variations in the C-terminal domains of xSMRT
Exon 37
The characterization of SMRT cDNA clones from 3' RACE reactions identified two variants that differed by the inclusion or exclusion of an additional 38 amino acids at the boundary between sequences predicted to be encoded by exons 37 and 38 (Figure 3A). Comparing the additional sequence to the hypothetical translation of the intron between these two exons in the mouse genome identified a region of extensive similarity that arises from read-through of the conventional exon 37 splice donor (Figure 3B). We have called this additional sequence exon 37b. The same organization is seen in the human genome (data not shown). RT–PCR with primers from exons 37 and 38 generated two bands using both Xenopus and mouse RNA of sizes predicted for exon 37b(+) and 37b(–) transcripts (Figure 3C) and this was confirmed by sequence analysis of the cloned fragments. The amino acid sequence encoded by exon 37b shows extensive similarity to the equivalent region in xNCoR (Figure 3D). This region in NCoR contains CoRNR box-3, IDVII, which shows enhanced affinity for THR (12). In xSMRT this region is IDAII and this sequence is conserved from frog to man. The sequence I.x.x.I.I conforms to the consensus sequence for corepressor NR interaction motifs (12). This region is not subject to alternative splicing in Xenopus NCoR as the internal splice donor is changed from GT to a non-functional GG. Hence, in response to alternative splicing xSMRT can encode either a 2-box or a 3-box form of corepressor.
Figure 3. Alternative splicing at exon 37 generates a third SMRT CoRNR box. Read-through of the standard splice donor in exon 37 generates exon 37b which encodes a sequence with extensive similarity to the region encoding NCoR CoRNR box-3 and includes a novel third SMRT CoRNR box. (A) Additional 38 amino acids identified in 3' RACE reactions that lie between exons 37 and 38. (B) Translation of the mouse genomic sequence beyond the exon 37 splice donor generates sequence with extensive similarity to the additional sequence seen identified in Xenopus cDNA clones and is followed by a new splice donor. (C) RT–PCR analysis of RNA from Xenopus blastula and mouse brain using primers from exons 37 and 38 generates two bands corresponding to 37b(+) and 37b(–) transcripts. (D) Alignment of sequences around CoRNR box-3 in SMRT and NCoR from human, mouse and Xenopus show extensive similarity. The CoRNR boxes are shown in boldface.
Exons 41, 42 and 43
Analysis of cDNA clones from 3' RACE reactions of the xSMRT cDNA clone identified transcripts in Xenopus that lack exon 41 indicating alternative splicing at this site (Figure 4A). This was confirmed by the amplification of two bands of the correct predicted sizes in an RT–PCR of Xenopus RNA using primers adjacent to the splice sites (Figure 4B) and the cloning and sequencing of the PCR products. In contrast, the same approach, using mouse RNA, generated a single band and this corresponded to the exon 41(+) transcript (Figure 4B) suggesting that alternative splicing at this site is not prevalent. Sequence analysis of exon 41 identified no known domains or motifs.
Figure 4. Exon 41 is alternatively spliced during Xenopus development. (A) Alignment of human, mouse and Xenopus SMRT sequences identifies the absence of exon 41 from some xSMRT transcripts. (B) RT–PCR using Xenopus and mouse RNA and specific primers that flank exon 41 identify two bands of the sizes that indicate alternative splicing in Xenopus but not in mouse. (C) Isoform specific primers (arrows) for exon 41(+) (top panel) and 41(–) (middle panel) transcripts used in RT–PCR reactions show that both transcript types are expressed throughout early development (numbers refer to Nieuwkoop and Faber stages). The additional band in the 41(–) panel (open arrow) represents alternatively spliced transcripts from exon 42b (see Figure 5). ODC primers (lower panel) were used as a control for the RNA levels.
Figure 5. Exon 42 is alternatively spliced from an internal exon donor. (A) The alternative splicing of exon 42 depends on an exonic GT (underlined and in boldface) donor that is part of codon 2216, but which is not found in the equivalent sequence (lower case) in NCoR. The shaded portion of exon 42 represent exon 42b. (B) Alignment of exon 42 sequences shows that the alternatively spliced sequence of exon 42b is extensively conserved from frog to man. (C) RT–PCR analysis demonstrates alternative splicing in Xenopus (left-hand panel), but not in mouse brain (right-hand panel) . (D) Developmental series of Xenopus RNA used in an RT–PCR reaction to identify alternative splicing of exon 42 shows that both isoforms are present during early development. The decreased levels at stages 11 and 13 are consistent with the overall lower levels of SMRT in gastrula and early neurula stages. ODC is presented as a loading control.
