A single m(6)A modification in U6 snRNA diversifies exon sequence at the 5’ splice site

N(6)-methyladenosine (m(6)A) is a modification that plays pivotal roles in RNA metabolism and function, although its functions in spliceosomal U6 snRNA remain unknown. To elucidate its role, we conduct a large-scale transcriptome analysis of a Schizosaccharomyces pombe strain lacking this modification and found a global change of pre-mRNA splicing. The most significantly impacted introns are enriched for adenosine at the fourth position pairing the m(6)A in U6 snRNA, and exon sequences weakly recognized by U5 snRNA. This suggests cooperative recognition of 5’ splice site by U6 and U5 snRNPs, and also a role of m(6)A facilitating efficient recognition of the splice sites weakly interacting with U5 snRNA, indicating that U6 snRNA m(6)A relaxes the 5’ exon constraint and allows protein sequence diversity along with explosively increasing number of introns over the course of eukaryotic evolution.