Mutational analysis of Schizosaccharomyces pombe U4 snRNA by plasmid exchange

We have developed a system for testing mutations by plasmid exchange in the fission yeast Schizosaccharomyces pombe. This system has been used to test the requirement for different regions of the small nuclear RNA U4 in S. pombe. Surprisingly, five of seven deletion and substitution mutations tested in different regions of U4 prevent the accumulation of the mutant RNA. Substitution of the U4 sequence in stem 1 of the U4/U6 interaction domain allows accumulation of the mutant U4, but does not support viability. Two sequences with homology to the Sm binding site are found in the 3' region of S. pombe U4; substitution of the 3' sequence of the two does not interfere with accumulation or function of U4, indicating that the 5' sequence is the functional Sm-binding site.