Alternative splicing of RPT5bWs. A, Hypothesis for the cause of alternative splicing at intron seven of RPT5b. Schematic representation of the Col intron with the different stages of splicing depicted: formation of a lariat between the 5' splice site and the branchpoint (1) and cleavage of the 3' splice site (2) before ligation of the two exon sequences. In the Ws intron, the SNP2 variant (U, in bold character) generates a new branchpoint sequence that activates the selection of a downstream 3' splice site. Exons are represented by boxes. The yellow box corresponds to the beginning of exon 8 that is spliced out in Ws. B, Graphic representation of mis-splicing occurring at RPT5b intron 7. Primers used for the cDNA amplification and for the splicing characterisation are represented (See methods). C, Strategy used to assess the level of mis-splicing at intron 7. Normal spliced forms are amplified with primers N and e10 while the mis-spliced form is detected by amplification with primers M and e10, as exemplified in D. E, Plasmids containing both e5-e10 fragments of the normally spliced and mis-spliced form of RTP5b intron 7 were mixed up to 10 ng, as indicated below the graph. The level of mis-splicing was then assessed as represented in Fig. 3C. At least 100 cloned cDNAs were tested per genotype. Results show that our test gives a good estimate of the mis-spliced form vs total form ratio. Hence, in our conditions, there is no significant bias of PCR amplification and/or cloning of one spliced form over the other.