BASE-PAIRING AT THE 5'-SPLICE-SITE WITH U1 SMALL NUCLEAR-RNA PROMOTESSPLICING OF THE UPSTREAM INTRON BUT MAY BE DISPENSABLE FOR SPLICING OF THE DOWNSTREAM INTRON

We previously reported that exon skipping in vivo due to point mutatio ns in the 5' splice site (5'ss) signal of an internal mammalian exon c an be prevented by coexpression of U1 small nuclear RNAs, termed shift -U1s, with complementarity to sequences upstream or downstream of the mutated site. We now show by SI nuclease protection experiments that a typical shift-U1 restores splicing of the upstream intron, but not ne cessarily of the downstream intron. This indicates that the normal 5's s sequence acts as an enhancer for splicing of the upstream intron, th at it owes this activity to base pairing with U1, and that the enhance r activity is reproduced by base pairing of U1 with other sequences in the area. Shift-U1s are dispensable when the 3'ss sequence of the ups tream intron is improved, which suggests that base pairing of U1 with sequences at or near the downstream end of the exon normally functions by compensating for a weakness in the upstream 3'ss. Accordingly, U1 appears to be involved in communication across the exon, but our data indicate at the same time that extensive base pairing between U1 and t he 5'ss sequence is not necessary for accurate splicing of the downstr eam intron. These findings are discussed in relation to the coordinate selection of exon termini proposed by the exon definition model.