Exon 41(+) and 41(–) transcripts are expressed throughout the early development (Figure 4C) and in all Xenopus tissues examined (data not shown). The specific primers used to detect exon 41(–) transcripts (Figure 4C, lower panel) generated two bands in RT–PCR reactions from Xenopus RNA, which suggests the alternative splicing of either exon 42 or exon 43. The size of the smaller band corresponds to the use of an internal splice donor in exon 42 and the use of this site was confirmed by cloning and sequence analysis (Figure 5A). This splicing event cannot occur in NCoR where the equivalent sequence is a CT, which is not active as a splice donor (Figure 5A).
Exon 42 can therefore be considered as a composite of exons 42 and 42b (Figure 5A). A BLAST search using exon 42b, or the alternative exons 42 and 43, sequence failed to identify sequences other than SMRT or NCoR and there is no apparent sequence motif within this region (Figure 5B). Alternative splicing in Xenopus was demonstrated by RT–PCR (Figure 5C), but the identification of a single band using equivalent primers and mouse brain RNA suggests that the alternative splicing of exon 42 is not prevalent in this tissue (Figure 5C) and this observation was extended to a range of other mouse tissues (data not shown). The ratio of exon 42b(+) and 42b(–) transcripts remains approximately constant during early Xenopus development (Figure 5D).
We did not identify alternatively spliced forms of exon 43 in Xenopus, but the screen of EST (expressed sequence tag) databases identified an exon 43(–) human SMRT clone (Image clone 1341723). Analysis of mouse RNA by RT–PCR confirmed alternative splicing at this site in each of several tissues (data not shown). Although the sequence of exon 43 is highly conserved between frog, mouse and human, there is no evidence that it encodes a functional motif.
Exon 44
The alternative splicing of exon 44 has been reported briefly in (27). An exon-splice donor within exon 44 can be recognized and will generate either a short exon 44 or a longer form, which we term exon 44b to maintain nomenclature with exons 37 and 42 (Figure 6A). Although the sequences of SMRT and NCoR are extensively conserved, the critical exon donor GT is changed to a GC in NCoR. The site of the internal exon 44 splice donor is adjacent to the 3' end of CoRNR box-1 in ID-C, and alternative splicing therefore changes the flanking sequence next to this motif (Figure 6B). The alternative splicing of exon 44 is found in both frog and mouse (Figure 6C) and is therefore a conserved event between two divergent species.
Figure 6. Alternative splicing of exon 44 in xSMRT. (A) Alternative splicing of xSMRT at exon 44 depends on the use of an exonic splice donor (GT) within codon 2294 (underlined and in boldface) that is not conserved in the equivalent sequence in NCoR (lower case). CoRNR box-1 is shown in diagonal stripes and exon 44b is shaded. (B) Alignment of sequences around the alternative splice site in exon 44 for 44b(+) and 44b(–) transcripts. The CoRNR box motif (LEAII) is shown in boldface. The flanking sequence in both transcripts shows conservation from frog to man. (C) RT–PCR using Xenopus and mouse cDNA with primers flanking exon 44b generate two bands of sizes corresponding to the 44b(+) (black arrow) and 44b(–) isoforms (open arrow). In Xenopus, the additional band (light shaded arrow) may indicate the production of further isoforms.
The SMRT paralogue, NCoR, does not undergo alternative splicing during early Xenopus development
NCoR-specific oligonucleotide primers located adjacent to exons that in xSMRT are alternatively spliced and were used in RT–PCR with Xenopus embryonic RNA (Figure 7A). In each case, the combination of primers gave a single band, indicating a lack of alternative splicing (Figure 7B). This confirms the prediction from sequences for exons 37, 42 and 44 where the critical splice donor GT in SMRT is altered in NCoR. During the development of the embryo, the xSMRT primary transcript is subject to extensive alternative splicing, however, this source of diversity is apparently unavailable to the NCoR gene.
