The CD44 molecule is known to display extensive size heterogeneity, which has been attributed both to alternative splicing and to differential glycosylation within the exracellular domain. Although the presence of several alternative exons has been partly inferred from cDNA sequencing, the precise intron-exon oration of the CD44 gene has not been described to date to our knowledge. In the present study we describe the structure of the human CD44 gene, which contains at least 19 exons s ning some 50 kiobases of DNA. We have identified 10 alternatively spliced exons within the extracellular domain, including 1 exon that has not been previously reported. In addition to the cluson or excusion of whole exons, more diversity is generated through the uztion of internal splice donor and acceptor sites within 2 of the individual exons. The variation previously reported for the cytoplasmic domain is shown to result from the alternative splicing of 2 exons. The genomic structure of CD44 reveals a remarkable degree of complexity, and we confirm the role of alternative splicing as the basis of the structural and functional diversity seen in the CD44 molecule.The human CD44 glycoprotein [Pgp-1 (1), HCAM (2), Hermes antigen (3), ECMR III (4)] has been proposed to function as a lymph node homing receptor on circulating lymphocytes. Expressed on a wide range of different tissues, the CD44 molecule also binds the extracellular matrix components hyaluronic acid (5), fibronectin (6), and collagen (4) as well as the cytoskeletal protein ankyrin (7). Several antibodies recognizing CD44 have been shown to induce lymphocyte activation (8,9) and to inhibit lymphopoiesis (10). In addition, CD44 can mediate both homotypic and heterotypic cell adhesion (11,12). The many functional roles of this molecule may relate to its considerable size heterogeneity, which cannot be accounted for simply by differences in glycosylation (5,13,14).Recently, we and others have isolated a number of different isoforms of CD44 (15-18). These have been characterized by cDNA sequencing and appear to arise by alternative splicing in two different regions, the membrane proximal extracellular domain and the cytoplasmic tail. Some of this variation has been shown to produce functional changes in the molecule. For example, the presence ofthe 396-base-pair (bp) insert in the epithelial variant of CD44 reduces the affinity for hyaluronic acid (5). Study ofthese cDNA variants has given some insight into the genomic organization of CD44 but has not allowed the precise characterization of the number and boundaries of exons that encode the variant region of the molecule.In the present paper we have cloned the gene for CD44 from a human yeast artificial chromosome (YAC) and characterized its genomic structure.* These studies reveal a remarkable degree of complexity within the structure of the CD44 gene and demonstrate conclusively the role of alternative splicing in generating the structural heterogeneity that is characteristic of the CD44 molecule. MATERIALS AND M...

SR proteins have a characteristic C‐terminal Ser/Arg‐rich repeat (RS domain) of variable length and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre‐mRNA splicing factors. We have cloned a cDNA encoding a novel human SR protein designated SRp30c, which has an unusually short RS domain. We also cloned cDNAs encoding the human homologues of Drosophila SRp55/B52 and rat SRp40/HRS. Recombinant proteins expressed from these cDNAs are active in constitutive splicing, as shown by their ability to complement a HeLa cell S100 extract deficient in SR proteins. Additional cDNA clones reflect extensive alternative splicing of SRp40 and SRp55 pre‐mRNAs. The predicted protein isoforms lack the C‐terminal RS domain and might be involved in feedback regulatory loops. The ability of human SRp30c, SRp40 and SRp55 to modulate alternative splicing in vivo was compared with that of other SR proteins using a transient contransfection assay. The overexpression of individual SR proteins in HeLa cells affected the choice of alternative 5′ splice sites of adenovirus E1A and/or human beta‐thalassemia reporters. The resulting splicing patterns were characteristic for each SR protein. Consistent with the postulated importance of SR proteins in alternative splicing in vivo, we demonstrate complex changes in the levels of mRNAs encoding the above SR proteins upon T cell activation, concomitant with changes in the expression of alternatively spliced isoforms of CD44 and CD45.

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Although the splicing of transcripts from most eukaryotic genes occurs in a constitutive fashion, some genes can undergo a process of alternative splicing. This is a genetically economical process which allows a single gene to give rise to several protein isoforms by the inclusion or exclusion of sequences into or from the mature mRNA. CD44 provides a unique example; more than 1,000 possible isoforms can be produced by the inclusion or exclusion of a central tandem array of 10 alternatively spliced exons. Certain alternatively spliced exons have been ascribed specific functions; however, independent regulation of the inclusion or skipping of each of these exons would clearly demand an extremely complex regulatory network. Such a network would involve the interaction of many exon-specific trans-acting factors with the pre-mRNA. Therefore, to assess whether the exons are indeed independently regulated, we have examined the alternative exon content of a large number of individual CD44 cDNA isoforms. This analysis shows that the downstream alternatively spliced exons are favored over those lying upstream and that alternative exons are often included in blocks rather than singly. Using a novel in vivo alternative splicing assay, we show that intron length has a major influence upon the alternative splicing of CD44. We propose a kinetic model in which short introns may overcome the poor recognition of alternatively spliced exons. These observations suggest that for CD44, intron length has been exploited in the evolution of the genomic structure to enable tissue-specific patterns of splicing to be maintained.Alternative pre-mRNA splicing allows a single gene to direct the synthesis of several structurally and functionally distinct protein isoforms. A number of patterns of alternative splicing have been described and include the use of alternative 5Ј or 3Ј splice sites, mutually exclusive use of exons, failure to remove introns, and the inclusion or skipping of whole exons (16,25,33). In some cases, alternative splicing results in dramatic changes in the polypeptide chain either by altering the transcriptional frame or by leading to premature termination. In other cases, the changes are more subtle, leading to the addition or deletion of one or more protein domains by exon skipping.A particularly noteworthy example is the case of the lymphocyte homing receptor CD44, which is expressed on lymphocytes and at a variety of nonlymphoid sites and is believed to act primarily as an adhesion molecule with an affinity for extracellular matrix components, including hyaluronic acid, collagen, and fibronectin (22,35,43). It is perhaps one of the most impressive examples of alternative splicing yet described because the central 10 of a total of 20 exons can be included or skipped to produce over 1,000 potential isoforms (Fig. 1) (31,32,41). The most frequently found isoform is the so-called hematopoietic variant (CD44H), in which all of the alternative exons are omitted. Isoforms containing alternative exons have been estimated to a...