Spliceosomal component PRP-40 is a central regulator of microexon splicing

Figures

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  Figure 1.. Spliceosomal component PRP-40 is required for cell-specific alternative splicing of unc-16/JIP3 (A) Gene model for unc-16, with alternative cassette exon highlighted in blue and dotted box indicating region cloned for minigene reporter. Not to scale (introns shortened). (B) Two-color fluorescent splicing reporter. Cassette exon (in blue) is engineered to encode a +1 nt frameshift, so that exon skipping will produce an in-frame GFP followed by a stop codon, but exon inclusion will shift GFP out of frame and without stop codons, followed by RFP translation in frame. (C) Whole-animal visualization of unc-16 splicing reporter in the nervous system. Splicing pattern is invariant between wild-type animals (n > 20). Scale bar, 50 μm. (D) Schematic of the splicing reporter across the entire nervous system. Dotted box indicates region of the ventral nerve cord examined in (E)–(G). (E–G) Motor neurons of the ventral nerve cord in wild-type, prp-40(−), and prp-40(−) + full-length prp-40 fosmid rescuing transgene. In wild-type conditions, excitatory neurons express only the included isoform, but in prp-40(−) they express both isoforms. Splicing patterns are invariant among individuals of a…

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  Figure 2.. PRP-40 genetically interacts with RNA binding protein exc-7/ELAV to control neuronal splicing of unc-16 (A) Schematic of unc-16 splicing reporter. Boxed region indicates nerve cord visualized throughout the figure. (B–L) unc-16 splicing reporter in motor neurons of the ventral nerve cord. Note that cell identity and numbers do not change in prp-40 mutants (see also Figures S2B and S2C). Scale bar, 10 μm. In wild-type (B), excitatory motor neurons express only the included isoform, while inhibitory motor neurons express both isoforms. In either unc-75 (D) or exc-7 (E) RNA binding protein mutants, excitatory motor neurons express both isoforms. prp-40 (C) mutants likewise express both isoforms in excitatory motor neurons (as negative control, same image as Figure 1F). Both unc-75; exc-7 (F) and unc-75; prp-40 (G) double mutants completely lose exon inclusion in all neurons, while exc-7; prp-40 (H) double mutants resemble either single mutant. (I) Genetic model of unc-16 splicing showing prp-40 in same genetic pathway as exc-7, parallel to unc-75. (L) Whole animal RT-PCR mirrors the results from cell-specific splicing reporters. (K) exc-7/+; +/prp-40 resemble either homozygous…

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  Figure 3.. PRP-40 regulates alternative splicing, but not constitutive splicing, transcriptome-wide (A) Sashimi plot of RNA-seq data confirming unc-16 cassette exon undergoes loss of inclusion in prp-40(−), but flanking introns and exons are spliced normally. (B) Percentage spliced in (PSI) values for the unc-16 cassette exon obtained from RNA-seq triplicates. (C) All significant (|ΔPSI| ≥ 20, q < 0.01) splicing differences between wild-type and prp-40(−), grouped by type of event. Cassette exons are the most dysregulated type of splicing event. (D) Among all detected cassette exons (at least five junction spanning reads for each isoform), PRP-40 regulates 16%. (E) Among PRP-40-regulated cassette exons (|ΔPSI| ≥ 20, q < 0.01), the majority (94%) exhibit decreased inclusion in the absence of PRP-40. (F) Volcano plot of all cassette exons detected. Colored data points indicate |ΔPSI| ≥ 20 and q < 0.005. (G) Sashimi plot of sad-1 33 nt cassette exon showing complete loss of exon inclusion in prp-40(−). (H) PSI values for the sad-1 cassette exon obtained from RNA-seq triplicates. (I) sad-1 RT-PCR further confirms that exon inclusion is strongly downregulated in prp-40(−). Internal primer within…

