Pre-mRNA splicing is a regulated step in eukaryotic gene expression in which introns are removed from primary transcripts and exons are joined together to form mature mRNAs that are subsequently exported to the cytoplasm for translation (Braunschweig et al., 2013). To carry out the splicing reaction, exon-intron boundaries in the pre-mRNA must be correctly identified. These boundaries consist of highly-conserved splice site sequences including intronic and exonic nucleotides. Apart from the splice sites, pre-mRNAs contain exonic and intronic sequences that interact with different splicing factors to regulate how often each exon is included in the final transcript (Wang and Burge, 2008). If this process is not properly defined, transcripts encoding altered amino acid sequences or premature stop codons can be produced. Depending on the location of the premature stop codon, this results in destruction of the mRNA by nonsense- mediated mRNA decay or production of a truncated protein (Lindeboom et al., 2019). mRNA degradation, truncated protein expression, abnormal protein sequences and altered isoform ratios can all disrupt cellular functions and cause disease (Anna and Monika, 2018; Scotti and Swanson, 2016; Sterne-Weiler and Sanford, 2014). Alterations in splicing contribute to diseases ranging from autoimmune diseases (Agrebi et al., 2017) to neurodevelopmental disorders (Xiong et al., 2015) and cancer (Bonnal et al., 2020; Cancer Genome Atlas Research Network et al., 2018; Rahman et al., 2020; Rhine et al., 2018; Supek et al., 2014). Indeed, in one analysis a third of disease-causing mutations were predicted to result in aberrant splicing (Lim et al., 2011) and in another 10% of 4964 disease-causing missense mutations altered the inclusion of cassette exons when tested in an episomal mini-gene construct (Soemedi et al., 2017).

To evaluate how often random mutations alter the inclusion of exons, several groups have recently subjected exons in mini-gene constructs to deep mutational scanning (DMS) (Kinney and McCandlish, 2019). In these DMS experiments, the effects of hundreds or thousands of mutations in or around an exon are quantified in parallel by selection and sequencing. Exons subjected to DMS include FAS exon 6 (Julien et al., 2016), whose inclusion is altered in autoimmune lymphoproliferative syndrome (Ben-Mustapha et al., 2018); RON exon 11 (Braun et al., 2018), whose skipping can promote oncogenesis (Collesi et al., 1996); and WT1 exon 5 (Ke et al., 2018), whose inclusion in leukemic cells confers resistance to chemotherapy drugs (Yang et al., 2007). These studies revealed that 60–70% of all single-nucleotide substitutions and mutations in over 90% of exon positions alter exon inclusion (Braun et al., 2018; Julien et al., 2016; Ke et al., 2018). One study where the flanking introns were also mutagenized (Braun et al., 2018) further revealed that mutations in ~80% of intronic positions flanking RON exon 11 also affect its inclusion.

However, these DMS experiments were performed on exons with intermediate or low inclusion levels: in the assayed conditions, wild-type (WT) FAS exon six had a percent spliced in (PSI, the percentage of mature transcripts that include the exon) of ~50%, RON exon 11 had a PSI of ~60%, and the different versions of WT1 exon five had PSI values ranging between ~0 and~70%. In contrast, in any given human tissue, most of the expressed exons are highly-included, for example ~60% of exons with a PSI > 10% actually have a PSI > 90% (Figure 4—figure supplement 2A). Notice that although a large fraction of exons in the human genome are included at very low levels (Figure 4—figure supplement 2A), these often correspond to pseudoexons or exons that are not usually expressed and may therefore be less physiologically relevant compared to other exons.

An alternative approach to random mutagenesis is to introduce a small number of mutations into an exon but in all possible combinations. For example, we recently quantified the effects on inclusion of all 3072 possible combinations of the 12 substitutions that accumulated in FAS exon six during its evolution from a constitutive exon in the last common ancestor of primates to an alternatively spliced exon in humans (Baeza-Centurion et al., 2019). This allowed us to quantify how the effect of each of these 12 mutations changes when it is made in different closely-related versions of exon six and revealed that the effects of mutations on exon inclusion ‘scale’ non-monotonically as the inclusion of an exon is increased. Specifically, as the PSI of an exon increases from 0 to 100%, the effect of a mutation increases to a maximum and then decreases again (Figure 1A), consistent with an underlying sigmoidal relationship between an additive trait affected by mutations and the inclusion level (Baeza-Centurion et al., 2019).

