Ancestral intronic splicing regulatory elements in the SCN α gene family

Abstract

SCNα genes encode components of voltage-gated sodium channels that are crucial for generating electrical signals. Humans have ten paralogous SCNα genes, some of which contain duplicated mutually exclusive exons 5a and 5b. In reconstructing their evolutionary history, we found multiple unannotated copies of exon 5 in distant species and showed that exon 5 duplication goes back to a common ancestor of the SCNα gene family. We characterized splicing patterns of exons 5a and 5b across tissues, tumors, and developmental stages, and demonstrated that the nonsense mediated decay (NMD) system is not the major factor contributing to their mutually exclusive choice. Comparison of SCN2A, SCN3A, SCN5A, and SCN9A intronic nucleotide sequences revealed multiple Rbfox2 binding sites and two highly conserved intronic splicing regulatory elements (ISRE) that are shared between paralogs. Minigene mutagenesis and blockage by antisense oligonucleotides showed that the formation of RNA structure between ISRE promotes exon 5b skipping in SCN9A. The inclusion of exon 5b is also suppressed in siRNA-mediated knockdown of Rbfox2, which makes the collective action of RNA structure and Rbfox2 compatible with the model of a structural RNA bridge. ISRE sequences are conserved from human to elephant shark and may represent an ancient, evolutionarily conserved regulatory mechanism. Our results demonstrate the power of comparative sequences analysis in application to paralogs for elucidating splicing regulatory programs.

Keywords: RNA structure; SCN; duplication; mutually exclusive exons; splicing.

Figure 1:

Transcript architecture in the human SCNα gene family (A) Exon-exon alignment of the human SCNα genes (first nine exons are shown). Duplications of exon 5 are marked as “a” and “b”. Rectangles on the gene line indicate the annotated exons. The shaded areas connecting exons in different genes illustrate sequence homology of their amino acid sequences, hence the X-like pattern demonstrates that exons 5a and 5b are homologous. (B) The alignment of aminoacid sequences of the human SCNα exons 5a and 5b. The intensity of the green background for each amino acid in the alignment reflects its degree of similarity.

Figure 2:

Exon 5 duplications in orthologs of human SCNα genes across species. (A) SCNα genes and exon 5 duplications in vertebrates. Asterisk indicates that zebrafish have an additional whole genome duplication, hence SCN8A and SCN4A orthologs have two duplicated genes, which are not shown here. (B) SCNα genes and their exon 5 duplications in animals. Bottom panel: The alignment of translations of the Cinav1a gene intron 5 with human SCN1A exon 5b, indicating partial exon duplication in Ciona.

Figure 3:

Splicing patterns in SCNα genes. (A) Splicing patterns in human tissues. The median Ψ value of each isoform (5a, 5b, 5a5b, and skip) is shown for each tissue. The color legend applies to all panels. The median expression level of a gene is represented by the TPM value (transcripts per million). (B) Ψ values of exon 5a and 5b in human cell lines (i3Neurons and A549) in NMD inhibition experiments. KD, Ctrl, and CHX denote the knockdown, the control, and the cycloheximide treatment experiments. (C) Developmental dynamics of SCN9A exons 5a and 5b in mouse. E — embryonic; P — postnatal. RNA-seq on postnatal stages were made for organs or structures that develop from embryonic ones. The contribution of 5a5b and skip isoforms was zero and is not shown. (D) Cancer-specific splicing of exons 5a and 5b in the breast cancer (BRCA) and lung squamous cell carcinoma (LUSC) cohorts from TCGA. Statistically significant differences at the 0.1% significance level are denoted by *** (two-tailed t-test with Bonferroni correction for testing 10 TCGA cohorts).

Figure 4:

Multiple sequence alignment of intronic nucleotide sequences spanning between exons 5b and 6 in the human SCN2A, SCN3A, SCN9A, and SCN5A genes. A part of the alignment is shown (see Figures S4 and S5 for details). ‘*’ indicates columns with the same nucleotide in all sequences; # indicates columns with the same nucleotides in all but one sequence; ‘.’ indicates other columns. Insets above the gene line indicate consensus sequences (TGCATG) representing the canonical Rbfox2 binding motif.

Figure 5:

Intronic regulatory elements. (A–D) Left panels: genomic organization of exons 4–6 in the human SCN2A, SCN3A, SCN9A, and SCN5A genes, the locations of ISRE1 and ISRE2, their free energy of hybridization (ΔG), support by RIC-seq or KARR-seq (data pooled across cell lines, see Methods), 100 vertebrates conservation score by PhastCons (green track). Conserved nucleotides in multiple sequence alignments are indicated by asterisks. Right panels: RNA secondary structure formed by ISRE1 and ISRE2 binding.

Figure 6:

Experimental validation of RNA structure in the SCN9A gene and its impact on splicing. (A) The genomic organization of SCN9A exons 4–6, ISRE2 location, sequence and its corresponding ASO2. ΔG denotes the predicted base pairing energy; 10 nts is the distance between exon 5b and ISRE1. (B) RT-PCR quantification of exon 5b/5a Ψ ratio under ASO2 treatment in the Huh7 cell line: gel image of RT-PCR products (bottom) and its densitometric quantitation (top). NT (non-treated control); Con (control ASO); 5, 25, 100 refer to ASO2 LNA concentration (nM). (C) RT-qPCR quantification of exon 5b/5a Ψ ratio under ASO2 treatment in the Huh7 cell line; x-axis as in panel (B). (D) Disruptive and compensatory mutations (sequence reversal) in ISRE1 and ISRE2. wt — the wild type; m1, m2 — either ISRE1 or ISRE2 sequence is reversed as indicated by arrows; m1m2 — both ISRE1 and ISRE2 are reversed to restore base pairing. ΔG denotes the predicted base pairing energy. (E) RT-PCR quantification of exon 5b/5a Ψ ratio in single (m1 and m2) and double (m1m2) mutants. The SCN9A minigene was cloned in HEK293T cells (as in panel B). (F) RT-PCR quantification of exon 5b/5a Ψ ratio in siRNA-mediated KD of Rbfox2 (siRBFOX2) vs. KD of the luciferase gene (siLUC). (G) The proposed model of RNA bridge in Rbfox2-mediated regulation of exon 5b suppression. ‘*’ and ‘**’ denote statistically significant differences at the 5% and 1% significance levels, respectively (two-sample t-test); non-significant differences are labeled ”ns”.

Figure 7:

Common RNA structures in vertebrate SCNα genes. (A) SCNα genes, their exon 5 duplications and presence of ISRE1/ISRE2 sequences. (B) Top: a heatmap representation of the alignment of the SCN1A intron between exons 5b and 6 in elephant shark with the SCN3A introns between exons 5b and 6 in human, mouse, chicken and frog. Shades of red color indicate similarity between the aligned positions. Bottom: the alignment of the elephant shark SCN1A and human SCN3A indicates the ancient origin of ISRE1, ISRE2, and the Rbfox2 binding site. ‘*’ and ‘.’ are as in Figure 4.