The combinatorial control of alternative splicing in C. elegans

Abstract

Normal development requires the right splice variants to be made in the right tissues at the right time. The core splicing machinery is engaged in all splicing events, but which precise splice variant is made requires the choice between alternative splice sites-for this to occur, a set of splicing factors (SFs) must recognize and bind to short RNA motifs in the pre-mRNA. In C. elegans, there is known to be extensive variation in splicing patterns across development, but little is known about the targets of each SF or how multiple SFs combine to regulate splicing. Here we combine RNA-seq with in vitro binding assays to study how 4 different C. elegans SFs, ASD-1, FOX-1, MEC-8, and EXC-7, regulate splicing. The 4 SFs chosen all have well-characterised biology and well-studied loss-of-function genetic alleles, and all contain RRM domains. Intriguingly, while the SFs we examined have varied roles in C. elegans development, they show an unexpectedly high overlap in their targets. We also find that binding sites for these SFs occur on the same pre-mRNAs more frequently than expected suggesting extensive combinatorial control of splicing. We confirm that regulation of splicing by multiple SFs is often combinatorial and show that this is functionally significant. We also find that SFs appear to combine to affect splicing in two modes-they either bind in close proximity within the same intron or they appear to bind to separate regions of the intron in a conserved order. Finally, we find that the genes whose splicing are regulated by multiple SFs are highly enriched for genes involved in the cytoskeleton and in ion channels that are key for neurotransmission. Together, this shows that specific classes of genes have complex combinatorial regulation of splicing and that this combinatorial regulation is critical for normal development to occur.

Fig 1. Predicting direct targets of 4 diverse SFs.

(A) The SFs in this study all contain one or more RRM domain(s). In comparison to all RRM domains encoded in the C. elegans genome, the RRMs in these SFs fall into distinct clades based on sequence similarity. The scale bar refers to the length of SF proteins. (B) Direct targets of each of the 4 SFs are predicted by identifying differentially spliced exons by RNA-seq and querying surrounding sequences for presence of conserved SF binding motifs.

Fig 2. Validation of AS changes using semi-quantitative RT-PCR.

(A) Semi-quantitative RT-PCR reproduces AS changes observed by RNA-seq. Independent RNA samples isolated from L4-sorted worms were used to validate RNA-seq data. (B) Changes in PSI values in wild-type and mutant worms show strong correlation with RNA-seq data. PSI values estimated from RT-PCR data was compared to RNA-seq derived PSI values and the strength of correlation was measured using Pearson’s correlation co-efficient.

Fig 3. Overlap of AS changes in two different mec-8 mutants.

(A) PSI values of L4-staged AS events in both mec-8 mutants are strongly correlated. The PSI values of all exons that have a PSI value of 1–99% in wild-type L4 worms were plotted and their PSI values in mec-8(u218) and mec-8(u303) mutant worms were compared using Pearson’s correlation. (B) Single and multiple exon skipping events overlap in both mec-8 mutants. Illustrated are the number of exons that are differentially spliced in the two different mec-8 mutants (p < 0.05, |ΔPSI| ≥ 15%) (C) Comparison of changes in PSI values for exons that are differentially spliced in one or both mec-8 mutants. Black points denote AS events that are differentially spliced in both mutants, orange for events only differentially spliced in mec-8(u218), and red for events only differentially spliced in mec-8(u303). Pearson’s correlation was measured for all splice changes observed in either mec-8 mutant.

Fig 4. Binding motifs for SFs are enriched at splice sites surrounding differentially spliced exons.

(A) RNAcompete-derived binding specificities of each SF. Motif logos were taken from the cisBP-RNA database [68]. (B) Positional bias for conserved binding motifs found at differentially spliced sites. Among the differentially spliced exons that have a conserved motif found at the alternative exon, flanking introns, or constitutive exons, the proportions of those AS events with one or more conserved motif(s) at that region are illustrated. The motif search was restricted to 300nt from the splice site and the Caenorhabditis species multiple alignment from the UCSC Genome Browser database [82] was used to identify conserved motifs, which we define here as the presence of the motif in 2 or more species other than C. elegans. (C) Conserved binding motifs are enriched in AS events that show increased inclusion in the asd-1 mutant. This enrichment is not present if all instances of motifs (conserved and not conserved) are considered. In all comparisons, a random set of exons with PSI values matched with the affected exons was used as the control, and the data represented was the average taken from 100 randomized samples. Enrichment of AS events with motifs relative to control was tested using Fisher’s Exact Test.

Fig 5. Enrichment of shared AS changes between SF mutants.

The overlap of differentially spliced exons identified in mec-8(u218) and various other SF mutants was compared to that expected from a background of L4 AS events. A one-tailed hypergeometric test was used to calculate the significance of enrichment of overlapping exons.

Fig 6. Loss of both EXC-7 and MEC-8 show an additive effect on shared targets.

Synchronized L4 worms were subjected to RNAi on NGM plates seeded with dsRNA-expressing bacteria. GFP was used as a non-targeting control. The L4 progeny of these worms were then collected using a COPAS worm sorter, and their RNA isolated for use in these RT-PCR experiments.