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This scaling of mutational effects predicts that, although DMS experiments have revealed that many mutations have large effects on the inclusion levels of exons with intermediate PSI values, this may not be the case for exons with PSI values close to 100%. Here we first test this prediction by performing mutational scans of two exons with high inclusion levels. We find that, consistent with the scaling of mutational effects, mutations rarely substantially alter the inclusion of these exons. Analyses of additional mutagenesis datasets and human genetic variation strongly support this conclusion and show that exonic and intronic mutations – except those affecting the splice sites themselves – only rarely alter the splicing of exons with high PSI values. Mutations that alter exon inclusion are therefore very non-uniformly distributed across the human transcriptome and are focussed in and around alternative exons with intermediate inclusion levels. This provides an important framework for prioritising synonymous and other variants as candidate disease-causing mutations and for predicting from sequence when a change in splicing is – or is not – likely to be the causal molecular mechanism linking a genetic variant to a human disease.

Systematic mutagenesis of three different alternatively spliced exons with intermediate inclusion levels has revealed that mutations at most positions in exons can have substantial effects on their inclusion levels (Braun et al., 2018; Julien et al., 2016; Ke et al., 2018). Considered in isolation, this finding might suggest that changes in splicing could be one of the major mechanisms by which exonic mutations cause phenotypic variation and human disease. However, the effects of mutations on inclusion levels have recently been proposed to scale with the level of inclusion, predicting mutations will only rarely have large effects on the inclusion levels of highly-included exons (Baeza-Centurion et al., 2019). Since highly-included exons make up most of the expressed exons in the genome in any cell type (Figure 4B and Chen, 2014), this leads to the prediction that exonic mutations will actually only rarely change exon inclusion and that mutations having large effects on splicing correspond to the minority of exons included at intermediate levels in a cell type.

To test these predictions, we performed deep mutagenesis of two highly-included exons. With the exception of mutations in canonical splice sites, we found that mutations generally had little influence on the inclusion of highly-included exons. Deep mutagenesis of a third exon supports this conclusion (Souček et al., 2019) as do the effects of thousands of mutations in libraries spanning thousands of different exons (Figure 3B–D) and the associations between common human genetic variation and changes in inclusion (Figure 4A). Analyses of mutations in introns suggest that the same conclusion applies to intronic mutations (Figure 5B–E) and further analyses suggest that it is also true for trans perturbations (Figure 6). The vast majority of single-nucleotide mutations in human exons and introns should therefore be expected to have negligible effects on exon inclusion.

Our conclusions may appear to be in conflict with previous reports of exonic splicing enhancers (exonic sequences that promote higher inclusion) in highly included exons. However, we do not think this is actually the case. In these studies, enhancers were often identified by deleting or substituting entire exonic domains (Liu et al., 1998; Watakabe et al., 1993) or by using antisense oligonucleotides to block binding to an entire domain (Aartsma-Rus et al., 2006). Moreover, studies reporting strong effects of single-nucleotide changes generally involved in vitro experiments in which the splicing reaction occurs less efficiently compared to in vivo experiments (Hicks et al., 2005), so the mutated exons were never highly included. Additionally, exons were often sensitized to the effects of perturbations by surrounding these exons with the genomic sequences of less included exons (Liu et al., 1998; Schaal and Maniatis, 1999; Shiga et al., 1997; Tanaka et al., 1994; Watakabe et al., 1993). In agreement with our conclusions, a previous study showed that single and double nucleotide substitutions had little effect on enhancer activity compared with more complex mutations (Schaal and Maniatis, 1999). Highly-included exons have been shown to have a higher density of enhancers when compared with alternative exons, which presumably contributes to their high inclusion levels (Ke et al., 2011; Wang et al., 2005). However, this does not mean that individual point mutations in these elements will produce large changes in inclusion. Indeed, our results revealed that, at the same inclusion level, mutations have a stronger effect in alternative exons than in constitutive exons (Figure 7). This may be, in part, precisely due to the higher density of enhancer elements in constitutive exons, which produces sequence redundancy in these exons.

Zhang et al., 2005 have argued against enhancer redundancy, proposing instead that most or all of the existing enhancers in a given exon are necessary to reach the inclusion levels of a constitutive exon. Indeed, individual cases of single-nucleotide substitutions able to disrupt the splicing of highly-included exons have been reported (de Boer et al., 2017; Zhang et al., 2005) and also exist in our systematic mutagenesis datasets. However, systematic experiments and genetic analyses argue that these effects are not prevalent. Figure 9 provides a possible rationale to reconcile these observations. Under most conditions, the high levels of inclusion of constitutive exons create, through the scaling of mutational effects, an apparent redundancy between individual elements, ‘locking’ the exon in a state of nearly 100% inclusion. When the same exons are included at lower levels, for example due to a change in conditions, the redundancy effect disappears and individual mutations display stronger effects.

