KIF1A, an axonal transporter of synaptic vesicles, is mutated in hereditary sensory and autonomic neuropathy type 2

Hereditary sensory and autonomic neuropathy type II (HSANII) is a rare autosomal-recessive disorder characterized by peripheral nerve degeneration resulting in a severe distal sensory loss. Although mutations in FAM134B and the HSN2 exon of WNK1 were associated with HSANII, the etiology of a substantial number of cases remains unexplained. In addition, the functions of WNK1/HSN2 and FAM134B and their role in the peripheral nervous system remain poorly understood. Using a yeast two-hybrid screen, we found that KIF1A, an axonal transporter of synaptic vesicles, interacts with the domain encoded by the HSN2 exon. In parallel to this screen, we performed genome-wide homozygosity mapping in a consanguineous Afghan family affected by HSANII and identified a unique region of homozygosity located on chromosome 2q37.3 and spanning the KIF1A gene locus. Sequencing of KIF1A in this family revealed a truncating mutation segregating with the disease phenotype. Subsequent sequencing of KIF1A in a series of 112 unrelated patients with features belonging to the clinical spectrum of ulcero-mutilating sensory neuropathies revealed truncating mutations in three additional families, thus indicating that mutations in KIF1A are a rare cause of HSANII. Similarly to WNK1 mutations, pathogenic mutations in KIF1A were almost exclusively restricted to an alternatively spliced exon. This study provides additional insights into the molecular pathogenesis of HSANII and highlights the potential biological relevance of alternative splicing in the peripheral sensory nervous system.     

Differential expression and developmental regulation of a novel alpha-dystrobrevin isoform in muscle

Alpha-dystrobrevin is a dystrophin-related protein expressed primarily in skeletal muscle, heart, lung and brain. In skeletal muscle, alpha-dystrobrevin is a component of the dystrophin-associated glycoprotein complex and is localized to the sarcolemma, presumably through interactions with dystrophin and utrophin. Alternative splicing of the alpha-dystrobrevin gene generates multiple isoforms which have been grouped into three major classes: alpha-DB1, alpha-DB2, and alpha-DB3. Various isoforms have been shown to interact with a variety of proteins; however, the physiological function of the alpha-dystrobrevins remains unknown. In the present study, we have cloned a novel alpha-dystrobrevin cDNA encoding a protein (referred to as alpha-DB2b) with a unique 11 amino acid C-terminal tail. Using RT PCR with primers specific to the new isoform, we have characterized its expression in skeletal muscle, heart, and brain, and in differentiating C2C12 muscle cells. We show that alpha-DB2b is expressed in skeletal muscle, heart and brain, and that exons 12 and 13 are alternatively spliced in alpha-DB2b to generate at least three splice variants. The major alpha-DB2b splice variant expressed in adult skeletal muscle and heart contains exons 12 and 13, while in adult brain, two alpha-DB2b splice variants are expressed at similar levels. This is consistent with the preferential expression of exons 12 and 13 in other alpha-dystrobrevin isoforms in skeletal muscle and heart. Similarly, in alpha-DB1 the first 21 nucleotides of exon 18 are preferentially expressed in skeletal muscle and heart relative to brain. We also show that the expression of alternatively spliced alpha-DB2b is developmentally regulated in muscle; during differentiation of C2C12 cells, alpha-DB2b expression switches from an isoform lacking exons 12 and 13 to one containing them. We demonstrate similar developmental upregulation of exons 12, 13, and 18 in alpha-DB1 and of exons 12 and 13 in alpha-DB2a. Finally, we show that alpha-DB2b protein is expressed in adult skeletal muscle, suggesting that it has a functional role in adult muscle. Together, these data suggest that alternatively spliced variants of the new alpha-dystrobrevin isoform, alpha-DB2b, are differentially expressed in various tissues and developmentally regulated during muscle cell differentiation in a fashion similar to that previously described for alpha-dystrobrevin isoforms.     

Structure and evolution of the alternatively spliced fast troponin T isoform gene.

The vertebrate fast skeletal muscle troponin T gene, TnTf, produces a complexity of isoforms through differential mRNA splicing. The mechanisms that regulate splicing and the physiological significance of TnTf isoforms are poorly understood. To investigate these questions, we have determined the complete sequence structure of the quail TnTf gene, and we have characterized the developmental expression of alternatively spliced TnTf mRNAs in quail embryonic muscles. We report the following: 1) the quail TnTf gene is significantly larger than the rat TnTf gene and has 8 non-homologous exons, including a pectoral muscle-specific set of alternatively spliced exons; 2) specific sequences are implicated in regulated exon splicing; 3) a 900-base pair sequence element, composed primarily of intron sequence flanking the pectoral muscle-specific exons, is tandemly repeated 4 times and once partially, providing direct evidence that the pectoral-specific TnT exon domain arose by intragenic duplications; 4) a chicken repeat 1 retrotransposon element resides upstream of this repeated intronic/pectoral exon sequence domain and is implicated in transposition of this element into an ancestral genome; and 5) a large set of novel isoforms, produced by regulated exon splicing, is expressed in quail muscles, providing insights into the developmental regulation, physiological function, and evolution of the vertebrate TnTf isoforms.     

Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane Ca2+ pump.

The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree. The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b. In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site. The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site. The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2. A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency. Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms.     

Four fibroblast tropomyosin isoforms are expressed from the rat alpha-tropomyosin gene via alternative RNA splicing and the use of two promoters

cDNA clones encoding four rat tropomyosin isoforms, termed TM-2, TM-3, TM-5a, and TM-5b, were isolated and characterized. All are derived from the alpha-tropomyosin gene via alternative RNA processing and the use of two alternate promoters. The cDNA sequences predict that TM-2 and TM-3 both contain 284 amino acids and differ from each other only at an internal region of the protein from amino acids 189 through 213, due to alternative splicing of exons 6a and 6b. TM-5a and TM-5b both contain 248 amino acids and differ from each other only at an internal exon encoding amino acids 153 through 177, also due to alternative splicing of exons 6a and 6b. The differences in the amino acid sequence encoded by these alternate exons affects the theoretical actin-binding pattern of the tropomyosins, such that TM-5b is expected to bind actin with greater affinity than TM-5a. TM-2 and TM-3 are transcribed from the upstream promoter, and TM-5a and TM-5b are transcribed from an internal promoter. In addition, all four isoforms contain the identical COOH-terminal coding region. RNA protection analyses revealed that the mRNA for each isoform is expressed in a number of different tissues and cell types, although the expression of some isoforms is restricted to particular cell types. Furthermore, the expression of mRNA encoding these isoforms was found to be altered in a number of different virally transformed cell lines. The changes in the expression of tropomyosin mRNAs in transformed cells reflect changes in the relative use of the two promoters, as well as the relative use of alternatively spliced exons 6a and 6b.