Fig 7. mec-8 and exc-7 combines to regulate splicing of unc-52 exons.

(A) mec-8 and exc-7 show antagonistic regulation of exons 17–18 in unc-52 at the L1 stage. (B) Reduction of activity of both mec-8 and exc-7 show additive effects on unc-52 splicing patterns at the L1 stage. (C) Study of unc-52 splicing patterns in vivo using a bichromatic splicing reporter. The bichromatic splicing reporter was constructed using the same method as described by Norris et al. [48]. Skipping of exons leads to expression of GFP, while inclusion of exon(s) results in a frameshift that leads to readthrough of the GFP reading frame, and resulting expression of mCherry. Worms were subjected to RNAi at the L4 stage, and late L1/L2 worms were imaged. A dsRNA that targets a C. briggsae gene was used as a non-targeting control. Transgenic mec-8 mutant worms show increased inclusion of the unc-52 minigene exons in hypodermal cells, while worms treated with exc-7 RNAi show increased exon skipping. Transgenic mec-8 worms treated with exc-7 RNAi show both exon-included and exon-skipped isoforms in hypodermal cells. Examples of hypodermal cells that express both isoforms are highlighted with white arrowheads.

Fig 8. The distribution patterns of binding motifs for co-occurring SF motifs vary among different SF pairs.

(A-B) Binding motifs for EXC-7 and SUP-12, and for FOX-1/ASD-1 and EXC-7 were more likely to be conserved across Caenorhabditis species if they were found closer together. The proportions of motif pairs where both motifs were conserved across species were plotted cumulatively, where pairs of motifs with inter-motif distances less than or equal to the x-axis values were included at each point. The black horizontal line represents the proportion of conserved motifs among all considered motif pairs. (C) While the distances between EXC-7 and MEC-8 recognition motifs fall within a wide range, the relative position of the EXC-7 motif as being upstream of the MEC-8 motif is conserved in other Caenorhabditis species. For each AS event with co-occurring EXC-7 and MEC-8 binding motifs, the position of a MEC-8 recognition motif was plotted at 0 on the x-axis of the density plot and the position of the co-occurring EXC-7 motif was plotted relative to that. A positive value indicates positioning of an EXC-7 motif downstream of a MEC-8 motif, while a negative value indicates positioning of an EXC-7 motif upstream of a MEC-8 motif. Significance was calculated using a binomial test (* = p < 0.05).

Fig 9. exc-7 and mec-8 genetically interact in a fitness assay.

An average of 10 L1 worms were grown in bacteria expressing dsRNA for 5 days with GFP used as a non-targeting control. The resulting population sizes were counted using a COPAS worm sorter, and values were normalized to the GFP control and used as a proxy for fitness as previously described [102,103]. (A) A multiplicative model was used to calculate the expected fitness value for each mutant/RNAi condition, and exc-7 mutant worms subjected to mec-8 RNAi exhibit a more severe fitness defect than expected (student’s t-test). A log fitness score of 0 implies no interaction, and a negative log score implies a negative interaction. (B) Time-of-flight (TOF) and extinction (EXT) values of worm populations were also measured using the worm sorter. Lower TOF and EXT values for exc-7 mutant worms under mec-8 RNAi condition suggest a growth defect more severe than that observed in worms with loss of either exc-7 or mec-8 alone.

Fig 10. Binding motifs for multiple SFs co-occur at introns flanking alternative exons.

Example of co-occurring motifs that are conserved at introns flanking alternatively spliced exons. Sequence alignments were taken from the UCSC Genome Browser. A vertical line indicates added bases not shown, with the number of bases indicated below. Each oval represents a conserved binding motif, and only motifs that are present in introns and within 300nt of each splice site are illustrated. The alternative exon was expanded for illustrative purposes and is not drawn to scale.

Fig 11. Combinatorial effects on movement and function of the neuromuscular system.

(A) Loss of asd-1 and asd-2 results in sterile animals. Wild-type and mutant L1 worms were subjected to RNAi on NGM plates seeded with dsRNA-expressing bacteria for 4 days. GFP was used as a non-targeting control. (B) Reduction in activity of both asd-1 and asd-2 results in an increased number of paralysed worms. As in (A), L1 worms were subjected to RNAi on NGM plates and phenotypes of these worms were scored 3 days past adulthood. Worms were considered to be paralysed if the body of the worm was non-responsive to prodding with a worm pick. (C) Loss of mec-8 and exc-7 result in increased defects in synaptic transmission. Wild-type and mutant L4 worms were subjected to RNAi on NGM plates, and after 2 days, their L1 progeny were used for the drug assay. ~100 L1 worms were treated with 2mM aldicarb in a liquid assay for 3h. After 3h, their movements were scored depending on whether they exhibited wild-type-like movement similar to that observed in the no drug (DMSO only) control. The proportion of worms in each well that exhibited normal movement were plotted, with a total of 20 wells scored across 4 biological replicates. A student’s t-test was used to compare differences in proportions.