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.2015 Sep 30;43(17):8488-501.
doi: 10.1093/nar/gkv763. Epub 2015 Aug 10.

Widespread alternative and aberrant splicing revealed by lariat sequencing

Affiliations

Widespread alternative and aberrant splicing revealed by lariat sequencing

Nicholas Stepankiw et al. Nucleic Acids Res..

Abstract

Alternative splicing is an important and ancient feature of eukaryotic gene structure, the existence of which has likely facilitated eukaryotic proteome expansions. Here, we have used intron lariat sequencing to generate a comprehensive profile of splicing events in Schizosaccharomyces pombe, amongst the simplest organisms that possess mammalian-like splice site degeneracy. We reveal an unprecedented level of alternative splicing, including alternative splice site selection for over half of all annotated introns, hundreds of novel exon-skipping events, and thousands of novel introns. Moreover, the frequency of these events is far higher than previous estimates, with alternative splice sites on average activated at ∼3% the rate of canonical sites. Although a subset of alternative sites are conserved in related species, implying functional potential, the majority are not detectably conserved. Interestingly, the rate of aberrant splicing is inversely related to expression level, with lowly expressed genes more prone to erroneous splicing. Although we validate many events with RNAseq, the proportion of alternative splicing discovered with lariat sequencing is far greater, a difference we attribute to preferential decay of aberrantly spliced transcripts. Together, these data suggest the spliceosome possesses far lower fidelity than previously appreciated, highlighting the potential contributions of alternative splicing in generating novel gene structures.

© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Figure 1.
Figure 1.
Intron lariat sequencing defines splicing patterns. (A) Image of two-dimensional gel electrophoresis of RNA isolated fromΔdbr1 S. pombe. Intron lariats (red-bounded region) were isolated and used as source material for sequencing. (B) Pie-chart summarizing allocation of lariat sequencing reads to indicated genomic regions. (C) Illustration of intron lariat and splice sites, depicting intron-mapping reads (in black) and branch-spanning reads (in red-orange). (D) Schematic of alignment strategy for candidate branch-spanning reads, together with illustration of aligned branch-spanning read. (E) Histogram of annotated introns counts (y-axis) separated by length (x-axis, 20 nucleotide bins), indicating introns recovered with intron-mapping reads (blue) and those not recovered (red). (F) Histogram of annotated introns counts (y-axis) separated by length (x-axis, 20 nucleotide bins), indicating introns precisely recovered with branch-spanning reads (blue) and those not recovered (red). (G) Intron-mapping reads (y-axis indicates read density) aligning (x-axis indicates alignment position) to indicatedS. pombe pre-mRNAs, together with branch-spanning reads (orange-red) aligned with split-read mapping strategy. Thebtf3 peak density truncated at +/-50 nt of intron boundaries. Theppk9 peak tapers just upstream of intron boundaries.
Figure 2.
Figure 2.
Global analysis of alternative and novel splice sites inS. pombe. (A–E) Distribution of splice-site strengths as boxplots (x-axis indicates PWM scores), together with examples of alternative site sequences. (A) Splice site scores (5′SS and BP) corresponding to annotated introns (annot.), and sites corresponding to annotated introns recovered with branch-spanning reads (recov.) (B) BP scores for indicated categories of alternative splicing events associated with alternative BPs that are paired with annotated 3′SSs. (C) Splice site scores (5′SS and BP) corresponding to alternative splice site scores partitioned into upstream and downstream alternative intron boundary sites, compared to annotated and recovered sites (annot. recov.). (D) Splice site scores (5′SS and BP) corresponding to splice site scores associated with exon-skipping events partitioned into sites participating in exon-skipping (used site) and those skipped (skipped site), compared to annotated and recovered sites (annot. recov.). (E) Splice site scores (5′SS and BP) corresponding to sites found in novel introns in indicated genomic regions, compared to annotated and recovered sites (annot. recov.).
Figure 3.
Figure 3.
Cross validation and comparisons of alternative splicing detected using RNAseq and lariat sequencing. (A) Venn diagram illustrating alternative 5′SSs identified by RNAseq, lariat sequencing, or both. (B) Web-logo comparisons of annotated 5′SSs compared to those identified by RNAseq, lariat sequencing, or both. (C) Intron length comparisons of annotated introns compared to those identified by RNAseq, lariat sequencing, or both. (D) qRT-PCR measurements of relative lariat levels compared for pairs of lariats from two-intron genes (blue), compared to RNAseq determinations of exon-exon junction reads for the corresponding splice junctions (SPAC1952.04c labeled as 1952). (E) Boxplots indicating distributions of fold ratios (y-axis) of branch-spanning read counts for pairs of introns from multi-intron genes, compared to ratios of exon-exon spanning read counts for splice junctions from multi-intronic genes with comparably sized intron lengths. (F) Scatter-plot of values shown in (e), relating RNAseq-derived ratios (y-axis) to lariat sequencing-derived ratios (x-axis) for genes whose introns are of comparable size.
Figure 4.
Figure 4.
Cross validation and comparisons of novel introns detected using RNAseq and lariat sequencing. (A) Venn diagram illustrating novel introns identified by RNAseq, lariat sequencing, or both. (B) Boxplot distributions of 5′SS strength (y-axis) for 5′SSs corresponding to alternative sites recovered using lariat sequencing (alt. recov.) for annotated introns, or for novel introns identified using: lariat sequencing (lar. seq); RNAseq; or both. (C) Boxplot distributions of BP sequence strength (y-axis) for categories indicated in (B). (D) Boxplot distributions of intron length (y-axis) for categories indicated in (B).
Figure 5.
Figure 5.
Extent of alternative splicing inS. pombe. (A) Percentage of reads corresponding to alternative splice products detected by RNAseq or lariat sequencing (lar. seq), and in lariat sequencing restricting the analysis to introns for which the annotated intron has a size optimal for detection by lariat sequencing (lar. seq opt. size; 70–150 nucleotides). (B) total RNAseq RPKM values (y axis) for intron containing transcripts separated into expression quartiles (x axis). (C) Percentage of reads corresponding to alternative splice products detected by RNAseq (y axis), shown by expression quartiles (x axis). (D) Inferred rate of alternative splicing, modeled using RNAseq data, shown by expression quartiles (x axis). (E) Percentage of reads corresponding to alternative splice products detected by lariat sequencing, shown by expression quartiles (x axis). (F) Boxplot distributions of 5′SS strength (y-axis) for upstream and downstream alternative 5′SSs for each expression quartile (x-axis; recovered), and for best-scoring upstream and downstream candidate sites whose usage was not observed (not recovered).
Figure 6.
Figure 6.
Comparative analyses ofS. pombe splice sites. (A) Cladogram illustratingSchizosaccharomyces species included in subsequent analyses. (B) Heat map and boxplots indicating relationship and distributions of annotated 5′SSs inS. pombe (x-axis) andS. octosporus (y-axis). (C) Recovered annotated intron BP sequences, plotted as in (B). (D) Counts of alternative 5′SSs inS. pombe exceeding a score of zero, together with counts for sites conserved in indicated species. (E–H) Comparison of 5'SS betweenS. pombe andS. octosporus for: alternate upstream 5′SSs (E), alternate downstream 5'SS (F), control upstream 5′SSs (G) and control downstream 5'SSs (H); plotted as in (B). (I–L) Comparison of BPs betweenS. pombe andS. octosporus for: alternate upstream BPs (I), alternate downstream BPs (J), control upstream BPs (K) and control downstream BPs (L); plotted as in (B).
Figure 7.
Figure 7.
Influence of splice site strength on frequency of alternative splicing. (A–D) Scatter-plots of proportion of alternative splicing events found in RNAseq data (x-axis) plotted against predicted strength of alternative 5′SS (y-axis). Alternative events analyzed separately for upstream (A,B) and downstream alterative 5′SSs (C,D), using RNAseq from wild-type (A,C) and NMD-deficient strains (B,D).
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