Correct ligation of exons in pre-mRNA splicing requires splice site 
juxtaposition (splice site pairing), usually involving a 5' splice site and a 
downstream 3' splice site. Splicing of a 5' splice site to an upstream 3' splice 
site, however, is predicted to result in a circular RNA. This mode of splice 
site pairing across the axon has been hypothesized to account for rare RNAs 
containing scrambled exons (Nigro JM et al., 1991, Celt 64:607-613; Cocquerelle 
C et al., 1992, EMBO J 11:1 095-1098). Additionally, this mode of splice site 
pairing has been postulated to explain the formation of SRY circular transcripts 
in mouse testis (Capel B et al., 1993, Celt 73:1019- 1030). Here we show that 
splice site pairing across the exon can result in exon circularization in vitro. 
These results indicate that spliceosome-mediated axon circularization indeed can 
account for the formation of scrambled exons and circular RNAs. Exon 
circularization efficiency decreased dramatically as the length of the exon was 
increased from 95 nt to 274 nt. Circularization of this longer exon was 
restored, however, when intronic complementary 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 splicing of this structured RNA recapitulated SRY-like exon 
circularization in vitro.