Short S et al. (2015), Short linear motif acquisition, exon formation ...

XB-ART-51158

Open Biol 2015 Aug 01;58:. doi: 10.1098/rsob.150063.

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Short linear motif acquisition, exon formation and alternative splicing determine a pathway to diversity for NCoR-family co-repressors.

Short S , Peterkin T , Guille M , Patient R , Sharpe C .

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Vertebrate NCoR-family co-repressors play central roles in the timing of embryo and stem cell differentiation by repressing the activity of a range of transcription factors. They interact with nuclear receptors using short linear motifs (SLiMs) termed co-repressor for nuclear receptor (CoRNR) boxes. Here, we identify the pathway leading to increasing co-repressor diversity across the deuterostomes. The final complement of CoRNR boxes arose in an ancestral cephalochordate, and was encoded in one large exon; the urochordates and vertebrates then split this region between 10 and 12 exons. In Xenopus, alternative splicing is prevalent in NCoR2, but absent in NCoR1. We show for one NCoR1 exon that alternative splicing can be recovered by a single point mutation, suggesting NCoR1 lost the capacity for alternative splicing. Analyses in Xenopus and zebrafish identify that cellular context, rather than gene sequence, predominantly determines species differences in alternative splicing. We identify a pathway to diversity for the NCoR family beginning with the addition of a SLiM, followed by gene duplication, the generation of alternatively spliced isoforms and their differential deployment.

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Species referenced: Xenopus laevis
Genes referenced: ncor1 ncor2

