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.

MicroRNAs (miRNAs) are short, single-stranded RNAs that silence gene expression by either degrading mRNA or repressing translation. Each miRNA regulates a specific set of mRNA ''targets'' by binding to complementary sequences in their 39 untranslated region. In this study, we examined the importance of the base-pairing strength of the miRNA-target duplex to repression. We hypothesized that if base-pairing strength affects the functionality of miRNA repression, organisms with higher body temperature or that live at higher temperatures will have miRNAs with higher G/C content so that the miRNA-target complex will remain stable. In the nine model organisms examined, we found a significant correlation between the average G/C content of miRNAs and physiological temperature, supporting our hypothesis. Next, for each organism examined, we compared the average G/C content of miRNAs that are conserved among distant organisms and that of miRNAs that are evolutionarily recent. We found that the average G/C content of ancient miRNAs is lower than recent miRNAs in homeotherms, whereas the trend was inversed in poikilotherms, suggesting that G/C content is associated with temperature, thus further supporting our hypothesis. In the organisms examined, the average G/C content of miRNA ''seed'' sequences was higher than that of mature miRNAs, which was higher than pre-miRNA loops, suggesting an association between the degree of functionality of the sequence and its average G/C content. Our analyses show a possible association between the base-pairing strength of miRNAtargets and the temperature of an organism, suggesting that base-pairing strength plays a role in repression by miRNAs.