Figure 9

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The exception to this is mutations that affect splice sites, which can change the inclusion of even highly-included exons (Figure 6). Our results reveal that splice site mutations in exons that are mostly skipped can also have an inclusion-promoting effect stronger than that of mutations elsewhere in the transcript. Therefore, the activation of a cryptic splice site (a sequence that resembles a splice site but which is not recognized as such) could be a common mechanism by which distal intronic mutations affect splicing. This is often the case with Alu elements (retrotransposons that make up over 10% of the human genome). When inserted in an antisense orientation, an Alu element contains a number of sequences resembling splice sites (and are therefore cryptic splice sites). Such an Alu element could therefore be thought of as an exon included at 0%. Mutations in these cryptic splice sites have been shown to activate them, resulting in the insertion of a new exon in the mature transcript and causing disease (Ule, 2013). Other distal intronic mutations have been shown to cause disease using a similar mechanism. For example, a mutation that strengthens a cryptic splice site was shown to activate a silent exon in the CAPN3 gene, resulting in limb-girdle muscular dystrophy type 2A (Blázquez et al., 2013). Indeed, the activation of cryptic splice sites is one of the most common mechanisms by which distal intronic mutations affect splicing (Vaz-Drago et al., 2017).

The small effects of mutations in highly-included exons have implications for the evolution of alternative splicing. The evolution of lower inclusion levels is likely to involve multiple mutations with each of these mutations individually seeming to have no or little effect. This suggests that the evolution of alternative splicing is often likely to initially involve neutral drift until the splicing efficiency of an exon drops enough for mutations to have a noticeable effect on inclusion, allowing skipping-promoting mutations to be selected for or against, if they have a fitness impact. For example, in our recent study a single mutation was never sufficient to decrease the inclusion of the ancestral FAS exon six to the inclusion levels of the human exon. Indeed only 8% of double mutants, 11% of triple mutants and 19% of genotypes containing 10 mutations reached the inclusion levels of the human exon (Baeza-Centurion et al., 2019).

Sequencing clinical samples frequently returns novel alternative alleles in known or candidate disease genes whose effects on disease are unclear (ClinGen et al., 2015). A major challenge in human genetics is to determine whether such variants of unknown significance (VUS) are disease-causing mutations. A second challenge is to understand the mechanisms by which alternative alleles cause disease. Our results suggest that VUS are most likely to alter exon inclusion, and therefore be candidate disease-causing alleles by this mechanism, if they are located in or close to an alternative exon with intermediate levels of inclusion, or in a splice site.

In summary, our results show that very few mutations in both exons and proximal intronic regions typically alter the inclusion of highly-included exons and even fewer impact the inclusion of constitutive exons. Indeed, the distribution of inclusion-altering mutations will be very non-uniform across the genome, focussed around alternatively spliced exons with intermediate levels of inclusion. The exception to this is mutations in splice sites, which can have large effects on inclusion even in highly-included exons. Exons with intermediate inclusion levels make up a minority of expressed exons in any human cell type. Thus, considering the entire human genome, most mutations in exons and introns will actually have negligible effects on exon inclusion. We therefore propose as a general rule to prioritise, as candidate causal disease mechanisms, mutations in canonical splice sites and mutations in and surrounding alternative exons with intermediate levels of inclusion.

Raw sequencing data have been submitted to GEO with accession number GSE151942. All scripts used in this study are available at https://github.com/lehner-lab/Constitutive_Exons (copy archived at https://archive.softwareheritage.org/swh:1:rev:b2e04359c9b615ee7a1b826ebaed15f073b87298/).

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Author details

1. Pablo Baeza-Centurion

   Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Belén Miñana

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8141-8075

2. Belén Miñana

   Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain

   Contribution

   Validation, Investigation, Methodology, Writing - review and editing

   Contributed equally with

   Pablo Baeza-Centurion

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7676-5788

3. Juan Valcárcel

   1. Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain
   2. Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

   Contribution

   Conceptualization, Supervision, Funding acquisition, Project administration, Writing - review and editing

   For correspondence

   juan.valcarcel@crg.eu

   Competing interests

   Reviewing editor, eLife

4. Ben Lehner

   1. Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain
   2. Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
   3. Universitat Pompeu Fabra (UPF), Barcelona, Spain

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   ben.lehner@crg.eu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8817-1124

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