Alternative splicing generates multiple SMRT transcripts encoding conserved repressor domains linked...

Alternative splicing generates multiple SMRTtranscripts encoding conserved repressordomains linked to variable transcriptionfactor interaction domainsMarianne Malartre, Stephen Short and Colin Sharpe*

Institute of Biomedical and Biomolecular Science, School of Biological Science, University of Portsmouth,King Henry I St, Portsmouth PO1 2DY, UK

Received May 14, 2004; Revised July 9, 2004; Accepted August 4, 2004

ABSTRACT

Silencing mediator for retinoid and thyroid hormonereceptor (SMRT) and nuclear receptor corepressorprotein (NCoR) are corepressors that interact with arange of transcription factors. They both consist ofN-terminal repressor domains that associate withhistone deacetylases and C-terminal interactiondomains (IDs) that contain CoRNR box motifs.These motifs mediate the interaction betweencorepressors and nuclear receptors (NRs), such asthe retinoid and thyroid hormone receptors.However, whilst NCoR produces a single transcriptduring Xenopus development, xSMRT is subject toalternative splicing at four sites in the 30 part of thetranscript, the region encoding the C-terminal IDs.Although this provides the potential to produce 16different transcripts, only five isoforms are found inearlyembryos.The sites ofalternative splicing predictthat the resultant isoforms will differ in their ability tointeract with NRs, as one site varies the number ofCoRNR boxes, the second site changes the sequenceflanking CoRNR box-1 and the other sites deleteamino acid residues between CoRNR boxes 1 and 2and so alter the critical spacing between these motifs.SMRT and NCoR therefore represent paralogues inwhich one form, SMRT, has evolved the ability togenerate 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 largeproteins of >2300 amino acid residues sharing 37% sequenceidentity and a conserved gene structure. They act as transcrip-tional corepressors [reviewed in (1,2)] and this is reflectedin 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 histonedeacetylases (HDACs) to the transcription complex (3,4), andtwo adjacent SANT domains target the HDAC activity to theextended tails of nearby histones and this renders the chromatintranscriptionally inactive (5,6). The C-terminal region interactswith a range of transcription factors including Su(H), SRF,NFkB and AP1 (2). However, probably the best-characterizedinteractions are with the nuclear receptors (NRs) retinoic acidreceptor (RAR) and thyroid hormone receptor (THR).

Nuclear receptors act as ligand-dependent regulators oftranscription and this depends on their ability to interactwith 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 complexthat stimulates transcription both through direct interactionwith the core transcription machinery (via the DRIP–TRAPand Mediator complexes) and through the acetylation of his-tone tails that make the adjacent chromatin transcriptionallycompetent (7). In the absence of the ligand, corepressors suchas SMRT and NCoR, are recruited to the DNA-bound NRs tonucleate the assembly of the repressor complex (7). The NRsassociate with IDs in SMRT that contain the CoRNR box motif(L/V.x.x.I/V.I) (8–10). Peptide sequences adjacent to the motifcontribute to the specificity of interaction between the corep-ressor and the various NRs suggesting that each box prefer-entially interacts with a specific NR (9,11). Until recently, amajor difference between NCoR and SMRT was the presenceof three CoRNR box motifs in NCoR (12) compared to justtwo in SMRT. However, here we show that the SMRT genehas the capacity to encode a third CoRNR box motif in anadditional sequence that arises from the read-through of theexon 37 splice donor to a site within the ensuing intron.

The importance of active repression by corepressors in NRsignalling has been demonstrated in Xenopus by the use ofdominant negative forms of SMRT, which disrupt normal headdevelopment and can be rescued by a dominant negative reti-noid receptor or retinoid antagonist (13). However, SMRT andNCoR do not have entirely redundant functions, as knockoutmice lacking the NCoR gene are embryonic lethal and show anumber of specific defects including impaired neural develop-ment (2). The ability of SMRT to interact with NRs is also animportant factor in a number of diseases. The PML–RARa

*To whom correspondence should be addressed. Tel: +44 23 92 84 2062; Fax: +44 23 92 84 2053; Email: [email protected]

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gene fusion that contributes to acute promyelocytic leukaemiaproduces a protein with an increased affinity for SMRT, whichaccounts for many of the observed molecular defects (14).Resistance to the thyroid hormone can also be due to mutationsin THR that increase its affinity to SMRT and which thenrequire elevated levels of thyroid hormone to activate targetgene expression (15).