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	Fig. 1. The NCoR-family conserved motifs and exon structure. (a) The NCoR-family proteins in the vertebrates typically contain two SANT domains (green bar), followed by three CoRNR boxes, nuclear receptor interaction motifs (yellow bars) and a carboxy-terminal SHARP-binding motif (red bar). Alignment of vertebrate NCoR1 and NCoR2 sequences identifies a further four conserved motifs (blue bars, lower diagram). Full sequence alignments are in the electronic supplementary material, figure S1. (b) Identity of conserved vertebrate sequences using the motif notation. Yellow bars overlie the consensus CoRNR box motif L/I.x.x.I/H.I.x.x.x.I/L [50,51] that is embedded in each of motifs 1, 2 and 5. The C-terminal SHARP-binding sequence is overlined in red as part of motif 8. (c) Exon organization of the 3â² end of representative NCoR-family genes. The regions encoding the motifs have been mapped onto the relevant exons maintaining the colour scheme in (a). In parentheses is the number of exons in this region of the gene. The C-terminal motifs are encoded by one large exon encoding 843 amino acids in the sea urchin, but 12 exons encoding 365 amino acids in the sea squirt. (d) Summary of C-terminal motif acquisition across the representative deuterostome panel. All contain motifs 1,2 (CoRNR boxes 1 and 2) and 8, but motif 5 (the third CoRNR box) is not present in the echinoderm and incomplete in the hemichordate and urochordate. Two of the four vertebrate specific motifs (3 and 4) are represented by partial motifs in the urochordate and cephalochordate (motif 4 only). Sequence alignments of the motifs are in the electronic supplementary martial, figure S2.
	Fig. 2. NCoR1 may have lost the capacity for alternative splicing in exon 37 after gene duplication. (a) Sequence alignments of Xenopus NCoR1 (XN1) and Ciona NCoR family (CNF) each with part of exon 37 of Xenopus NCoR2 (XN2). Vertical lines mark identical residues, the green box encodes CoRNR box 1. The sequences are conserved around the internal splice donor of NCoR2, except for the GT splice donor (red) that is a GA in NCoR1. (b) Plot of MAXENTSCAN splice score against position in exon 37 for all dinucleotides that can be changed by point mutation to a GT. Each bar represents the score for a nine-base sequence centred on the GT dinucleotide. The horizontal lines represent the mean and standard deviation calculated from all other characterized splice donors in the Xenopus NCoR1 gene. Blue bars mark splice donors that maintain the reading frame with exon 38 while red are out of frame. The black arrow marks the position of the site equivalent to the internal splice donor in NCoR2. The open arrow marks the position of the upstream potential splice donor used as a control for specificity. (c) Splicing constructs used to test the efficiency of the splice donors. Exon 37 from NCoR2 (grey and white box for exon 37b), NCoR1 and two point mutated forms of NCoR1 (grey boxes) were cloned along with flanking intron sequence between two human exons (black boxes) in the vector pTBNde1. A vertical dotted line marks the position of the internal splice donor equivalent to that found in NCoR2. Actual and potential GT splice donors are marked by a thick line. (d) RT-PCR analysis of transcripts from the splicing vector injected into Xenopus embryos. L marks the size ladder, U indicates uninjected control. The NCoR2 construct undergoes alternative splicing of exon 37 (open arrows), while the wild-type NCoR1 and the upstream specificity control (NCoR1con) do not, and produce a single band of the length expected for the inclusion of the entire exon 37. In contrast, the NCoR1 construct containing the point mutation at the equivalent internal site to NCoR2 (NCoR1spl) produces two bands (black arrows), the smaller representing the exclusion of exon 37b.
	Fig. 3. The conservation of NCoR2 exon 37 alternative splicing. NCoR2 exon 37 in Xenopus and mouse has two splice donors (red bars), site 1 is internal and site 2 is at the end of the extended exon. Exon 37b, between the two sites, encodes the CoRNR box 1 motif. (a) The potential site 1 GT splice donor is present in at least one representative of each vertebrate group and when present is part of a high-scoring consensus. A site 1 GT is present in the sea squirt, but is part of a poor consensus splice donor. (b) RT-PCR analysis of exon 37 alternative splicing across a range of species. The size of the PCR product generated from lamprey cDNA indicates that it includes exon 37b while that from 48 h zebrafish embryos corresponds predominantly to 37bâtranscripts. Xenopus, in contrast, generates both isoforms as shown by the two bands in the RT-PCR.
	Fig. 4. Evidence for the alternative splicing of exon 37 in zebrafish NCoR2. (a) RT-PCR analysis of RNA taken from a timecourse of zebrafish early development. The 37bâ isoform is predominant and the 37b(+) isoform is only detectable in later development. (b) Adult zebrafish were dissected into different parts and extracted RNA assayed by RT-PCR. The 37b+ isoform is only detected in brain and eye suggesting the tissue-specific control of the alternative splicing of this exon. A similar pattern is seen in mice but not frogs [27].
