IMPLICATIONS OF DIFFERENTIAL SPLICING

In eukaryotes, alternative splicing has an important role in tissue specific and developmentally regulated gene

expression [Maniatis, 1991]. Other membrane skeleton

components produce multiple transcripts from single genes, via alternative splicing and utilisation of alternative

promoters, which may reflect the multiple functions that such proteins must perform in a vast number of cell types.

It has been suggested that dystrophin and spectrin are part of a super family of cytoskeletal proteins [Davison e t al., 1988, Koenig et al., 1988, Byers et al., 1989, Luna et al., 1992 re vie w , Love et al., 1993 re view , Bennett et al., 1993 review ]. Dystrophin and spectrin have homologous amino terminal actin binding regions [Byers et al., 1989, Dubreuil

et a l, 1990], both proteins have central rod domain

comprising a structurally comparable series of repeat units [Koenig et a/., 1988a, Cross et a l, 1990, Koenig et a l, 1990] and both have a possible calcium binding site at the carboxy terminus [Koenig et al., 1988a, Moncrief et a l, 1990,

Nakayama et a l, 1992]. Spectrin itself is part of a

multigene family, an a-chain which is coded for by at least two genes and a p-chain which is coded for by at least five genes. The a-chain isoforms are of similar sizes and each comprises 22 repeats of 106 amino acids, an src-homology 3 (SH3) domain and the predicted calcium binding site at the carboxy terminus. The p-spectrins have an amino terminus with actin and protein 4.1 binding sites and 17 repeat

segments in the rod domain, but vary in size and have different carboxy termini [Bennett et a l, 1990, Kennedy e t

a l, 1991, Hu et a l, 1992].

Spectrin loci also demonstrate alternative splicing.

In fibroblasts, erythroid a-spectrin is expressed along with alternatively spliced forms. Relative to erythroid a-

spectrin, one of these isoforms has an insertion of SObp between the SH3 motif and domain 11 and another form involves the deletion of 18bp in domain 21 at the C-

terminal [Moon et a l, 1990]. Erythrocyte p-spectrin (Pr) is expressed in cardiac muscle (along with non-erythroid Per spectrin) and has several alternatively spliced isoforms in this tissue which have different subcellular localisations

[Vybiral et a l, 1992].

Many other genes have also diversified by a combination of gene duplications to give rise to mulitple loci and by differential splicing to give rise to multiple products from the same gene. For example, tropomyosin is a cytoskeletal component that closely associates with actin filaments. It is a rod-shaped dimeric molecule that lies head to tail in the groove of the actin filament [Smillie, 1979]. In muscle, tropomyosin mediates muscle contraction via calcium, troponin and actomyosin ATPase. It is also expressed in non-muscle cells and may participate in regulation of cell shape [Liu et a l, 1989]. At least five different isoforms

have been detected in nonmuscle cells named Tm1, Tm2, Tm3, Tm4 and Tm5, which are encoded by a multigene family [Matsumura et al., 1983, 1985, Lees-Miller et a!.,

1990]. In nonmuscle cells, four tropomyosin genes have been characterised with the p-T m gene coding for Tm 1, a- Tmf encoding Tm2, 3, 5a and 5b, and the Tm 4 gene encoding Tm 4 and hTmnm. in addition to aTms (which is also

transcribed in slow twitch muscle) which codes for Tm5 [Helfman et a!., 1986, Reinach et a!., 1986, MacLoed et al., 1987, Yammawaki-Kataoka et al., 1987, Clayton et al., 1988, Goodwin et al., 1991, Gunning et al., 1990, Lees- Miller et al., 1990].

The p-tropomyosin gene is 10Kb in length, has 11 exons and two isoforms transcribed from the same gene locus. Differential splicing occurs in a tissue specific manner such that exons 1 to 5, 8 and 9 are present in all species of transcript, whereas exons 6 and 11 are present in the

fibroblast and smooth muscle specific forms, with exons 7 and 10 found only in skeletal muscle specific transcripts [Guo et al., 1993].

Ankyrin gene products link the spectrin based membrane skeleton to the plasma membrane and are encoded by

members of a large multigene family [Bennett et al., 1991, review ]. The best characterised members of this family are the erythroid and brain ankyrins which are coded for by different genes, but each codes for a number of

alternatively spliced transcripts [Peters, et al., 1991b, White et al., 1992]. The erythroid ankyrin has multiple amino termini and therefore is likely to be regulated by multiple promoters. It also has alternative polyadenylation sites, a variety of small insertions and deletions which would produce proteins of identical size, but of different functions and transcripts with large deletions identified in the spleen, skeletal muscle and heart (although their

primary structure has not been determined) [Berkenmeier e t al., 1993].

Protein 4.1 was initially detected in the erythrocyte membrane skeleton and interacts with spectrin, f-actin and

transmembrane proteins giycophorin and band 3. It is thought to act as a linking molecule attaching the

cytoskeleton to the plasma membrane and so stabilizing it [Bennet, 1985, Marchesi, 1985 reviews ]. The protein 4.1 gene locus spans over 90Kb and has at least 23 exons [Huang et a!., 1993]. It also shows multiple isoforms generated by differential splicing. Isoforms have been identified in human erythroid and nonerythroid cells [Tang et al., 1988, 1990, Conboy et al., 1991] which vary in size, subcellular localization and levels of abundance. Recently, four

transcripts have been identified in human endothelial cells and in mouse four mRNA species have been detected in the embryo, five in adult brain and one in adult bone marrow, some of which were the same as those identified in human tissue. It seems that the exons that are preferentially utilised in some specific tissues are clustered into a specific function domain and this may be related to alteration of function in different tissues [Huang et al., 1993].

Most of the genes coding for cytoskeletal proteins are part of multigene families with related products being transcribed from entirely seperate loci. Gene duplication allows for diversification of both the gene promoter and the gene product, by mutation. In the case of dystrophin, the DMD locus provides a great number of alternatively spliced mRNAs and mRNAs transcribed from alternative promoters, which may provide dystrophin proteins, with the functional diversity required, without usage of a large number of separate loci. Since it seems likely that members of multigene families arise from gene duplication, it may be that the DMD locus is too large to be successfully

duplicated very often and therefore has attained functional diversity by utilisation of a number of promoters, within the same locus, with different temporal and spatial s p e c ific itie s .