The alternative splicing of primary transcripts generatesdifferent but related proteins from the same gene and mayaccount for much of the additional diversity in the vertebrateproteome that compensates for the relatively low number ofgenes. It has been estimated that up to 60% of human genesmay undergo some form of alternative splicing (16,17) and insome cases the isoforms produced have opposing functions(18–20), whilst in others alternative splicing generatesvast numbers of isoforms that provide exceptional levels ofprotein diversity (21,22). However, it is also likely thatclosely related isoforms fine-tune the efficiency of cellularprocesses by producing related proteins with subtly differentabilities.

As SMRT and NCoR have a similar gene organization andprotein architecture, we decided to examine the extent ofalternative splicing in each gene. In SMRT, we identifiedone difference in the 50 part of the transcript that encodesthe RDs, but this is not due to alternative splicing. However,there is one site of alternative splicing in this region of themouse gene that is not found in the frog gene. In contrast, wefound extensive alternative splicing in the SMRT C-terminaldomain in both frog and mouse. However, we did notdetect alternative splicing in the same region of NCoR.Consequently, the introduced variability from alternativesplicing is confined to just one paralogue. If alternativesplicing in Xenopus SMRT was not regulated, the foursites would produce 16 distinct isoforms. However,examination of the SMRT transcript profile identifies justfive significant alternatively spliced isoforms in the earlyembryo.

MATERIALS AND METHODS

Animals

Xenopus embryos were grown in 0.1· MBS (23) and col-lected at specific stages (24). Embryos were manually dissectedin 1· MBS.

Cloning of SMRT

A Xenopus neurula stage cDNA library in lambda phage (25)was screened with an xNCoR cDNA clone derived fromIMAGE clone 347360 that encodes the N-terminal RDs.One clone was isolated and subsequently identified as aSMRT cDNA clone. The full sequence of xSMRT was derivedfrom 50 and 30 RACE reactions (First Choice RLM, Ambion).

Analysis of alternative splicing by RT–PCR

Total nucleic acid was extracted from embryos and the DNAwas removed by RNAse-free DNase. RNA was converted tocDNA using Superscript II (Invitrogen) and stored at �20�C.cDNA from the equivalent of 0.1 embryos was used for eachsemiquantitative PCR using specific primers and the productsrun on 2% agarose gels. The standard programme involved 32cycles, which was within the logarithmic phase of amplifica-tion for SMRT, and an annealing temperature of 53�C. ForRT–PCR transcript profile analysis, cDNA from 0.1 embryoswas amplified with primers adjacent to the alternativelyspliced sites and 5 mCi of [32P]dATP in a 25 ml reactionand the products resolved on a denaturing 6% polyacrylamidegel. The amplified DNA was visualized on a Fujifilm FLA5000 phosphorimager.

Cloning and characterization of the transcript profileproducts

An unlabelled RT–PCR using RNA from the equivalent of 0.1embryos 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 characterizedby automated sequencing (MWG Corp.). Clones correspond-ing to the radiolabelled bands on the transcript profile wereidentified by PCR using the same primers and comparison byelectrophoresis on polyacrylamide gels. Each clone corres-ponded to an element of the profile pattern and the abundanceof each clone corresponded to the approximate intensity of itsband in the RT–PCR transcript profile. Although this approachis semiquantitative, it provides an indication of the relativeabundance of the different transcripts. This is supported by theobservation that the same pattern with the same relativeintensities was seen in both Xenopus tropicalis and Xenopuslaevis and that the pattern and intensities vary consistentlyduring development (C. Sharpe and S. Short, unpublisheddata).

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 consistingof 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 thirdCoRNR 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 beenhighlighted.

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RESULTS

A neurula stage cDNA library was screened with a probe forthe corepressor gene, xNCoR. Of the clones identified, oneencoded the closely related protein, xSMRT. The fullsequence of xSMRT was determined from this cDNA cloneand from clones encoding the 50 and 30 end of the transcriptobtained by RACE reactions. These sequences show thatxSMRT has extensive similarity to human and mouseSMRT with >70% conservation of amino acid sequence inthe N-terminal RD1. In contrast, xSMRT differs significantlyfrom xNCoR (with which it shares 37% similarity) (26) indi-cating that the frog does encode both corepressor genes.

