Alternative splicing within the chicken c-ets-1 locus: implications for transduction within the E26 retrovirus of the c-ets proto-oncogene.

Two overlapping c-ets-1 cDNA clones were isolated which contained the alpha and beta genomic sequences homologous to the 5' end of v-ets not detected in the previously described c-ets RNA species or proteins. Nucleotide sequencing demonstrated that these cDNAs corresponded to the splicing of alpha and beta to a common set of 3' exons (a through F) already found in the p54c-ets-1 mRNA. They contained an open reading frame of 1,455 nucleotides which could encode a polypeptide of 485 amino acids with a predicted molecular mass of 53 kilodaltons. However, when expressed in COS-1 cells, the cDNAs directed the synthesis of a protein with an apparent molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 68 kilodaltons, p68c-ets-1, comigrating with a protein expressed at low levels in normal chicken spleen cells. These two proteins were shown to be identical by partial digestion with protease V8. Northern (RNA) blot hybridization analysis with the p68c-ets-1 -specific sequence and RNase protection experiments showed that the corresponding mRNA was expressed in normal chicken spleen and not in normal chicken thymus or in various T lymphoid cell lines. Thus, two closely related proteins, having distinct amino-terminal parts, are generated within the same locus by alternative addition of different 5' exons, alpha and beta or I54, respectively, onto a common set of 3' exons (a to F). Finally, we demonstrate that an aberrant splicing event between a cryptic splice donor site in c-myb exon E6 and the normal splice acceptor site of c-ets-1 exon alpha involved in the genesis of the E26 myb-ets sequence.     

How prevalent is functional alternative splicing in the human genome?

Comparative analyses of ESTs and cDNAs with genomic DNA predict a high frequency of alternative splicing in human genes. However, there is an ongoing debate as to how many of these predicted splice variants are functional and how many are the result of aberrant splicing (or 'noise'). To address this question, we compared alternatively spliced cassette exons that are conserved between human and mouse with EST-predicted cassette exons that are not conserved in the mouse genome. Presumably, conserved exon-skipping events represent functional alternative splicing. We show that conserved (functional) cassette exons possess unique characteristics in size, repeat content and in their influence on the protein. By contrast, most non-conserved cassette exons do not share these characteristics. We conclude that a significant portion of cassette exons evident in EST databases is not functional, and might result from aberrant rather than regulated splicing.     

The human CYP2C locus: a prototype for intergenic and exon repetition splicing events

In human there are four known CYP2C genes that have been mapped to chromosome 10q24 with the order Cen-2C18-2C19-2C9-2C8-Tel. Previously we have shown that splicing events joining exons from the neighboring 2C18 and 2C19 genes occur in human liver and epidermis. Here evidence is presented that the terminal genes of this cluster, 2C18 and 2C8, are also involved in intergenic splicing. Most interestingly, several of these 2C18/2C8 RNAs were composed of all nine exons, thus conceivably having the potential for coding functional proteins. Moreover, chimeric RNA species consisting of exons originating not only from the CYP2C8 and CYP2C18 genes, but also from the CYP2C19 gene were detected. In all cases the exons from the different CYP2C genes were joined at the correct canonical splice sites. However, the closely linked RBP4 gene is not participating in intergenic splicing with the CYP2C genes. In addition, CYP2C8 gene expression was found to generate a variety of scrambled RNA molecules including species that contained repetitions of certain exons.