Widespread adenine N6-methylation of active genes in fungi.

N6-methyldeoxyadenine (6mA) is a noncanonical DNA base modification present at low levels in plant and animal genomes, but its prevalence and association with genome function in other eukaryotic lineages remains poorly understood. Here we report that abundant 6mA is associated with transcriptionally active genes in early-diverging fungal lineages. Using single-molecule long-read sequencing of 16 diverse fungal genomes, we observed that up to 2.8% of all adenines were methylated in early-diverging fungi, far exceeding levels observed in other eukaryotes and more derived fungi. 6mA occurred symmetrically at ApT dinucleotides and was concentrated in dense methylated adenine clusters surrounding the transcriptional start sites of expressed genes; its distribution was inversely correlated with that of 5-methylcytosine. Our results show a striking contrast in the genomic distributions of 6mA and 5-methylcytosine and reinforce a distinct role for 6mA as a gene-expression-associated epigenomic mark in eukaryotes.

Diverse mechanisms for spliceosome-mediated 3' end processing of telomerase RNA.

The 3' end of Schizosaccharomyces pombe telomerase RNA (SpTER1) is generated by spliceosomal cleavage, a reaction that corresponds to the first step of splicing. The observation that the spliceosome functions in 3' end processing raised questions about the evolutionary origin and conservation of this mechanism. We now present data in support of spliceosomes generating 3' ends of telomerase RNAs in other fungi. Strikingly, the mechanistic basis for restricting spliceosomal splicing to the first transesterification reaction differs substantially among species. Unlike S. pombe, two other fission yeasts rely on hyperstabilization of the U6 snRNA-5' splice site interaction to impede the 2nd step of splicing. In contrast, a non-canonical 5' splice site blocks the second transesterification reaction in Aspergillus species. These results demonstrate a conserved role for spliceosomes functioning in 3' end processing. Divergent mechanisms of uncoupling the two steps of splicing argue for multiple origins of this pathway.