As the SMRT corepressor is thought to interact with manydifferent transcription factors (2) and has been shown to con-tain at least one site of alternative splicing (27), we looked forevidence of additional isoforms across the transcript usingthree approaches. First, we compared the xSMRT sequenceto that found in other species, aligned according to the anno-tated exon boundaries of the human gene. Second, we lookedfor differences amongst the 50 and 30 RACE clones that againmapped to exon boundaries and finally we evaluated candidatesites by RT–PCR using primers from adjacent exons. Thisstrategy identified two sites in the N-terminal repressor regionand four around the C-terminal IDs.

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 toalternative splicing. (A) Alignment of exon 25 showing extensive conservation of the predicted mouse sequence with the identified sequences in Xenopus andhumans. (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 bysplice 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 exon25(+) transcripts, is detected during Xenopus development (numbers represent stages of development).

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Variation in the N-terminal domains of SMRT

Compared to human and mouse, the Xenopus SMRT cDNAclone lacks exon 19 that encodes a short, 17 amino acid pep-tide. However, we found no evidence for the alternative spli-cing of this exon (data not shown). Similarly, comparison ofhuman, mouse and Xenopus cDNA sequences identified thelack of exon 25 in the published mouse cDNA sequence (28)(Figure 2A). However, hypothetical translation of the intronsequence between the adjacent exons in the mouse genomeidentifies a sequence with extensive similarity to exon 25,bounded by splice acceptor and donor sequences, which hasso far escaped annotation (Figure 2B). The alternative splicingof exon 25 in the mouse SMRT transcript has been confirmedby RT–PCR and additionally shows that the exon 25(+) iso-form is predominant in all tissues examined (Figure 2C).

However, RT–PCR analysis using Xenopus mRNA fails todetect exon 25(�) transcripts during early development(Figure 2D). This exon encodes part of RD3, though thespecific contribution of this peptide to HDAC recruitmenthas not been thoroughly evaluated (29).

Variations in the C-terminal domains of xSMRT

Exon 37. The characterization of SMRT cDNA clones from 30

RACE reactions identified two variants that differed by theinclusion or exclusion of an additional 38 amino acids at theboundary between sequences predicted to be encoded by exons37 and 38 (Figure 3A). Comparing the additional sequence tothe hypothetical translation of the intron between these twoexons in the mouse genome identified a region of extensivesimilarity that arises from read-through of the conventional

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 whichencodes a sequence with extensive similarity to the region encoding NCoR CoRNR box-3 and includes a novel third SMRT CoRNR box. (A) Additional 38 aminoacids identified in 30 RACE reactions that lie between exons 37 and 38. (B) Translation of the mouse genomic sequence beyond the exon 37 splice donor generatessequence 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 ofRNA from Xenopus blastula and mouse brain using primers from exons 37 and 38 generates two bands corresponding to 37b(+) and 37b(�) transcripts. (D) Alignmentof sequences around CoRNR box-3 in SMRT and NCoR from human, mouse and Xenopus show extensive similarity. The CoRNR boxes are shown in boldface.




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exon 37 splice donor (Figure 3B). We have called this addi-tional sequence exon 37b. The same organization is seen in thehuman genome (data not shown). RT–PCR with primers fromexons 37 and 38 generated two bands using both Xenopus andmouse RNA of sizes predicted for exon 37b(+) and 37b(�)transcripts (Figure 3C) and this was confirmed by sequenceanalysis of the cloned fragments. The amino acid sequenceencoded by exon 37b shows extensive similarity to the equiva-lent region in xNCoR (Figure 3D). This region in NCoR con-tains CoRNR box-3, IDVII, which shows enhanced affinity forTHR (12). In xSMRT this region is IDAII and this sequence isconserved from frog to man. The sequence I.x.x.I.I conformsto the consensus sequence for corepressor NR interactionmotifs (12). This region is not subject to alternative splicingin Xenopus NCoR as the internal splice donor is changed fromGT to a non-functional GG. Hence, in response to alternativesplicing xSMRT can encode either a 2-box or a 3-box form ofcorepressor.

