Figure 1

Figure 1

Zebrafish nebulin structure and nebulin mutations. (A) Diagram of zebrafish nebulin with position of mutations. N-terminus of nebulin consists of a glutamic acid rich sequence followed by several distinctive domains that mediate interactions with tropomodulin. Central region of nebulin has a repetitive, modular structure, and, in zebrafish, is organized into 21 super-repeats (SR). The C-terminus of nebulin consists of several linker modules, a serine-rich and an SH3 domains, which mediate interactions with proteins in the Z-disk. (B) Diagram of two SR. Each SR is made up of seven simple repeats (R1-R7) and contains a conserved WLKGIGW motif (troponin/tropomyosin binding site) in R3 (star). Each simple repeat contains an actin-binding motif (SDXXYK) (dots). (C) Percent spliced in index (PSI) showing alternative splicing in nebulin. Skipping of exons 33–37 was observed only in neb mutants, whereas splicing of exons 49–73 and 107 was observed in both wt and mutant zebrafish. Several splicing events were observed at the C-terminus of nebulin. (D–K) Splicing analysis and variants identified by RNAseq in zebrafish splice mutants. Four wild-type and four mutant replicates (rep1-rep4) have been analyzed and the PSI has been calculated for each. Light and darkblue arrowheads indicate the location of the alternative splice site/s used. Exons are represented by grey rectangles, introns by thick black lines, and intron inclusion by black rectangles. (D and E) Diagram (D) and table (E) illustrating alternative splicing events in neb^hu28 mutant. One splice variant was identified in neb^hu28 (alt 1), which was generated by the usage of an alternative splice donor site located in intron 46. This caused a partial intron retention, a frameshift and the formation of a stop codon in exon 47. (F and G) Diagram (F) and table (G) illustrating two alternative splicing events in neb³⁴ mutant (alt 1 and alt 2). In the alt 1 variant, an alternative splice donor site in exon 54 is used, which resulted in skipping the last 18 base pairs from exon 54, and generation of an in-frame transcript. In the alt 2 variant, the full intron was retained. (H and I) Diagram (H) and table (I) illustrating three alternative splicing events in neb²¹ mutant (alt 1, alt 2, and alt 3). In the alt 1 and 2 variants, usage of two different alternative splice acceptor sites located in intron 105 resulted in addition of 34 base pairs and out-of-frame transcript (alt 1, light blue arrowhead), or in addition of 3 base pairs and in-frame transcript (alt 2, dark blue arrowhead). In the alt 3 variant, the full intron was retained. (J and K) Diagram (J) and table (K) illustrating two alternative splicing events in neb³⁰ mutant (alt 1 and alt 2). A new splice acceptor site was generated by the mutation, located just one base pair downstream of the original site (alt 1). This caused a frameshift and the formation of a premature stop in exon 135, which is the last exon in zebrafish nebulin. In the alt 2 variant, the full intron was retained.