	The pathway to diversity for the NCoR family of co-repressors. Strongylocentrotus purpuratus (sea urchin) encodes two CoRNR boxes, but this increases to three in the cephalochordate amphioxus. Further motifs, identified by similarity to those in vertebrates, are found in the urochordate Ciona intestinalis. An additional CoRNR box motif will increase the range of nuclear receptors to which the co-repressor can bind. While the C-terminal interaction domains are encoded by a single exon in amphioxus they are encoded by at least 10 exons in Ciona and the vertebrates (for clarity, not all exons are shown). The ability to restore exon 37b alternative splicing in NCoR1 suggests that alternative splicing of this exon arose in the NCoR-family gene before duplication, which happened during the vertebrate genome duplication event after the divergence of Ciona and the vertebrates [69]. Of the two resulting paralogues, NCoR1 lost the alternative splicing of exon 37b by point mutation of the splice donor. Mammals, however, have recovered the capacity for alternative splicing to generate an isoform that lacks the first CoRNR box but which employs a different splice donor (blue lines). The alternative splicing of NCoR2 exon 37 is apparent in the teleost zebrafish, where, like mammals such as the mouse, the presence of the long form (red lines) including CoRNR box1 is tissue-specific, being found only in neural tissue. In addition Xenopus and the mouse undergo alternative splicing to generate isoforms of exon 44 [27]. Zebrafish lack the capacity for exon 44 alternative splicing. CoRNR boxes are in yellow and the SHARP domain in red.
	Figure 1. The NCoR-family conserved motifs and exon structure. (a) The NCoR-family proteins in the vertebrates typically contain two SANT domains (green bar), followed by three CoRNR boxes, nuclear receptor interaction motifs (yellow bars) and a carboxy-terminal SHARP-binding motif (red bar). Alignment of vertebrate NCoR1 and NCoR2 sequences identifies a further four conserved motifs (blue bars, lower diagram). Full sequence alignments are in the electronic supplementary material, figure S1. (b) Identity of conserved vertebrate sequences using the motif notation. Yellow bars overlie the consensus CoRNR box motif L/I.x.x.I/H.I.x.x.x.I/L [50,51] that is embedded in each of motifs 1, 2 and 5. The C-terminal SHARP-binding sequence is overlined in red as part of motif 8. (c) Exon organization of the 3â² end of representative NCoR-family genes. The regions encoding the motifs have been mapped onto the relevant exons maintaining the colour scheme in (a). In parentheses is the number of exons in this region of the gene. The C-terminal motifs are encoded by one large exon encoding 843 amino acids in the sea urchin, but 12 exons encoding 365 amino acids in the sea squirt. (d) Summary of C-terminal motif acquisition across the representative deuterostome panel. All contain motifs 1,2 (CoRNR boxes 1 and 2) and 8, but motif 5 (the third CoRNR box) is not present in the echinoderm and incomplete in the hemichordate and urochordate. Two of the four vertebrate specific motifs (3 and 4) are represented by partial motifs in the urochordate and cephalochordate (motif 4 only). Sequence alignments of the motifs are in the electronic supplementary martial, figure S2.
	Figure 2. NCoR1 may have lost the capacity for alternative splicing in exon 37 after gene duplication. (a) Sequence alignments of Xenopus NCoR1 (XN1) and Ciona NCoR family (CNF) each with part of exon 37 of Xenopus NCoR2 (XN2). Vertical lines mark identical residues, the green box encodes CoRNR box 1. The sequences are conserved around the internal splice donor of NCoR2, except for the GT splice donor (red) that is a GA in NCoR1. (b) Plot of MaxEntScan splice score against position in exon 37 for all dinucleotides that can be changed by point mutation to a GT. Each bar represents the score for a nine-base sequence centred on the GT dinucleotide. The horizontal lines represent the mean and standard deviation calculated from all other characterized splice donors in the Xenopus NCoR1 gene. Blue bars mark splice donors that maintain the reading frame with exon 38 while red are out of frame. The black arrow marks the position of the site equivalent to the internal splice donor in NCoR2. The open arrow marks the position of the upstream potential splice donor used as a control for specificity. (c) Splicing constructs used to test the efficiency of the splice donors. Exon 37 from NCoR2 (grey and white box for exon 37b), NCoR1 and two point mutated forms of NCoR1 (grey boxes) were cloned along with flanking intron sequence between two human exons (black boxes) in the vector pTBNde1. A vertical dotted line marks the position of the internal splice donor equivalent to that found in NCoR2. Actual and potential GT splice donors are marked by a thick line. (d) RT-PCR analysis of transcripts from the splicing vector injected into Xenopus embryos. L marks the size ladder, U indicates uninjected control. The NCoR2 construct undergoes alternative splicing of exon 37 (open arrows), while the wild-type NCoR1 and the upstream specificity control (NCoR1con) do not, and produce a single band of the length expected for the inclusion of the entire exon 37. In contrast, the NCoR1 construct containing the point mutation at the equivalent internal site to NCoR2 (NCoR1spl) produces two bands (black arrows), the smaller representing the exclusion of exon 37b.