Exons 41, 42 and 43. Analysis of cDNA clones from 30 RACEreactions of the xSMRT cDNA clone identified transcripts inXenopus that lack exon 41 indicating alternative splicing atthis site (Figure 4A). This was confirmed by the amplificationof two bands of the correct predicted sizes in an RT–PCRof Xenopus RNA using primers adjacent to the splice sites(Figure 4B) and the cloning and sequencing of the PCRproducts. In contrast, the same approach, using mouse RNA,generated a single band and this corresponded to the exon41(+) transcript (Figure 4B) suggesting that alternativesplicing at this site is not prevalent. Sequence analysis ofexon 41 identified no known domains or motifs.

Exon 41(+) and 41(�) transcripts are expressed throughoutthe early development (Figure 4C) and in all Xenopus tissuesexamined (data not shown). The specific primers used to detectexon 41(�) transcripts (Figure 4C, lower panel) generatedtwo bands in RT–PCR reactions from Xenopus RNA, whichsuggests the alternative splicing of either exon 42 or exon 43.

Figure 4. Exon 41 is alternatively spliced during Xenopus development. (A) Alignment of human, mouse and Xenopus SMRT sequences identifies the absence ofexon 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 thatindicate alternative splicing in Xenopus but not in mouse. (C) Isoform specific primers (arrows) for exon 41(+) (top panel) and 41(�) (middle panel) transcripts usedin RT–PCR reactions show that both transcript types are expressed throughout early development (numbers refer to Nieuwkoop and Faber stages). The additionalband 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 forthe RNA levels.




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The size of the smaller band corresponds to the use of an internalsplice donor in exon 42 and the use of this site was confirmedby cloning and sequence analysis (Figure 5A). This splicingevent cannot occur in NCoR where the equivalent sequence isa CT, which is not active as a splice donor (Figure 5A).

Exon 42 can therefore be considered as a composite ofexons 42 and 42b (Figure 5A). A BLAST search usingexon 42b, or the alternative exons 42 and 43, sequence failedto identify sequences other than SMRT or NCoR and there isno apparent sequence motif within this region (Figure 5B).Alternative splicing in Xenopus was demonstrated by

RT–PCR (Figure 5C), but the identification of a singleband using equivalent primers and mouse brain RNA suggeststhat the alternative splicing of exon 42 is not prevalent in thistissue (Figure 5C) and this observation was extended to a rangeof other mouse tissues (data not shown). The ratio of exon42b(+) and 42b(�) transcripts remains approximately constantduring early Xenopus development (Figure 5D).

We did not identify alternatively spliced forms of exon 43 inXenopus, but the screen of EST (expressed sequence tag)databases identified an exon 43(�) human SMRT clone(Image clone 1341723). Analysis of mouse RNA by

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 analysisdemonstrates alternative splicing in Xenopus (left-hand panel), but not in mouse brain (right-hand panel) [Ld, size ladder; SM, size marker of 164 bp to identify the42b(�) isoform of 161 bp; PCR, RT–PCR reactions]. (D) Developmental series of Xenopus RNA used in an RT–PCR reaction to identify alternative splicing of exon42 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 ingastrula and early neurula stages. ODC is presented as a loading control.




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RT–PCR confirmed alternative splicing at this site in each ofseveral tissues (data not shown). Although the sequence ofexon 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 reportedbriefly in (27). An exon-splice donor within exon 44 can berecognized and will generate either a short exon 44 or a longerform, which we term exon 44b to maintain nomenclature withexons 37 and 42 (Figure 6A). Although the sequences ofSMRT and NCoR are extensively conserved, the criticalexon donor GT is changed to a GC in NCoR. The site ofthe internal exon 44 splice donor is adjacent to the 30 endof CoRNR box-1 in ID-C, and alternative splicing thereforechanges the flanking sequence next to this motif (Figure 6B).

The alternative splicing of exon 44 is found in both frog andmouse (Figure 6C) and is therefore a conserved event betweentwo divergent species.

The SMRT paralogue, NCoR, does not undergoalternative splicing during early Xenopus development

NCoR-specific oligonucleotide primers located adjacent toexons that in xSMRT are alternatively spliced and wereused in RT–PCR with Xenopus embryonic RNA(Figure 7A). In each case, the combination of primers gave asingle band, indicating a lack of alternative splicing (Figure 7B).This confirms the prediction from sequences for exons 37, 42and 44 where the critical splice donor GT in SMRT is alteredin NCoR. During the development of the embryo, the xSMRT

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 codon2294 (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 isshaded. (B) Alignment of sequences around the alternative splice site in exon 44 for 44b(+) and 44b(�) transcripts. The CoRNR box motif (LEAII) is shown inboldface. The flanking sequence in both transcripts shows conservation from frog to man. (C) RT–PCR using Xenopus and mouse cDNA with primers flanking exon44b 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.




