Exon junction complex shapes the transcriptome by repressing recursive splicing

Recursive splicing (RS) starts by defining an “RS-exon,” which is then spliced to the preceding exon, thus creating a recursive 5′ splice site (RS-5ss). Previous studies focused on cryptic RS-exons, and now we find that the exon junction complex (EJC) represses RS of hundreds of annotated, mainly constitutive RS-exons. The core EJC factors, and the peripheral factors PNN and RNPS1, maintain RS-exon inclusion by repressing spliceosomal assembly on RS-5ss. The EJC also blocks 5ss located near exon-exon junctions, thus repressing inclusion of cryptic microexons. The prevalence of annotated RS-exons is high in deuterostomes, while the cryptic RS-exons are more prevalent in Drosophila, where EJC appears less capable of repressing RS. Notably, incomplete repression of RS also contributes to physiological alternative splicing of several human RS-exons. Finally, haploinsufficiency of the EJC factor Magoh in mice is associated with skipping of RS-exons in the brain, with relevance to the microcephaly phenotype and human diseases

RES complex is required during zebrafish brain development.

<p><b>(A)</b> Acridine orange (ao) staining of zebrafish mutant embryos for <i>bud13</i> (30–32 hpf), <i>rbmx2</i> and <i>snip1</i> (48 hpf). Mutants show a marked degree of cells with nuclear uptake of ao compared to WT sibling most predominantly in the head. WT: represent phenotypically wild type sibling from the same mutant fish line. <b>(B)</b> Maximum intensity projections of individual and merged channels (GFP and dsRed) of 3D confocal images of <i>bud13</i>^Δ7/Δ7 and their wild-type siblings (scale bar 20 μm) in transgenic lines that label GABAergic neurons and precursors (Tg[dlx6a-1.4kbdlx5a/ dlx6a:GFP]) and glutamatergic neurons (Tg[vglut:DsRed]). WT: represent phenotypically wild type sibling from the same mutant fish line. <b>(C)</b> Total number of dlx5a/6a:GFP+ cells (GABAergic neurons and precursors) and vglut:DsRed+ cells (glutamatergic neurons) in the forebrain of the <i>bud13</i>^Δ7/Δ7 (n = 3) and WT sibling (n = 4) were quantified. *** vglut: <i>P</i> = 2 x 10⁻⁴; ***dlx5a/6a: <i>P</i> = 3 x 10⁻⁴ (one-way ANOVA).</p

RES complex is essential for early vertebrate development.

<p><b>(A)</b> Schematic model of the RES complex adapted from Brooks et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007473#pgen.1007473.ref008" target="_blank">8</a>]. Rbmx2 (light blue) is the core subunit with an RRM-domain structure. Bud13 (orange) and Snip1 (pink) interact with Rbmx2 (light blue). <b>(B)</b> In situ hybridization showing spatial and temporal expression of RES complex members. <b>(C-E)</b> Gene models of the mutant allele generated using CRISPR-Cas9-nanos. <b>(C)</b> <i>bud13</i>, 7 nt deletion in exon 6 generated a premature stop codon. <b>(D)</b> <i>rbmx2</i>, 16 nt deletion removed exon-intron boundary at exon 2 (exon capital letter, intron lower letter). <b>(E)</b> <i>snip1</i>, 11 nt deletion in exon 1 generated a premature stop codon. f-g) Lateral view of RES complex mutant embryos, their corresponding WT sibling and mutants injected with the cognate mRNA. <b>(F)</b> <i>bud13</i> mutant at 32 hpf (scale bar: 0.35mm) and at 48 hpf (scale bar: 0.5mm). <b>(G, H)</b> <i>rbmx2</i> and <i>snip1</i> mutant at 48 hpf respectively. WT: represent phenotypically wild type sibling from the same mutant fish line.</p

RES complex mutants show mild widespread intron mis-splicing.

<p><b>(A)</b> Biplot illustrating percent intron retention (PIR) in each of the RES mutants and the corresponding phenotypically wild type (WT) siblings. Blue and red dots correspond to introns with higher inclusion in the mutant and WT, respectively, using a cutoff of ∆PIR >15. Insets show exon gene expression levels in the same conditions (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007473#pgen.1007473.s011" target="_blank">S4 Fig</a> for details). <b>(B)</b> Stacked barplot showing the number of mis-regulated events upon RES loss-of-function. Clearly, most changes are intron retention supporting the role of the RES complex in splicing. <b>(C)</b> Scheme showing how PIR was measured (adapted from Braunschweig et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007473#pgen.1007473.ref022" target="_blank">22</a>]; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007473#sec007" target="_blank">Methods</a> for details). <b>(D)</b> Euler diagram showing the number of retained introns (∆PIR>5) in the three mutants and the inter-mutant overlaps. <b>(E)</b> RNA-seq read density across the <i>rrp8</i> gene in <i>bud13</i>, <i>rbmx2</i> and <i>snip1</i> mutants and their corresponding phenotypically WT siblings. Intronic signal increases in RES mutants (∆PIR>15) (dark blue) (dotted square box). <b>(F)</b> RT-PCR assays validate the increased retention of an <i>rrp8</i> intron (from panel E) in <i>bud13</i>, <i>rbmx2</i> and <i>snip1</i> mutants compared to the corresponding phenotypically WT siblings.</p

Logistic regression model can accurately classify RES-dependent and non-dependent introns.

