Introns are distributed unevenly along genes — common in the coding sequence but rare in the 3′ untranslated region (3′UTR). It is proposed that the positions of introns are maintained by natural selection because introns in the 3′UTRs can reduce expression through nonsense-mediated mRNA decay1. In contrast, introns in the mRNA leaders of complex eukaryotes are common but often overlooked2. These introns are spliced out before translation, leaving exon-exon junctions in the mRNA leaders (5′EEJ). A cross species and multi-omic approach was taken to study exon structure of the mRNA leaders, inferring functions and selective constraints. Among the features of mRNA leaders examined, the number of 5′EEJ is inversely correlated with the main protein translation, as does the number of uORFs. Across the five species studied, the lowest levels of translation were observed for mRNAs with both 5′EEJ and uORFs. This class of mRNAs also have ribosome footprints on uORFs, with strong triplet periodicity indicating uORF translation. Comparative genomic analysis shows that the positions of both 5′EEJ and uORFs are conserved in human-mouse divergence. Thus the uORFs of the mRNAs with 5′EEJ are more likely to be functional and have regulatory roles.