A single m⁶A modification in U6 snRNA diversifies exon sequence at the 5' splice site

N⁶-methyladenosine (m⁶A) 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 m⁶A 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 m⁶A facilitating efficient recognition of the splice sites weakly interacting with U5 snRNA, indicating that U6 snRNA m⁶A relaxes the 5' exon constraint and allows protein sequence diversity along with explosively increasing number of introns over the course of eukaryotic evolution.