EXON CIRCULARIZATION IN MAMMALIAN NUCLEAR EXTRACTS

Correct ligation of exons in pre-mRNA splicing requires splice site ju xtaposition (splice site pairing), usually involving a 5' splice site and a downstream 3' splice site. Splicing of a 5' splice site to an up stream 3' splice site, however, is predicted to result in a circular R NA. This mode of splice site pairing across the exon has been hypothes ized to account for rare RNAs containing scrambled exons (Nigro JM et al., 1991, Cell 64:607-613; Cocquerelle C et al., 1992, EMBO J11:1095- 1098). Additionally, this mode of splice site pairing has been postula ted to explain the formation of SRY circular transcripts in mouse test is (Capel B et al., 1993, Cell 73:1019-1030). Here we show that splice site pairing across the exon can result in exon circularization in vi tro, These results indicate that spliceosome-mediated exon circulariza tion indeed can account for the formation of scrambled exons and circu lar RNAs. Exon circularization efficiency decreased dramatically as th e length of the exon was increased from 95 nt to 274 nt. Circularizati on of this longer exon was restored, however, when intronic complement ary sequences were included in the RNA substrate, These complementary sequences could form a stem that served to bring the splice sites into proximity and thereby promote splice site pairing, Therefore, the spl icing of this structured RNA recapitulated SRY-like exon circularizati on in vitro.