Abstract
Many genes of eukaryotes and viruses contain introns. The roles of introns are not fully understood because many of them seem dispensable. To address this long-standing question, comparative genomics and ‘multi-omic’ approaches were taken.
Firstly, the evolutionary history of introns was examined using 335 fungal species. The number of introns per one kb protein-coding sequence for the last fungal ancestor was estimated to be 6.1-8.9 intron/kb. Strikingly, over one-third of species analyzed have undergone massive loss of introns — falling below 10% of the intron/kb of the last fungal ancestor. Widespread intron loss has occurred independently in Microsporidia, Ustilaginomycotina, Saccharomycotina, and Pezizomycotina. To study selection and roles of introns, eight intron-poor species were chosen. Notably, their first introns are distributed toward the 5′ ends of the genes (i.e., the length of the first exon tends to be shorter than half of the gene length). Their introns are abundant in some classes of genes, including the ribosomal protein genes, but depleted in others, such as the base-excision repair genes. Interestingly, these intron-containing genes were expressed and translated at higher levels than the intronless genes.
Secondly, the distribution and patterns of introns in five intron-rich eukaryotes were investigated. The five organisms chosen were human, mouse, zebrafish, fruit fly and Arabidopsis thaliana. Similarly to the surviving fungal introns, the complex eukaryotic introns are also distributed toward the 5′ ends of the mRNAs. The exon-exon junctions (EEJs; after intron removal) are commonly present in the mRNA leaders (5′UTRs), ranging from 24% in A. thaliana to 53% in human. Strikingly, their first EEJs are most frequently located just upstream of the translation initiation codons. More importantly, the frequency of the EEJs in the mRNA leaders (leader EEJs) anti-correlates with translation. Furthermore, the leader EEJ is a stronger predictor of low translation rate compared to uORF. When combined together, the leader EEJ and uORF predict lower expression for nearly one-third of eukaryotic proteins.
Thirdly, the introns in the virus with the smallest DNA genome, hepatitis B virus (HBV; 3.2-kb), were explored. Alternative splicing and its regulation in HBV infection are poorly understood. Four HBV genotypes, A2, B2, C2, and D3 were chosen for transcriptomic analysis. Here complex alternative splicing patterns in HBV RNAs were revealed. In particular, two main alternative splicing patterns are conserved across the genotypes whereas some other patterns are genotype specific. Interestingly, the major intron has a suboptimal 5′ splice donor site across the genotypes. To investigate this, the HBV RNA elements associated with splicing and nuclear export, collectively known as the Post-transcriptional Regulatory Element (PRE), were analyzed. In particular, two RNA stem-loops of the PRE are conserved not only across the genotypes but also in all known hepadnaviruses found in human, woodchuck, non-human primates and ground squirrel. These results suggest that splicing is tightly regulated by the PRE.
Finally, the results show that the positions of introns are predictive of translation in eukaryotes. Introns in the coding regions correlate with main ORF translation whereas introns in the 5′UTRs correlate with uORF translation. These relationships are likely due to intricate links between splicing and translation.