Figure 7. NCoR transcripts are not alternatively spliced across the 3' regions during early Xenopus development. (A) NCoR and SMRT are paralogues that share a related exon organization and CoRNR box (black boxes) distribution. Although alternative splicing from the equivalent of exons 37, 42 and 44 (cross-hatched in SMRT) is unlikely as the important donor GT sequences are mutated in NCoR, we checked for alternative splicing across the 3' region of the coding sequence using a series of primers (F1-F4 and R). (B) Each set of primers gave a single band of expected size by RT–PCR indicating the presence of a single transcript in Xenopus embryos.
Transcript profiles for the region encoding the C-terminal isoforms
If alternative splicing occurred independently at each identified site in the 3' part of the gene, it would generate 16 different SMRT transcripts. However, it is possible that not all isoforms are produced or that some may be more abundant than others, indicating either that some sites are inherently more prone to alternative splicing or that the process is regulated. To determine the xSMRT transcript profile, primers were used that flank all the variable 3' exons in the X.tropicalis gene and the RT–PCR products resolved on acrylamide gels (Figure 8A). At the blastula stage, there are five expressed SMRT isoforms. The same profile was seen using X.laevis embryos (data not shown) and when an independent upstream primer was used. This demonstrates that some SMRT isoforms are preferentially expressed in the early embryo. The same approach using NCoR-specific primers detected just one isoform corresponding to the entire 3' coding region, confirming the lack of alternative splicing across this region of NCoR (Figure 8B). The predominant SMRT isoforms were cloned, sequenced and compared to the original RT–PCR to identify the bands (Figure 8A). Five isoforms are consistently found, of which four (SMRTc, e, j and m) are prevalent and one (SMRTa) is less abundant in the blastula embryo.
Figure 8. Only a subset of xSMRT transcript isoforms are expressed in the blastula stage embryo. (A) Xenopus RNA was subject to RT–PCR using primers that span the 3' region that harbours the four alternatively spliced exons. This generates a series of bands corresponding to the isoforms expressed at the blastula stage of development. The bands were isolated and cloned and the clones analysed by PCR using the same primer set. These clones were aligned with the bands in the RT–PCR reaction to identify the expressed transcript isoforms. On denaturing acrylamide gels, the DNA strands dissociate and run as doublets. The 16 possible isoforms are named in the order of decreasing sizes from SMRT-a to SMRT-p and the expressed isoforms and their composition are indicated. (B) Primers that span the equivalent 3' region of NCoR generate a single isoform (seen as a strand-separated doublet) confirming the lack of alternative splicing in NCoR.
DISCUSSSION
SMRT and NCoR are paralogues that act as corepressors for a range of transcription factors including a number of NRs (2). In the absence of ligand, they repress transcriptional activation by recruiting a multicomponent complex that includes HDAC proteins (4). Although SMRT and NCoR have similar protein architecture, we show that they differ in the number of protein isoforms each produces. xSMRT has four sites of alternative splicing that increase diversity in the C-terminal ID region. This could potentially generate 16 related proteins. In contrast, xNCoR is not alternatively spliced during early development and so produces a single isoform.
Variation within the N-terminal repressor domain
Two sites of variation corresponding to exons 19 and 25 (all numbering from the human genome) were identified. Exon 19 is apparently absent in Xenopus but is present in human and mouse genomes, but is not subject to alternative splicing (data not shown). The NCoR gene also lacks the equivalent exon in frog, mouse and human, suggesting that exon 19 was added after the duplication of the SMRT/NCoR precursor and after the divergence of amphibians from the line leading to mammals. Exon 19 is located between repressor domains RD1 and RD2, 51 residues C-terminal to the second SANT domain, but does not encode a peptide with a known functional motif. Exon 19 may represent an example of exon shuffling during evolution (30).
In contrast, exon 25 was identified in frog cDNA and in human genome and cDNA sequences, but not in published mouse cDNA sequences or in its annotated genome. However, a BLAST search of sequence from the appropriate mouse intron identified an open reading frame bounded by splice acceptor and donor sequences that shows extensive similarity to human exon 25. This exon is alternatively spliced in the mouse transcriptome, but there is no evidence for this in Xenopus. Although the exon 25-encoded peptide is in RD3, it maps to a region not directly involved in HDAC binding (29) and it would be interesting to compare the repressive abilities of SMRT isoforms that either lack or contain this sequence.