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  Figure 4.. PRP-40 is a central regulator of microexon splicing (A) Percentage of cassette exons affected by prp-40(−) binned by size: 1–27 nt (microexons), 28–50 nt (small exons), and ≥51 nucleotides (conventionally sized exons), at either Δ15 or Δ30 PSI (q < 0.01). (B) Magnitude (ΔPSI) of change between wild-type and prp-40(−) for exons binned by exon size. Whiskers represent 5th and 95th percentiles. (C–E) PSI values for each exon in wild-type and in prp-40(−) for microexons (C), small exons (D), and conventional exons (E). (F) Sashimi plot showing complete loss of a 9 nt microexon in the unc-13 gene in a prp-40 mutant. (G) PSI values for the unc-13 microexon obtained from RNA-seq triplicates. (H) In vivo splicing reporter for unc-13 microexon shows that in wild-type animals, the microexon is included in many neurons, and skipped in many others, with few neurons expressing both isoforms simultaneously. In prp-40(−), all neurons lose expression of the included isoform. Splicing patterns are invariant within a given genotype (n ≥ 7 biological replicates). Scale bar, 10 μm. (I) RT-PCR confirms that the unc-13 microexon is lost in prp-40(−). See also Figure S4.

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  Figure 5.. PRP-40-regulated microexons define a network of neuronal transcripts (A) Gene Ontology biological process analysis for genes containing PRP-40-regulated microexons (|ΔPSI| ≥ 20, q < 0.01) visualized by Enrichment Map plugin for Cytoscape. (B) Gene Ontology cellular component analysis on genes containing PRP-40-regulated microexons. (C) Histogram of PRP-40-regulated microexon size. Microexons with sizes divisible by 3 indicated in orange. (D) A substantial minority of PRP-40-regulated microexons < 30 nt (30%) are previously unannotated, and a small number (4%) are annotated as constitutive exons. (E) prp-40(−) L3-stage animals have defects in locomotion, as measured by rate of thrashing in liquid (M9 buffer). n = 20 animals per genotype. (F) prp-40(−) L3-stage animals are resistant to 0.2 mM aldicarb. n = 20 animals per genotype per replicate. Statistical test; paired t test with unequal variance and one way ANOVA test. Mean ± SEM (*p < 0.001).

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  Figure 6.. Molecular mechanisms underlying PRP-40-mediated microexon inclusion (A) Analysis of motif enrichment using MEME suite reveals no strongly enriched splicing factor cis-elements flanking PRP-40-regulated microexons but finds motifs (adjusted p = 0.07) for exc-7 and asd-1. (B) RNA-seq analysis shows that exc-7, fox-1, and asd-1 all regulate a small subset of PRP-40-regulated microexons, but no single splicing factor plays a dominant role. (C) unc-13 microexon was expanded into a 55 nt “conventionally sized” exon. (D) Inclusion of the unc-13 9 nt microexon is completely dependent on PRP-40, as shown in more detail in Figure 4H. (E) Increasing the size of the unc-13 microexon does not substantially alter its cell-specific splicing pattern but completely relieves its dependence on PRP-40. Splicing patterns are invariant among individuals of a given genotype (n ≥ 7). (F) RT-PCR confirms that the unc-13 microexon, but not the engineered “macroexon,” is completely dependent on PRP-40. (G) Both exceptional microexons that are independent of PRP-40 have unusually short introns. (H) The magnitude of splicing dysregulation (ΔPSI) of small exons upon loss of PRP-40 depends on the size of the…

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  Figure 7.. Mammalian PRPF40A regulates microexon splicing (A) PRPF40A siRNA knockdown caused increased skipping in seven of ten of microexons tested, detected by RT-PCR. (B) Three of ten microexons did not undergo appreciable increase in skipping. (C) Summary of RT-PCRs indicating fraction of microexons or conventionally sized exons regulated by PRPF40A. (D) Model for PRP-40-mediated regulation of microexons. For conventionally sized exons, spliceosomal components initially assemble and interact across an exon via exon definition, before subsequently remodeling to splice across the intron. Exon definition is sterically infeasible across microexons because of the prohibitively small sequence causing steric interference between spliceosomal components. Instead, PRP-40 facilitates intron definition across long introns through its protein-protein interactions between the U1 snRNP at the 5′ splice site and the BBP and the branchpoint. See also Figure S6.