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primary transcript is subject to extensive alternative splicing,however, this source of diversity is apparently unavailable tothe NCoR gene.

Transcript profiles for the region encoding theC-terminal isoforms

If alternative splicing occurred independently at each identi-fied site in the 30 part of the gene, it would generate 16different SMRT transcripts. However, it is possible thatnot all isoforms are produced or that some may be moreabundant than others, indicating either that some sites areinherently more prone to alternative splicing or that the pro-cess is regulated. To determine the xSMRT transcript profile,primers were used that flank all the variable 30 exons in theX.tropicalis gene and the RT–PCR products resolved onacrylamide gels (Figure 8A). At the blastula stage, thereare five expressed SMRT isoforms. The same profile wasseen using X.laevis embryos (data not shown) and whenan independent upstream primer was used. This demonstratesthat some SMRT isoforms are preferentially expressed in theearly embryo. The same approach using NCoR-specific pri-mers detected just one isoform corresponding to the entire 30

coding region, confirming the lack of alternative splicingacross this region of NCoR (Figure 8B). The predominantSMRT isoforms were cloned, sequenced and compared to theoriginal 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 inthe blastula embryo.

DISCUSSSION

SMRT and NCoR are paralogues that act as corepressors fora range of transcription factors including a number of NRs(2). In the absence of ligand, they repress transcriptionalactivation by recruiting a multicomponent complex thatincludes HDAC proteins (4). Although SMRT and NCoRhave similar protein architecture, we show that they differin the number of protein isoforms each produces. xSMRT hasfour sites of alternative splicing that increase diversity in theC-terminal ID region. This could potentially generate 16related proteins. In contrast, xNCoR is not alternativelyspliced during early development and so produces a singleisoform.

Variation within the N-terminal repressor domain

Two sites of variation corresponding to exons 19 and 25 (allnumbering from the human genome) were identified. Exon 19is apparently absent in Xenopus but is present in human andmouse genomes, but is not subject to alternative splicing (datanot shown). The NCoR gene also lacks the equivalent exon in

Figure 7. NCoR transcripts are not alternatively spliced across the 30 regions during early Xenopus development. (A) NCoR and SMRT are paralogues that share arelated exon organization and CoRNR box (black boxes) distribution. Although alternative splicing from the equivalent of exons 37, 42 and 44 (cross-hatched inSMRT) is unlikely as the important donor GT sequences are mutated in NCoR, we checked for alternative splicing across the 30 region of the coding sequence using aseries 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 Xenopusembryos.




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frog, mouse and human, suggesting that exon 19 was addedafter the duplication of the SMRT/NCoR precursor and afterthe divergence of amphibians from the line leading to mam-mals. Exon 19 is located between repressor domains RD1 andRD2, 51 residues C-terminal to the second SANT domain, butdoes not encode a peptide with a known functional motif.Exon 19 may represent an example of exon shuffling duringevolution (30).

In contrast, exon 25 was identified in frog cDNA and inhuman genome and cDNA sequences, but not in publishedmouse cDNA sequences or in its annotated genome. However,a BLAST search of sequence from the appropriate mouseintron identified an open reading frame bounded by spliceacceptor and donor sequences that shows extensive similarityto human exon 25. This exon is alternatively spliced in themouse transcriptome, but there is no evidence for thisin Xenopus. Although the exon 25-encoded peptide is inRD3, it maps to a region not directly involved in HDACbinding (29) and it would be interesting to compare the repres-sive abilities of SMRT isoforms that either lack or contain thissequence.

Variation in the C-terminal interaction domains

Previous analysis of the CoRNR boxes of SMRT and NCoRhas proposed three mechanisms by which they achieve differ-ential affinity for specific NRs (Figure 9). These are the use ofmultiple CoRNR boxes (12), the contribution of regionsflanking the CoRNR boxes (9,11,31) and the spacing betweenCoRNR boxes (11). In this paper, we show that alternativesplicing in SMRT affects each of these parameters and couldtherefore have a significant impact on the activity of SMRT asa corepressor.

