Complete genomic structure of the human nebulin gene and identification of alternatively spliced transcripts

Abstract

The giant nebulin protein is a fundamental structural component of the thin filaments of the striated muscle sarcomere. Nebulin binds to actin and the size of nebulin correlates with actin filament length, suggesting that nebulin may determine the length of the thin filaments during myofibrillogenesis. We have previously described the genomic organization of the 3 0 end of the nebulin gene (NEB), and identified 18 different NEB mutations in patients with autosomal recessive nemaline myopathy. Here we present the genomic organization of the entire nebulin gene, and the identification of numerous alternatively spliced mRNAs. The gene comprises 183 exons spanning 249 kb of the genomic sequence. The translation initiation codon is in exon 3, and the stop codon and the 3 0 UTR are in exon 183. There are four regions with alternatively spliced exons, that is, exons 63 -66, 82-105, 143 -144 and 166 -177, giving rise to a number of different transcripts. The alternatively spliced exons 143 -144 give rise to two different transcripts varying between muscle types and between muscles of different developmental stages. The alternatively spliced exons 166 -177 express at least 20 different transcripts in adult human tibialis anterior muscle alone. Preliminary results show several transcripts in both of the two remaining alternatively spliced regions. Extensive alternative splicing of NEB may explain why nemaline myopathy patients with homozygous truncating mutations show expression of the carboxy-terminus of the nebulin protein contrary to expectations . The use of alternative transcripts might also explain why severe phenotypes are rare among patients with two truncating mutations.

Nebulin (NEB) mutations in a childhood onset distal myopathy with rods and cores uncovered by next generation sequencing

“…The heterozygous NEB mutation c.24372_24375dup (p.Val8126fs) in exon 172 is predicted to result in a frame shift and a premature stop codon. Donner et al 17 demonstrated that there are four regions of the Nebulin gene which exhibit alternative splicing. Exon 172 is located in the furthest distal of these, exons 166-177, which exhibits an extremely high degree of isoform variability in some tissues.…”

“…NEB encodes nebulin, a protein of B800 KDa. 1,2 Although the precise function of nebulin is not fully understood, observations in gene knock-out animals suggest that it has a role in the regulation of thin filament length and muscle contraction. 3 Typical NEB-related NM is of infantile-onset and static or only slowly progressive, affecting the axial, facial, bulbar and proximal limb muscles.…”

Nebulin: A Study of Protein Repeat Evolution

“…We are still confident that most are coding exons, since a region composed of 11 of these exons was identified by Donner et al in a minor splice form. 13 …”

“…Donner et al suggested that this LINE-2 could be responsible for the duplication event. 13 It should be noted that the duplicated region is not part of a major splice form and that it has only been seen in fetal transcripts. 13 Thus, the functional implications of this duplication are unclear.…”

“…In Nebulin, there is a clear SR pattern, with more than 20 SRs flanked by simple repeat nebulin domains and an SH3 domain at the C-terminus. 13 The Nebulin protein undergoes alternative splicing, which regulates its length, thereby affecting the size of muscle thin filaments. 11 NRAP (Nebulin-related anchoring protein) is the only other protein that contains nebulin SRs, although only five copies.…”