Mutations primarily alter the inclusion of alternatively spliced exons
- Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Spain
- Institució Catalana de Recerca i Estudis Avançats (ICREA), Spain
- Universitat Pompeu Fabra (UPF), Spain
Figures
Figure 1 with 1 supplement
Scaling of mutation effects.
(A) Mutation-induced changes in exon inclusion (ΔPSI) depend on the initial inclusion levels (starting PSI or PSIs), but the underlying additive effect (A, which can also be interpreted as the …
Figure 1—figure supplement 1
Predicted ΔPSI distributions using mutagenesis data from different alternative exons.
(A) Predictions based on the FAS exon six dataset. (B) Predictions based on the WT1 exon five dataset in the presence of hexamer B (see Materials and methods). (C) Predictions based on the WT1 exon …
Figure 2 with 1 supplement
Deep mutagenesis of highly-included exons.
(A) The inclusion levels of FAS exon 6 (gel image adapted from Julien et al., 2016), the ancestral FAS exon 6, and PSMD14 exon 11. All inclusion levels were measured in HEK293 cells. (B) …
Figure 2—figure supplement 1
Experimental validation of PSI values determined in our DMS experiments.
(A) Experimentally-validated PSI values for a subset of variants in the ancestral FAS exon six library. (B) Experimentally-validated PSI values for a subset of variants in the PSMD14 exon 11 …
Figure 3 with 2 supplements
Across thousands of exons, exonic mutations have a stronger effect on the inclusion of exons with intermediate inclusion levels.
(A) Cartoon highlighting the major difference between a deep mutagenesis experiment and a multiplexed experiment. Deep mutagenesis assays involve the analysis of many different mutations in the same …
Figure 3—figure supplement 1
Distribution of mutation effects in different multiplexed libraries.
(A) Mutation effects in the Vex-seq library. (B) Mutation effects in the SRE library. (C) Mutation effects in the SNV library.
Figure 3—figure supplement 2
Effects of exonic mutations in the multiplexed datasets, binned by the starting PSI.
(A) Effects of exonic mutations in the Vex-seq library transfected into K562 cells. (B) Effects of exonic mutations in the SRE library inserted into an SMN1 minigene construct. The numbers above the …
Figure 4 with 4 supplements
Common alternative alleles have a stronger effect on the inclusion of exons with intermediate inclusion levels.
(A) Distribution of exonic allele effects in all exons and all tissues in the GTEx population. The data was split into 25 equally-populated bins according to the inclusion levels of each exon in the …
Figure 4—figure supplement 1
Effects of common exonic alternative alleles in different human tissues, binned by the starting PSI.
Figure 4—figure supplement 2
Distribution of exon inclusion levels in the human genome.
(A) All exons in all tissues. (B) All exons, split by tissue.
Figure 4—figure supplement 3
Distribution of genome-wide nucleotide inclusion levels.
Red histogram shows the distribution of splice site inclusion levels. (A) All nucleotides in all exons in all tissues. (B) All nucleotides in all exons, split by tissue.
Figure 4—figure supplement 4
The distribution of splice-altering effects of mutations in all human exons, divided by tissue.
Predicted distributions shown with coloured lines. The distributions of common alternative allele-associated splicing changes is overlaid (dashed black line).
Figure 5 with 4 supplements
Intronic mutations have a stronger effect on the inclusion of exons with intermediate inclusion levels.
(A) The distribution of intronic mutation effects in the RON exon 11 dataset can be converted into a distribution of effects on splicing efficiency, which can, in turn, be used to predict the …
Figure 5—figure supplement 1
Effects of intronic mutations in the multiplexed datasets, binned by the starting PSI.
(A) Effects of intronic mutations in the Vex-seq library transfected into K562 cells. (B) Effects of intronic mutations in the SRE library inserted into an SMN1 minigene construct. The numbers above …
Figure 5—figure supplement 2
Effects of common intronic alternative alleles in different human tissues, binned by the starting PSI.
Figure 5—figure supplement 3
Splice-altering effects of changing the flanking introns, binned by the initial inclusion levels.
