THE PHYLOGENETICALLY INVARIANT ACAGAGA AND AGC SEQUENCES OF U6 SMALL NUCLEAR-RNA ARE MORE TOLERANT OF MUTATION IN HUMAN-CELLS THAN IN SACCHAROMYCES-CEREVISIAE

U6 small nuclear RNA (snRNA) is the most highly conserved of the five spliceosomal snRNAs that participate in nuclear mRNA splicing. The pro posal that U6 snRNA plays a key catalytic role in splicing [D. Brow an d C. Guthrie, Nature (London) 337:14-15, 19891 is supported by the phy logenetic conservation of U6, the sensitivity of U6 to mutation, cross -linking of U6 to the vicinity of the 5' splice site, and genetic evid ence for extensive base pairing between U2 and U6 snRNAs. We chose to mutate the phylogenetically invariant 41-ACAGAGA-47 and 53-AGC-55 sequ ences of human U6 because certain point mutations within the homologou s regions of Saccharomyces cerevisiae U6 selectively block the first o r second step of mRNA splicing. We found that both sequences are more tolerant to mutation in human cells (assayed by transient expression i n vivo) than in S. cerevisiae (assayed by effects on growth or in vitr o splicing). These differences may reflect different rate-limiting ste ps in the particular assays used or differential reliance on redundant RNA-RNA or RNA-protein interactions. The ability of mutations in U6 n ucleotides A-45 and A-53 to selectively block step 2 of splicing in S. cerevisiae had previously been construed as evidence that these resid ues might participate directly in the second chemical step of splicing ; an indirect, structural role seems more likely because the equivalen t mutations have no obvious phenotype in the human transient expressio n assay.