A ROLE FOR U2 U6 HELIX IB IN 5'-SPLICE-SITE SELECTION/

Selection of pre-mRNA splice sites is a highly accurate process involv ing many trans-acting factors. Recently, we described a role for U6 sn RNA position G52 in selection of the first intron nucleotide (+1G). Be cause some U2 alleles suppress U6-G52 mutations, we investigated wheth er the corresponding U2 snRNA region also influenced 5' splice site se lection. Our results demonstrate that U2 snRNAs mutated at position U2 3, but not adjacent nucleotides, specifically affect 5' splice site cl eavage. Furthermore, all U2 position U23 mutations are synthetic letha l with the thermosensitive U6-G52U allele. Interestingly, the U2-U23C substitution has an unprecedented hyperaccurate splicing phenotype in which cleavage of introns with a +1G substitution is reduced, whereas the strain grows with wild-type kinetics, U2 position U23 forms the fi rst base pair with U6 position A59 in U2/U6 helix Ib. Restoration of t he helical structure suppresses 5' splice site cleavage defects, showi ng an important role for the helix Ib structure in 5' splice site sele ction, U2/U6 helix Ib and helix II have recently been described as bei ng functionally redundant. This report demonstrates a unique role for helix Ib in 5' splice site selection that is not shared with helix II.