Variation in the C-terminal interaction domains
Previous analysis of the CoRNR boxes of SMRT and NCoR has proposed three mechanisms by which they achieve differential affinity for specific NRs (Figure 9). These are the use of multiple CoRNR boxes (12), the contribution of regions flanking the CoRNR boxes (9,11,31) and the spacing between CoRNR boxes (11). In this paper, we show that alternative splicing in SMRT affects each of these parameters and could therefore have a significant impact on the activity of SMRT as a corepressor.
Figure 9. The alternative splicing of xSMRT may determine its interactions with NRs by altering three CoRNR box parameters. Between exons 37 and 45 in xSMRT, there are four sites of alternative splicing. At exon 37, this results in the presence or absence of CoRNR box-3 and the formation of SMRT-3-box and SMRT-2-box isoforms. In NCoR, CoRNR box-3 has been shown to have a preference for specific NRs and it is likely that the complement of CoRNR boxes present in the protein will influence its interaction capability (12). The alternative splicing at exon 42 alters the spacing between CoRNR boxes 1 and 2, at least in the primary sequence, and it has been speculated that this parameter can modulate interactions with specific NRs (11). Finally, the alternative splicing of exon 44 results in the combination of CoRNR box-1 with different flanking regions, which may determine the specificity of interaction with NRs (9,11).
The first mechanism is the inclusion or exclusion of a CoRNR box. Alternative splicing at exon 37 involves the read-through of the normal exon donor to a second donor 114 bp downstream. The additional sequence (exon 37b) encodes a third CoRNR box, which is located within ID-N such that SMRT exists in both 2-box and 3-box isoforms where the 3-box isoforms are similar in architecture to NCoR (12). The CoRNR box-3 in NCoR is important for interactions with THR (12), and the sequence around this motif is conserved in SMRT and NCoR.
The second mechanism depends on alternative splicing at exon 44 (27), which generates isoforms that differ in amino acid sequence flanking CoRNR box-1. Mutational analyses of CoRNR box flanking sequences have demonstrated their importance for the specificity of interaction with NRs (9,11). The different flanking sequences provided by exon 44b and by exon 45 are each conserved between frog, mouse and human and this may reflect their function.
The final mechanism depends on alternative splicing of Xenopus exons 41 and 42. There are no identified functional domains in either exon and the alternatively spliced isoforms of exons 41 and 42 do not directly affect the CoRNR motifs or their flanking sequences. However, they do affect the spacing between the boxes and it has been suggested that this parameter may influence the heterodimeric NR complexes with which SMRT may bind (11). We have been unable to identify exon 41(–) or 42b(–) transcripts in mice. However, exon 43 is alternatively spliced in humans where 43(+) and 43(–) transcripts are represented in the EST databases though we have not been able to detect this transcript in Xenopus. It is likely though that there are other sites of alternative splicing in this region of the mouse transcript (C. Sharpe and S. Short, unpublished data).
SMRT and NCoR are paralogues of which one, SMRT, is subject to alternative splicing whilst the other is not. It is possible that this difference represents an evolutionary strategy that allows diversification without compromising the fundamental activity of the protein. This would allow the rapid evolution of diverse isoforms with a range of related functions. A similar relationship is seen in the p53 family. p53 itself is not alternatively spliced but the closely related genes, p63 and p73, generate multiple isoforms (32). Till date, the functional analysis of SMRT in Xenopus (13) has used a mouse dominant negative with an exon composition equivalent to SMRT-c: 37b(–)/41(+)/42b(+)/43(+)/44b(+). Although this is one of the prevalent transcripts in the early embryo, it may not act as a dominant negative for all SMRT–transcription factor interactions. The analysis of dominant negative transcripts of the other isoforms may be informative.
A common and highly successful approach to examine the response of a cell to a particular stimulus, or to identify tissue-specific gene expression, is to examine changes in the transcriptome using microarrays based on sequences from the 3' end of transcripts. However, this approach would fail to appreciate the diversity of SMRT protein isoforms. Given that some estimates suggest that up to 60% of human genes may show alternative splicing, to ignore this phenomenon may be to overlook a vital aspect of complexity within the vertebrate genome.
ACKNOWLEDGEMENTS
Mouse tissue samples were a kind gift from Chun Fu and Darek Gorecki, University of Portsmouth. We would like to thank members of the IBBS Genes and Development division for helpful discussion and Matt Guille and Sarah Brickwood for comments on the paper. M.M. was supported by a University of Portsmouth IBBS Graduate Bursary, S.S. by a BBSRC studentship and C.S. by a BBSRC Project Grant.
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