The mouse adducin gene family: alternative splicing and chromosomal localization

Mouse cDNA sequences encoding α, β, and γ adducins were cloned from a mouse reticulocyte cDNA library. The purified clones contain alternatively spliced exons from all three adducin genes. In the case of α and β, the inclusion of the alternatively spliced exons results in truncated polypeptide isoforms (called α-2 and β-2). The mouse predicted amino acid sequences are compared with published rat and human sequences. For completion of this comparison, cDNA encoding the rat β-1 carboxy terminus was cloned by PCR. The carboxy terminal region containing MARCKS homology, calmodulin-binding region-2, and spectrin-actin-binding site, is conserved among α-1, β-1, and γ-1 isoforms in mouse, rat, and humans. We also report here the localization of the gene encoding γ adducin (Add3) to murine Chr 19, in a region that shows conserved synteny with human Chr 10. Received: 1 June 1999 / Accepted: 25 August 1999     

Genomic organisation and alternative splicing of mouse and human thioredoxin reductase 1 genes

Background  

Thioredoxin reductase (TR) is a redox active protein involved in many cellular processes as part of the thioredoxin system. Presently there are three recognised forms of mammalian thioredoxin reductase designated as TR1, TR3 and TGR, that represent the cytosolic, mitochondrial and novel forms respectively. In this study we elucidated the genomic organisation of the mouse (Txnrd1) and human thioredoxin reductase 1 genes (TXNRD1) through library screening, restriction mapping and database mining.     

Bioinformatics analysis of alternative splicing

Over the past few years, the analysis of alternative splicing using bioinformatics has emerged as an important new field, and has significantly changed our view of genome function. One exciting front has been the analysis of microarray data to measure alternative splicing genome-wide. Pioneering studies of both human and mouse data have produced algorithms for discerning evidence of alternative splicing and clustering genes and samples by their alternative splicing patterns. Moreover, these data indicate the presence of alternative splice forms in up to 80 per cent of human genes. Comparative genomics studies in both mammals and insects have demonstrated that alternative splicing can in some cases be predicted directly from comparisons of genome sequences, based on heightened sequence conservation and exon length. Such studies have also provided new insights into the connection between alternative splicing and a variety of evolutionary processes such as Alu-based exonisation, exon creation and loss. A number of groups have used a combination of bioinformatics, comparative genomics and experimental validation to identify new motifs for splice regulatory factors, analyse the balance of factors that regulate alternative splicing, and propose a new mechanism for regulation based on the interaction of alternative splicing and nonsense-mediated decay. Bioinformatics studies of the functional impact of alternative splicing have revealed a wide range of regulatory mechanisms, from NAGNAG sites that add a single amino acid; to short peptide segments that can play surprisingly complex roles in switching protein conformation and function (as in the Piccolo C2A domain); to events that entirely remove a specific protein interaction domain or membrane anchoring domain. Common to many bioinformatics studies is a new emphasis on graph representations of alternative splicing structures, which have many advantages for analysis.     

The impact of alternative splicing in vivo: mouse models show the way

Alternative splicing is widely believed to have a major impact on almost all biological processes since it increases proteome complexity and thereby controls protein function. Recently, gene targeting in mice has been used to create in vivo models to study the regulation and consequences of alternative splicing. The evidence accumulated so far argues for a nonredundant, highly specific role of individual splicing factors in mammalian development, and furthermore, demonstrates the importance of distinct protein isoforms in vivo. In this review, we will compare phenotypes of mouse models for alternative splicing to crystallize common themes and to put them into perspective with the available in vitro data.     

cDNA cloning of mouse myelin-associated glycoprotein: a novel alternative splicing pattern

The structures of three forms of mouse myelin-associated glycoprotein mRNAs were determined from full-length cDNA clones. Two forms of mRNAs have been reported to be different by alternate inclusion of exon 2 and 12 in rat brain. One of the three forms of clones obtained here appeared to be a novel mRNA which lacked both the exon 2 and 12 portions, although others were identical splicing patterns to those of rat. Northern blot analysis using specific probes to mRNAs with or without the exon 2 portion in normal and quaking mouse confirmed that the splicing of exon 2 and 12 occurred independently.     

Expression and alternative splicing of N-RAP during mouse skeletal muscle development

N-RAP alternative splicing and protein localization were studied in developing skeletal muscle tissue from pre- and postnatal mice and in fusing primary myotubes in culture. Messages encoding N-RAP-s and N-RAP-c, the predominant isoforms of N-RAP detected in adult skeletal muscle and heart, respectively, were present in a 5:1 ratio in skeletal muscle isolated from E16.5 embryos. N-RAP-s mRNA levels increased three-fold over the first 3 weeks of postnatal development, while N-RAP-c mRNA levels remained low. N-RAP alternative splicing during myotube differentiation in culture was similar to the pattern observed in embryonic and neonatal muscle, with N-RAP-s expression increasing and N-RAP-c mRNA levels remaining low. In both developing skeletal muscle and cultured myotubes, N-RAP protein was primarily associated with developing myofibrillar structures containing alpha-actinin, but was not present in mature myofibrils. The results establish that N-RAP-s is the predominant spliced form of N-RAP present throughout skeletal muscle development.     

Structure of mouse calpastatin isoforms: implications of species-common and species-specific alternative splicing.

Mouse calpastatin cDNAs were cloned by the method of RT-PCR using RNA isolated from myoblast C2C12 cells. Nucleotide sequencing of the isolated clones revealed an in-frame ATG codon upstream of the previously assigned translation initiation methionine. Except for the N-terminal segment, the new translatable region (domain XL) was similar to the sequence of bovine calpastatin in which domain XL was first identified. Among the isolated mouse calpastatin cDNA clones, three isoforms (mCS-a, mCS-b, and mCS-c) were identified. In domain L, mCS-b had a deletion of the region corresponding to exon 3 of the human calpastatin gene. RT-PCR analyses of various mouse tissues revealed that mCS-b was the major form and that the content of mCS-a, nondeleted form, was 5-10% in tissues including skeletal muscle, liver, brain, etc. and about 30% in the myoblast C2C12 cells. Unlike human and rat cDNAs, no other deletions were detected in mouse calpastatin domain L. Isolation of the cDNA clone of mCS-c, which lacked regions corresponding to exons 3 and 12, was obtained by chance because its expression level was under the detectable level in the mouse tissues and even in C2C12 cells.