High-throughput analysis revealed mutations' diverging effects on SMN1 exon 7 splicing

Abstract

Splicing-affecting mutations can disrupt gene function by altering the transcript assembly. To ascertain splicing dysregulation principles, we modified a minigene assay for the parallel high-throughput evaluation of different mutations by next-generation sequencing. In our model system, all exonic and six intronic positions of the SMN1 gene's exon 7 were mutated to all possible nucleotide variants, which amounted to 180 unique single-nucleotide mutants and 470 double mutants. The mutations resulted in a wide range of splicing aberrations. Exonic splicing-affecting mutations resulted either in substantial exon skipping, supposedly driven by predicted exonic splicing silencer or cryptic donor splice site (5'ss) and de novo 5'ss strengthening and use. On the other hand, a single disruption of exonic splicing enhancer was not sufficient to cause major exon skipping, suggesting these elements can be substituted during exon recognition. While disrupting the acceptor splice site led only to exon skipping, some 5'ss mutations potentiated the use of three different cryptic 5'ss. Generally, single mutations supporting cryptic 5'ss use displayed better pre-mRNA/U1 snRNA duplex stability and increased splicing regulatory element strength across the original 5'ss. Analyzing double mutants supported the predominating splicing regulatory elements' effect, but U1 snRNA binding could contribute to the global balance of splicing isoforms. Based on these findings, we suggest that creating a new splicing enhancer across the mutated 5'ss can be one of the main factors driving cryptic 5'ss use.

Keywords: 5′ss; SMN1; U1 snRNA; cryptic splice sites; splicing-affecting mutation.

Figure 1.

SMN1 exon 7 in the experimental system. (A) Scheme of minigene containing exon 7 of the SMN1 gene. Boxes represent exons, lines introns. Part of the first exon is shown as a nucleotide sequence, where pl corresponds to exonic sequence before barcoding and bc after barcoding. Underlined nucleotides represent restriction sites used during plasmid library construction. BglII and Hind III restriction sites are used for exon 1 replacement. During this procedure, original exon 1 is replaced with the barcoded one and NheI and AscI restriction sites are lost, which can be used to control successful barcoding. Arrows represent primers used for amplification preceding NGS. Primers fpet and rint are designed for mutant characterization (after preceding intron excision by PacI and AsiSI) and primers fpet and rpet for splicing analysis. Cryptic 5′ss are depicted including their positions. (B) Schematic representation of detected transcripts. (C) Frequency of particular transcript isoforms in wt minigenes detected using NGS. A frequency is depicted as a bar-plot, where boxes correspond to quartiles and whiskers to 0.1 and 0.9 percentiles, respectively. (D) Wt transcript profiling using agarose electrophoresis for endogenous SMN1 and a minigene. SMN1 exon 7 skipping is visible only as a very blurred band.

Figure 2.

The effect of single nucleotide substitutions on splicing. A diagram depicts frequencies of ES (at the upper panel) and authentic 5′ss usage (at the lower panel) in transcripts containing SMN1 gene exon 7 of all analysed mutations as a heat map, where red and blue colours represent aberrant transcripts (ES at the upper panel and usage of cryptic and de novo 5′ss at the lower panel). Mutations are positioned in the panel according to their position inside the mutated region (depicted along the x-axis with marked wt nucleotide) and mutation (y-axis). Black colour in the panel represents wt nucleotides, green colour non-analysed mutations. Checkmark indicates a statistically significant decrease in the frequency of appropriate transcript variants (P < 0.01) and asterisks mutations with a statistically significant decrease of frequency above the threshold of 1%.

Figure 3.

Proportion of transcripts in mutants causing de novo and cryptic 5′ss usage. Exon skipping (ES), exon inclusion (EI; normal splicing isoform only), cryptic 5′ss (Cr1, 2, 3) and de novo 5′ss (Dn) splicing variants frequencies are depicted. Corresponding statistics, incl. standard deviations and p-values for differences among mutations, is available in Supplementary Table S1.

Figure 4.

Comparison of transcript levels in different cells. The results were acquired with standard minigene assay and exon skipping (ES) and exon inclusion (EI) rates were measured using capillary electrophoresis.

Figure 5.

The effect of chosen double mutations on splicing. A significant decrease in exon inclusion was observed in variants containing one mutation revealed among single mutations affecting splicing (A) or in variants where no mutation affected splicing (B). For comparison, appropriate single mutations were also included (grey column in A). To predict which region is responsible for predominant ES, the number of mutations in exon positions contributing to enhanced ES were counted (C). Error bars represent SD.

Figure 6.

RNA structures of studied RNA duplexes from molecular dynamics simulations after 1 microsecond (A). The structures contain one strand from 5ʹ-mRNA and a second one from 5ʹ-U1 snRNA. Time development of Rmsd values for these systems (B). The plots reveal RNA conformation stability in the course of the simulations, i.e. small Rmsd values indicate good agreement with the initial A-RNA geometry (e.g. systems e+2U>C, A, G), larger Rmsd values indicate RNA conformation instability (e.g. e+1G>C,A,U). Instabilities in these models can be interpreted as unsuccessful duplex formation events.

Figure 7.

Predicting SRE motifs in SMN1 exon7 variants containing intronic 5′ss mutations. HEXplorer score and ESRseq score were calculated for all single and double mutations identified in the NGS experiment and plotted against the frequency of all transcripts using cryptic 5′ss to disclose the potential dependence of cryptic 5′ss usage on splicing regulators binding across former authentic 5′ss and 3ʹ end of exon 7. HEXplorer scores and ESRseq scores were counted for these mutations’ positions for every mutation or mutation combination separately and in the case of double mutants, the average values of both scores for the position of these mutations were counted. HEXplorer score (A) and ESRseq score (B) of wt variant was subtracted from corresponding mutations scores (mut-wt) for all available mutations to reflect local SRE changes.

Figure 8.

Modifying effect of exonic mutations on HEXplorer and ESRseq scores of intronic mutants. To elucidate how exonic mutations modify the effect of intronic mutations in e+1, e+2 and e+3 positions, HEXplorer scores (A) and ESRseq (B) scores of double mutants were compared with the appropriate single intronic mutants (double mut – single mut) and plotted against changes in cryptic 5′ss frequency in comparison with this frequency determined for appropriate single intronic mutant. The correlation shows the diversifying effect of exonic mutations on intronic mutations caused by SRE motif creation.