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.