Human-mouse comparative genomics is an informative tool to assess sequence functionality as inferred from its conservation level. We used this approach to examine dependency among different positions of the 5 splice site. We compiled a data set of 50,493 homologous human-mouse internal exons and analyzed the frequency of changes among different positions of homologous human-mouse 5 splice-site pairs. We found mutual relationships between positions +4 and +5, +5 and +6, −2 and +5, and −1 and +5. We also demonstrated the association between the exonic and the intronic positions of the 5 splice site, in which a stronger interaction of U1 snRNA and the intronic portion of the 5 splice site compensates for weak interaction of U1 snRNA and the exonic portion of the 5 splice site, and vice versa. By using an ex vivo system that mimics the effect of mutation in the 5 splice site leading to familial dysautonomia, we demonstrated that U1 snRNA base-pairing with positions +6 and −1 is the only functional requirement for mRNA splicing of this 5 splice site. Our findings indicate the importance of U1 snRNA base-pairing to the exonic portion of the 5 splice site.

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