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. 2015 May 19;10(5):e0125998.
doi: 10.1371/journal.pone.0125998. eCollection 2015.

Neural differentiation modulates the vertebrate brain specific splicing program

Alicia Madgwick  1 , Philippe Fort  2 , Peter S Hanson  3 , Philippe Thibault  4 , Marie-Claude Gaudreau  5 , Georges Lutfalla  6 , Tarik Möröy  5 , Sherif Abou Elela  4 , Bill Chaudhry  1 , David J Elliott  1 , Christopher M Morris  3 , Julian P Venables  1
Affiliations

Neural differentiation modulates the vertebrate brain specific splicing program

Alicia Madgwick et al. PLoS One. .

Abstract

Alternative splicing patterns are known to vary between tissues but these patterns have been found to be predominantly peculiar to one species or another, implying only a limited function in fundamental neural biology. Here we used high-throughput RT-PCR to monitor the expression pattern of all the annotated simple alternative splicing events (ASEs) in the Reference Sequence Database, in different mouse tissues and identified 93 brain-specific events that shift from one isoform to another (switch-like) between brain and other tissues. Consistent with an important function, regulation of a core set of 9 conserved switch-like ASEs is highly conserved, as they have the same pattern of tissue-specific splicing in all vertebrates tested: human, mouse and zebrafish. Several of these ASEs are embedded within genes that encode proteins associated with the neuronal microtubule network, and show a dramatic and concerted shift within a short time window of human neural stem cell differentiation. Similarly these exons are dynamically regulated in zebrafish development. These data demonstrate that although alternative splicing patterns often vary between species, there is nonetheless a core set of vertebrate brain-specific ASEs that are conserved between species and associated with neural differentiation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Genomewide screen for brain-specific ASEs in mouse.
PCR primers were designed across all the simple ASEs in the mouse RefSeq collection [10] and RT-PCR was performed on three mouse tissue cDNA;. 809 PCRs gave good data; the psi values are shown for mouse brain, kidney and liver. The ASEs have been clustered according to the shift between brain and the two other tissues, indicated by the difference in psi values. 93 ASE psi values shifted more than 50% between brain and both of the other tissues (see the top and bottom of the heat map). Numerical data are given in S1 Table.
Fig 2
Fig 2. Cross-evolutionary screen for brain-specific ASEs found in human.
Of the 93 mouse candidate ASEs for conserved brain-specificity, 89 had orthologous regions in human. Of these, 62 ASEs gave good data by PCR on 6 human tissue cDNAs: brain, heart, muscle, kidney, liver and lung. The psi values are shown in the heat map; the genes are clustered on both axes according to their psi values. X-axis clustering shows the brain, heart/muscle and liver/kidney/lung have 3 distinct splicing profiles. Y axis clustering groups the genes based on their psi values’ patterns across tissues; the brain-specific ASEs cluster at the bottom of the heat map but the clustering also distinguishes some ASEs in the centre that splice similarly in brain/heart/muscle as distinct from liver/kidney/lung.
Fig 3
Fig 3. Switch-like tissue-specific ASEs are conserved in all vertebrates.
RT-PCR was performed on 15 genes across human, mouse and zebrafish. The 9 genes shown have conserved switch-like splicing in all three vertebrate species. Brain-specific splice forms are indicated with a red arrow. The alternative kidney/liver-specific forms are indicated with a double-headed blue arrow. The different, expected and found, PCR sizes for the long and short form of each gene in each species are given in S1 Table.
Fig 4
Fig 4. Nine vertebrate brain specific alternative splice events.
a. Primary structures of proteins encoded by the 9 human genes with vertebrate conserved brain-specific splicing. Shown are the annotated human proteins with the regions of the nine splice events indicated by red boxes. TM: transmembrane region; β-APP C-ter: C-terminus of the β Amyloid Precursor Protein (pf10515); CAP-Gly: Cytoskeletal-Associated protein (pf00225); HELP: Hydrophibic EMAP-Like Protein (pf03451); WD40: β-transducin repeat (pf00400); SiP: Signal peptide; Ig: Immunoglobulin-like domain (pf00047); FN3: Fibronectin type III domain (pf00041); AT-hook: DNA-binding for A/T-rich regions (pf02178); PHD finger: Plant HomeoDomain (Cys)4-His-(Cys)3 (pf00628). Gene names are labelled with a 1 if exclusion of the alternatively splice regions directly affects structural domains. Note all ASEs are multiples of 3 nucleotides, thus all the alternative splicing events confer in frame peptide omission or insertion. b. Brain-specific alternative splicing is conserved in vertebrates, and possibly beyond, in microtubule-associated genes. Metazoan genomes in Ensembl were searched for paralogs and orthologs of each _target gene and for the presence (yellow) or absence (gray) of potential ASEs (Accession numbers are shown in S1 Table). Yellow indicates that alternatively spliced mRNAs were detected in EST databases. Orange indicates that there were too few ESTs to conclude. Green indicates the absence of both genomic and EST data. Duplications are indicated by thick lines along with the names of the duplicated genes. An indication of the function of each gene is given on the right.
Fig 5
Fig 5. Neural splicing dynamics.
a. The 9 conserved brain-specific ASEs shifts during zebrafish embryogenesis. Time-course of splicing of the nine vertebrate brain-specific ASEs during the first two days of zebrafish embryogenesis, spanning 2 to 48 hours post fertilisation (hpf). The red arrows indicate the commencement of expression of the brain-specific AS transcript. b. Splicing of 13 genes pivots from predominantly one AS transcript isoform to the other upon neural differentiation of stem cells between days 6–10. 93 ASEs were assayed by PCR and their psi values were evaluated during neural stem cell differentiation. 56 ASEs gave good data for all time points (see S1 Table). Chart showing the psi values of 13 ASEs whose psi values shift more than 50% during the time-course.
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