How have the branchpoint motifs evolved in organisms of different complexity? Here we identified and examined the consensus motifs (R₁C₂T₃R₄A₅Y₆, R: A or G, Y: C or T) of 898 fungal genomes. In Ascomycota unicellular yeasts, the G₄/A₄ ratio is mostly (98%) below 0.125 but increases sharply in multicellular species by about 40 times on average, and in the more complex Basidiomycota, it increases further by about 7 times. The global G₄ increase is consistent with A₄ to G₄ transitions in evolution. Of the G₄/A₄-interacting amino acids of the branchpoint binding protein MSL5 (SF1) and the HSH155 (SF3B1), as well as the 5ʹ splice sites (SS) and U2 snRNA genes, the 5ʹ SS G³/A³ co-vary with the G₄ to some extent. However, corresponding increase of the G₄-complementary GCAGTA-U2 gene is rare, suggesting wobble-base pairing between the G₄-containing branchpoint motif and GTAGTA-U2 in most of these species. Interestingly, the G₄/A₄ ratio correlates well with the abundance of alternative splicing in the two phyla, and G₄ enriched significantly at the alternative 3' SS of genes in RNA metabolism, kinases and membrane proteins. Similar wobble nucleotides also enriched at the 3' SS of multicellular fungi with only thousands of protein-coding genes. Thus, branchpoint motifs have evolved U2-complementarity in unicellular Ascomycota yeasts, but have gradually gained more wobble base-pairing nucleotides in fungi of higher complexity, likely to destabilize branchpoint motif-U2 interaction and/or branchpoint A protrusion for alternative splicing. This implies an important role of relaxing the branchpoint signals in the multicellularity and further complexity of fungi.

How have the branchpoints evolved in organisms of different complexity? Here we identified and examined the consensus motifs (R₁C₂T₃R₄A₅Y₆, R: A or G, Y: C or T) of 898 fungal genomes. In Ascomycota unicellular yeasts, the G₄/A₄ ratio is mostly (98%) below 0.125 but increases sharply in multicellular species by about 40 times on average, and in the more complex Basidiomycota it increases further by about 7 times. The global G₄ increase is consistent with A₄ to G₄ transitions in evolution. Of the G₄/A₄-interacting amino acids of the branchpoint binding protein MSL5 (SF1) and the HSH155 (SF3B1), as well as the 5ʹ splice sites (SS) and U2 snRNA genes, the 5ʹ splice site G³/A³ co-vary with the G₄. However, corresponding increase of the G₄-complementary GCAGTA-U2 gene is rare, suggesting wobble-base pairing between the branchpoint motif and U2 in most species. Interestingly, the G₄/A₄ ratio correlates well with the abundance of alternative splicing in the two phyla, and G₄ enriched significantly in the alternative splice sites of genes in RNA metabolism, kinases and membrane proteins. Similar wobble nucleotides also enriched in the splice sites of multicellular fungi with only thousands of protein-coding genes. Thus, branchpoint motifs have evolved U2-complementarity in unicellular Ascomycota yeasts, but have gradually gained more wobble base-pairing nucleotides in fungi of higher complexity, likely to destabilize branchpoint-U2 interaction and/or branchpoint A protrusion for alternative splicing. This implies an important role of the relaxation of branchpoint signals in the multicellularity and further complexity of fungi.

How have the branchpoint motifs evolved in organisms of different complexity? Here we identified and examined the consensus motifs (R₁C₂T₃R₄A₅Y₆, R: A or G, Y: C or T) of 898 fungal genomes. In Ascomycota unicellular yeasts, the G₄/A₄ ratio is mostly (98%) below 0.125 but increases sharply in multicellular species by about 40 times on average, and in the more complex Basidiomycota, it increases further by about 7 times. The global G₄ increase is consistent with A₄ to G₄ transitions in evolution. Of the G₄/A₄-interacting amino acids of the branchpoint binding protein MSL5 (SF1) and the HSH155 (SF3B1), as well as the 5ʹ splice sites (SS) and U2 snRNA genes, the 5ʹ SS G³/A³ co-vary with the G₄ to some extent. However, corresponding increase of the G₄-complementary GCAGTA-U2 gene is rare, suggesting wobble-base pairing between the G₄-containing branchpoint motif and GTAGTA-U2 in most of these species. Interestingly, the G₄/A₄ ratio correlates well with the abundance of alternative splicing in the two phyla, and G₄ enriched significantly at the alternative 3' SS of genes in RNA metabolism, kinases and membrane proteins. Similar wobble nucleotides also enriched at the 3' SS of multicellular fungi with only thousands of protein-coding genes. Thus, branchpoint motifs have evolved U2-complementarity in unicellular Ascomycota yeasts, but have gradually gained more wobble base-pairing nucleotides in fungi of higher complexity, likely to destabilize branchpoint motif-U2 interaction and/or branchpoint A protrusion for alternative splicing. This implies an important role of relaxing the branchpoint signals in the multicellularity and further complexity of fungi.