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PLoS Genet
2007 Oct 01;310:1838-47. doi: 10.1371/journal.pgen.0030173.
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Evolution of Nova-dependent splicing regulation in the brain.
Jelen N
,
Ule J
,
Zivin M
,
Darnell RB
.
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A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters) show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs.
Figure 1. Analysis of Nova Protein in the Brain and Liver of Different Vertebrate Species(A) Protein alignment of the 100% conserved region of the Nova1 KH1 domain.(B) Immunoblot analysis of brain and liver extract from mouse, chicken, and zebrafish using polyclonal Nova and the eIF3a loading control antibody. Protein (50 μg) was loaded in each lane. Mouse brain contains an additional slower migrating Nova band that is not detected in chick and zebrafish brain (but is present in human brain). In another study, we cloned this slower migrating band and found that it binds to similar YCAY clusters and regulates the same exons as the previously cloned Nova1 and Nova2 isoforms (unpublished data).The abbreviations for the following species are used: H. sapiens (hs), M. musculus (mm), G. gallus (gg), and D. rerio (dr).
Figure 2. Conservation of Mouse YCAY Clusters and Nova-Regulated Alternative Exons in Different VertebratesWe analyzed conservation of 49 mouse YCAY clusters that were predicted by microarray. We observed a high degree of conservation of YCAY clusters across species, with 94% conservation in human and 41% conservation in zebrafish. We also analyzed genomic conservation of 88 mouse Nova-regulated alternative exons in vertebrates, based on cDNA/EST data, genomic alignment, and sequencing. The results were similar to conservation of YCAY clusters, with 56% of exons conserved in zebrafish and 99% in human. Because cDNA/EST data is not complete and may contain errors, we may have overestimated or underestimated the level of alternative exon conservation, but in general our sequencing results agreed very well with cDNA/EST or genomic prediction.
Figure 3. Comparison of YCAY Cluster Score to Brain-Specific Splicing in Chicken and ZebrafishThe graph shows relative exon inclusion levels against calculated YCAY cluster scores. Dashed lines indicate cut-off values for conserved YCAY clusters (score > 0.6) and |ΔI| (20% change in exon inclusion between brain and liver). Data for chicken are marked with full triangles and data for zebrafish with empty triangles. From 24 cases in which exons had conserved YCAY clusters, 17 were splicing enhancers (upper right part of the graph, denoted with dotted line) and seven were splicing silencers (lower left part of the graph, denoted with dotted line). Note that the direction of splicing always correlates with the type of YCAY cluster (enhancer versus silencer) present; therefore, the upper left and the lower right part of the graph are empty. From 15 cases where YCAY clusters were not conserved, seven showed no alternative splicing (center of the graph, denoted with dotted square). Overall, we observed 31 cases where the presence of YCAY clusters matched with splicing patterns in vivo. The exceptions that did not match with our prediction were eight cases in which brain-specific splicing was present in the absence of conserved YCAY clusters. The data drawn in this graph are also shown in Table 1.
Figure 4. Five Examples of YCAY Cluster Conservation and RT-PCR Analysis of Associated ExonsMultiple nucleotide alignment of five YCAY clusters. Introns are in lowercase, exons in uppercase, YCAY silencer motifs in bold blue, and YCAY enhancer motifs in bold red. The abbreviations for the following species are used: H. sapiens (hs), M. musculus (mm), M. domestica (md), G. gallus (gg), and D. rerio (dr). Next to the alignment are RT-PCR data from wild-type and Nova2 knockout brain, and below are RT-PCR data from chicken and zebrafish brain and liver and diagrams of the splicing pattern. Blue represents silencing, red enhancement by Nova, rectangles represent exons, and circles represent the position of Nova binding.(A) Nova-dependent splicing silencer in neogenin (Neo1) pre-mRNA and brain-specific splicing of exon 27 conserved.(B) Nova-dependent splicing silencer in syntaxin binding protein 2 (Stxbp2/Munc18–2) pre-mRNA and brain-specific splicing of exon 3 are not conserved.(C) Nova-dependent splicing enhancer in protein tyrosine phosphatase, receptor type, F (Ptprf) pre-mRNA and brain-specific splicing of exon 6a are conserved.(D) Nova-dependent splicing enhancer is not conserved in zebrafish amyloid beta precursor-like protein 2 (Aplp2) pre-mRNA, in contrast with conserved brain-specific splicing of exon 12a.(E) Nova-dependent splicing enhancer in suppression of tumorigenicity 7 (St7) pre-mRNA and brain-specific splicing of exon 12a are not conserved.(F) The diagram obtained from the human genome browser shows the human St7 mRNAs that either contain or exclude the alternative exon 12a. Underneath, alignment of vertebrate genomes demonstrates lack of conservation in the region containing exon 12a in the genomes of oppossum, chicken, and zebrafish.
Figure 5. Conservation of YCAY Cluster Can Provide Insight into Evolution of Functionally Coherent Coregulated NetworkThe diagram shows a subset of proteins encoded by Nova-regulated transcripts for which we were able to obtain orthologous sequences for analysis of YCAY cluster conservation. The color coding represents the evolutionarily most distant species from mouse that contains a conserved YCAY cluster. High YCAY cluster conservation is seen in RNAs encoding adhesion and cytoskeletal scaffold proteins, ion channels, and signaling proteins. In comparison, RNAs with lower YCAY cluster conservation (limited to mammals) most often encode neurotransmitter receptors (such as glycine [GlyRα2] and kainate [GluR6] receptors) and signaling proteins (such as neurochondrin, Lrp12, and Gpr45).
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