The first mechanism is the inclusion or exclusion of aCoRNR box. Alternative splicing at exon 37 involves theread-through of the normal exon donor to a second donor114 bp downstream. The additional sequence (exon 37b)encodes a third CoRNR box, which is located within ID-Nsuch that SMRT exists in both 2-box and 3-box isoformswhere the 3-box isoforms are similar in architecture toNCoR (12). The CoRNR box-3 in NCoR is important forinteractions with THR (12), and the sequence around thismotif is conserved in SMRT and NCoR.

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 spanthe 30 region that harbours the four alternatively spliced exons. This generates a series of bands corresponding to the isoforms expressed at the blastula stage ofdevelopment. 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–PCRreaction to identify the expressed transcript isoforms. On denaturing acrylamide gels, the DNA strands dissociate and run as doublets. The 16 possible isoforms arenamed 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 equivalent30 region of NCoR generate a single isoform (seen as a strand-separated doublet) confirming the lack of alternative splicing in NCoR.




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The second mechanism depends on alternative splicing atexon 44 (27), which generates isoforms that differ in aminoacid sequence flanking CoRNR box-1. Mutational analyses ofCoRNR box flanking sequences have demonstrated theirimportance for the specificity of interaction with NRs(9,11). The different flanking sequences provided by exon44b 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 ofXenopus exons 41 and 42. There are no identified functionaldomains in either exon and the alternatively spliced isoformsof exons 41 and 42 do not directly affect the CoRNR motifs ortheir flanking sequences. However, they do affect the spacingbetween the boxes and it has been suggested that this para-meter may influence the heterodimeric NR complexes withwhich SMRT may bind (11). We have been unable to identifyexon 41(�) or 42b(�) transcripts in mice. However, exon 43 isalternatively spliced in humans where 43(+) and 43(�) tran-scripts are represented in the EST databases though we havenot been able to detect this transcript in Xenopus. It is likelythough that there are other sites of alternative splicing in thisregion of the mouse transcript (C. Sharpe and S. Short, unpub-lished data).

SMRT and NCoR are paralogues of which one, SMRT, issubject to alternative splicing whilst the other is not. It ispossible that this difference represents an evolutionary strat-egy that allows diversification without compromising the fun-damental activity of the protein. This would allow the rapidevolution of diverse isoforms with a range of related functions.A similar relationship is seen in the p53 family. p53 itself isnot alternatively spliced but the closely related genes, p63and p73, generate multiple isoforms (32). Till date, the func-tional analysis of SMRT in Xenopus (13) has used a mousedominant negative with an exon composition equivalent to

SMRT-c: 37b(�)/41(+)/42b(+)/43(+)/44b(+). Although thisis one of the prevalent transcripts in the early embryo, itmay not act as a dominant negative for all SMRT–transcriptionfactor interactions. The analysis of dominant negative tran-scripts of the other isoforms may be informative.

A common and highly successful approach to examine theresponse of a cell to a particular stimulus, or to identify tissue-specific gene expression, is to examine changes in the tran-scriptome using microarrays based on sequences from the 30

end of transcripts. However, this approach would fail toappreciate the diversity of SMRT protein isoforms. Giventhat some estimates suggest that up to 60% of human genesmay show alternative splicing, to ignore this phenomenon maybe to overlook a vital aspect of complexity within the verte-brate genome.

ACKNOWLEDGEMENTS

Mouse tissue samples were a kind gift from Chun Fu andDarek Gorecki, University of Portsmouth. We would like tothank members of the IBBS Genes and Development divisionfor helpful discussion and Matt Guille and Sarah Brickwood forcomments on the paper. M.M. was supported by a University ofPortsmouth IBBS Graduate Bursary, S.S. by a BBSRC student-ship and C.S. by a BBSRC Project Grant.

REFERENCES

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2. Jepsen,K. and Rosenfeld,M.G. (2002) Biological roles and mechanisticactions of corepressor complexes. J. Cell. Sci., 115, 689–698.

3. Heinzel,T., Lavinsky,R.M, Mullen,T.M., Soderstrom,M., Laherty,C.D.,Torchia,J., Yang,W.M., Brard,G., Ngo,S.D., Davie,J.R., Seto,E.,

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-boxisoforms. 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 proteinwill 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, andit has been speculated that this parameter can modulate interactions with specific NRs (11). Finally, the alternative splicing of exon 44 results in the combination ofCoRNR box-1 with different flanking regions, which may determine the specificity of interaction with NRs (9,11).




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