<p><b>(A)</b> Classification performance of logistic regression models for different data sets of differentially retained vs. Ctr introns. ROC curves are averaged over 10,000 repeated holdout experiments where models have been trained with randomly sampled subsets of 90% (1,268) of the RESdep introns versus 1,268 Ctr introns with 30 features (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007473#pgen.1007473.s003" target="_blank">S3 Table</a>) and Lasso feature selection. Classification performance was estimated using the remaining 10% (141) RESdep introns and 141 randomly sampled Ctr introns. Having held fixed parameters, the same model was used to estimate classification performance with randomly sampled 141 introns from the other RES-dependent data sets, namely: (i) “RESdep (∆PIR>10)” introns from the “RESdep” set with ∆PIR > 10 in all three mutants (871 introns); (ii) “RESdep (NMD)”, introns from the “RESdep” set predicted to trigger NMD when retained (574 introns); (iii) “<i>bud13</i>^{<i>∆7/∆7</i>}”, introns with ∆PIR>15 upon <i>bud13</i> mutation at 32 hpf (2,363 introns); (iv) “<i>rbmx2</i>^{<i>∆16/∆16</i>}”, introns with ∆PIR>15 upon <i>rbmx2</i> mutation at 48 hpf (2,186 introns); and (v) “<i>snip1</i>^{<i>∆11/∆11</i>}”, introns with ∆PIR>15 upon <i>snip1</i> mutation at 48 hpf (2,675 introns). 95% confidence interval of reported average AUCs corresponds to AUC ± 0.001. <b>(B)</b> Capacity of each feature to discriminate between “RESdep” and “Ctr” introns, measured as AUC (average area under ROC curve) when used as the only feature in a one-feature logistic regression model. <b>(C)</b> Schematic of the experiment performed to validate the predicted features, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007473#sec007" target="_blank">Material and Methods</a> for details. <b>(D)</b> RES dependent but not RES independent intron was retained in <i>bud13</i>^{<i>∆7/∆7</i>} mutant as the regression model predicted. The validation experiment was done using two independent biological replicates (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007473#pgen.1007473.s019" target="_blank">S12A Fig</a>).</p

Genome-wide analysis of intron retention in RES complex mutants.

<p><b>(A)</b> Schematic representation of intron features analyzed in (B) and (C). Top: Intron position. “Internal” introns correspond to all introns excluding the first two and last three introns. Bottom: NMD vs non-NMD triggering introns. Introns predicted to cause NMD upon retention introduce a premature termination codon (PTC) further than 50 nucleotides upstream of an exon-exon junction. Introns predicted not to cause NMD (noNMD) may correspond to: (i) last introns, (ii) introns in UTRs or non-coding genes, or (iii) introns that preserved the ORF upon retention (multiple of three nucleotides with no in-frame stop codons). <b>(B)</b> Box plots showing the ∆PIR of last and internal introns in the three different mutant of the RES complex. Only genes with more than 10 introns were considered for the analysis. <i>bud13</i> *** <i>P</i> = 4.37x10^-201; <i>rbmx2</i> *** <i>P</i> = 2.98x10^-202; <i>snip1</i> *** <i>P</i> = 4.76x10^-195 (Wilcoxon rank sum test). <b>(C)</b> Box plots showing the ∆PIR of introns predicted to trigger nonsense mediated decay (NMD) upon retention and those predicted not to trigger NMD (no-NMD) in the three different mutant of the RES complex. <i>bud13</i> *** <i>P</i> = 5.83x10^-208; <i>rbmx2</i> *** <i>P</i> = 4.94x10^-184; <i>snip1</i> *** <i>P</i> = 1.74x10^-183 (Wilcoxon rank sum test).</p

Evolutionary recruitment of flexible Esrp-dependent splicing programs into diverse embryonic morphogenetic processes

Epithelial-mesenchymal interactions are crucial for the development of numerous animal structures. Thus, unraveling how molecular tools are recruited in different lineages to control interplays between these tissues is key to understanding morphogenetic evolution. Here, we study Esrp genes, which regulate extensive splicing programs and are essential for mammalian organogenesis. We find that Esrp homologs have been independently recruited for the development of multiple structures across deuterostomes. Although Esrp is involved in a wide variety of ontogenetic processes, our results suggest ancient roles in non-neural ectoderm and regulating specific mesenchymal-to-epithelial transitions in deuterostome ancestors. However, consistent with the extensive rewiring of Esrp-dependent splicing programs between phyla, most developmental defects observed in vertebrate mutants are related to other types of morphogenetic processes. This is likely connected to the origin of an event in Fgfr, which was recruited as an Esrp target in stem chordates and subsequently co- opted into the development of many novel traits in vertebrates