Genome-wide evolution of wobble base-pairing nucleotides of branchpoint motifs with increasing organismal complexity

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.

不同复杂度生物的分支点基序（branchpoint motif）是如何演化的？本研究对898个真菌基因组的共有基序（consensus motif）（R₁C₂T₃R₄A₅Y₆，其中R代表A或G，Y代表C或T）进行了鉴定与分析。在子囊菌门的单细胞酵母中，G₄/A₄比值大多（98%）低于0.125，但在多细胞物种中该比值平均骤增约40倍；在更复杂的担子菌门中，该比值进一步提升约7倍。全局范围内的G₄占比升高与演化过程中A₄向G₄的转变相一致。针对分支点结合蛋白MSL5（SF1）、HSH155（SF3B1）中与G₄/A₄互作的氨基酸，以及5'剪接位点（SS）和U2小核RNA基因的分析显示，5' SS的G₃/A₃比例在一定程度上与G₄占比共变异。不过，与G₄互补的GCAGTA-U2基因的相应扩增却十分罕见，这表明在多数此类物种中，含G₄的分支点基序与GTAGTA-U2之间存在摆动碱基配对。值得注意的是，在这两个真菌门中，G₄/A₄比值与可变剪接的丰度呈现显著正相关，且G₄在参与RNA代谢、激酶及膜蛋白编码基因的可变3' SS位点处显著富集。在仅拥有数千个蛋白编码基因的多细胞真菌中，类似的摆动核苷酸也在其3' SS位点处富集。由此可见，分支点基序在单细胞子囊菌酵母中演化出了与U2互补的序列，但在复杂度更高的真菌中逐渐获得了更多摆动碱基配对的核苷酸，这可能是为了削弱分支点基序与U2的相互作用，或/且削弱分支点A突出结构，从而实现可变剪接。这暗示放松分支点信号的选择压力，在真菌的多细胞化及进一步复杂度提升过程中发挥了重要作用。