	Figure 3. The conservation of NCoR2 exon 37 alternative splicing. NCoR2 exon 37 in Xenopus and mouse has two splice donors (red bars), site 1 is internal and site 2 is at the end of the extended exon. Exon 37b, between the two sites, encodes the CoRNR box 1 motif. (a) The potential site 1 GT splice donor is present in at least one representative of each vertebrate group and when present is part of a high-scoring consensus. A site 1 GT is present in the sea squirt, but is part of a poor consensus splice donor. (b) RT-PCR analysis of exon 37 alternative splicing across a range of species. The size of the PCR product generated from lamprey cDNA indicates that it includes exon 37b while that from 48 h zebrafish embryos corresponds predominantly to 37bâtranscripts. Xenopus, in contrast, generates both isoforms as shown by the two bands in the RT-PCR.
	Figure 4. Evidence for the alternative splicing of exon 37 in zebrafish NCoR2. (a) RT-PCR analysis of RNA taken from a timecourse of zebrafish early development. The 37bâ isoform is predominant and the 37b(+) isoform is only detectable in later development. (b) Adult zebrafish were dissected into different parts and extracted RNA assayed by RT-PCR. The 37b+ isoform is only detected in brain and eye suggesting the tissue-specific control of the alternative splicing of this exon. A similar pattern is seen in mice but not frogs [27].
	Figure 5. NCoR2 exon 37 splicing patterns are determined by cellular context. (a) Xenopus NCoR2 exon 37 (light grey box, dark grey box marks exon 37b) with approximately 250 base pairs of upstream and downstream flanking intron was cloned into the splicing vector pTBNde1 between two human exons (black boxes). Zebrafish exon 37 was similarly cloned but owing to the close proximity of exon 36 (white box) to exon 37 both exons, the intervening intron and 250 base pairs upstream of exon 36 and approximately 460 bp downstream were used. (b) Plasmids were injected into early stage Xenopus (left) and zebrafish (right) embryos and then grown to post-gastrula stages. (c) RNA extracted from the embryos was subject to RT-PCR using specific primers shown as small arrows in (a). Xenopus and zebrafish clones both gave two bands indicative of exon 37 alternative splicing in Xenopus. Injection into zebrafish embryos resulted in splicing primarily from the internal site to give the shorter 37bâ isoform. This indicates that the pattern of alternative splicing is strongly influenced by the cellular context. S, size markers; U, uninjected; X, injected Xenopus construct; Z, injected zebrafish construct.
	Figure 6. The pathway to diversity for the NCoR family of co-repressors. Strongylocentrotus purpuratus (sea urchin) encodes two CoRNR boxes, but this increases to three in the cephalochordate amphioxus. Further motifs, identified by similarity to those in vertebrates, are found in the urochordate Ciona intestinalis. An additional CoRNR box motif will increase the range of nuclear receptors to which the co-repressor can bind. While the C-terminal interaction domains are encoded by a single exon in amphioxus they are encoded by at least 10 exons in Ciona and the vertebrates (for clarity, not all exons are shown). The ability to restore exon 37b alternative splicing in NCoR1 suggests that alternative splicing of this exon arose in the NCoR-family gene before duplication, which happened during the vertebrate genome duplication event after the divergence of Ciona and the vertebrates [69]. Of the two resulting paralogues, NCoR1 lost the alternative splicing of exon 37b by point mutation of the splice donor. Mammals, however, have recovered the capacity for alternative splicing to generate an isoform that lacks the first CoRNR box but which employs a different splice donor (blue lines). The alternative splicing of NCoR2 exon 37 is apparent in the teleost zebrafish, where, like mammals such as the mouse, the presence of the long form (red lines) including CoRNR box1 is tissue-specific, being found only in neural tissue. In addition Xenopus and the mouse undergo alternative splicing to generate isoforms of exon 44 [27]. Zebrafish lack the capacity for exon 44 alternative splicing. CoRNR boxes are in yellow and the SHARP domain in red.

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