(A) The effect of substituting the DHFR introns (the initial PSI condition) with the SMN1 introns (the final PSI condition). (B) The effect of substituting the SMN1 introns (the initial PSI …
Figure 5—figure supplement 4
Genome-wide distribution of splice-altering effects of common intronic alternative alleles.
In yellow, the distribution predicted using the RON exon 11 dataset. In black, the observed distribution in the GTEx population.
Figure 6
Splice-altering effects of a complex perturbation in trans binned by the initial inclusion levels.
(A) The effect of moving from HepG2 cells (the initial PSI condition) to K562 cells (the final PSI condition). (B) The effect of moving from K562 cells (the initial PSI condition) to HepG2 cells …
Figure 7 with 14 supplements
The effects of common alternative alleles in constitutive vs alternative exons.
(A) At the same starting PSI, the effects of skipping-promoting exonic alleles in alternative exons (blue) are stronger than in constitutive exons (black). Data summarised with loess curves and 95% …
Figure 7—figure supplement 1
The effects of common exonic alternative alleles in constitutive (black) and alternative (blue) exons in different human tissues.
Exons classified as in Figure 6.
Figure 7—figure supplement 2
The effects of common intronic alleles in constitutive (black) and alternative (blue) exons in different human tissues.
Exons classified as in Figure 6.
Figure 7—figure supplement 3
Using different thresholds to define constitutive and alternative exons.
(A) The difference in exonic mutation effects in constitutive vs alternative exons, using different PSI thresholds to define constitutive and alternative exons. The strongest difference is observed …
Figure 7—figure supplement 4
Density of ESEs in constitutive and alternative exons (included at >90% in at least one tissue).
Figure 7—figure supplement 5
Density of ESEs in constitutive and alternative exons with a PSI > 90% in each human tissue.
Figure 7—figure supplement 6
Density of ESEs in constitutive and alternative exons, accounting for splice site strength.
(A) All exons were binned into 10 groups depending on their density of exonic splicing enhancers (ESE), as in Figure 7B. Exons were further subdivided into whether they had strong or weak 3’ and 5’ …
Figure 7—figure supplement 7
Density of ESS hexamers.
(A) All exons were binned into 10 groups depending on their density of exonic splicing silencers (ESS), similar to Figure 7B. Constitutive exons (blue) were enriched in the bins with a lower density …
Figure 7—figure supplement 8
Density of suboptimal ESEs in constitutive and alternative exons (included >90% in at least one tissue).
Figure 7—figure supplement 9
Density of suboptimal ESEs in constitutive and alternative exons with a PSI > 90% in each human tissue.
Figure 7—figure supplement 10
Enrichment of ESEs in constitutive exons vs. ESE robustness (considering only alternative exons with a PSI > 90% in at least one tissue), for all exons in all human tissues.
Figure 7—figure supplement 11
Robustness of ESEs in constitutive exons vs. ESE robustness in each human tissue (considering only alternative exons with a PSI > 90% in that tissue).
Figure 7—figure supplement 12
Robustness of ESEs in constitutive exons vs robustness of ESEs in alternative exons, accounting for splice site strength and exon inclusion levels.
(A) Same analysis as Figure 7F, but further subdividing exons into those having strong or weak 3’ and 5’ splice sites. (B) Same analysis as Figure 7—figure supplement 10, but further subdividing …
Figure 7—figure supplement 13
Nucleotide distance between consecutive ESEs.
Constitutive exons are enriched among exons with ESEs closer to each other.
Figure 7—figure supplement 14
We counted the number of ESSs created upon disrupting each ESE in an exon with a point mutation (without allowing for the creation of ESEs that occupy the exact same six nucleotides as the disrupted ESE).
There is no correlation between the number of ESS created and the exon type.
Figure 8
The effects of splice site mutations.
(A) Position-dependent distribution of biophysical mutation effects in the RON exon 11 dataset. Loess curves and their 95% confidence bands are shown in red (all positions) and blue (all positions …
Figure 9
The scaling of mutational effects can create what appears to be sequence ‘redundancy’ as the exon inclusion level approaches 100 or 0%.
(A) Two exons can be included at nearly 100%, but be spliced with very different efficiencies. (B) A mutation that decreases the efficiency of splicing will have a strong effect on the inclusion of …
