Universally conserved and yeast-specific U1 snRNA sequences are important but not essential for U1 snRNP function

Abstract

To study the contribution of the large, 568-nucleotide yeast (Saccharomyces cerevisiae] U1 snRNA to pre-mRNA splicing, we generated mutations in two regions of the molecule and introduced each mutant gene back into yeast as the sole copy of the U1 snRNA gene. We mutagenized the "A loop," a subregion highly conserved in primary sequence in all U1 snRNA molecules analyzed to date. We also mutagenized a portion of the yeast core subdomain, a region conserved in primary and secondary structure among several yeast species but absent from the much smaller metazoan U1 molecule. Surprisingly, mutations in these two regions had little or no effect on growth rate, yet several of them affected an inefficiently spliced reporter gene construct. In addition, combinations of mutants in both regions gave rise to reduced growth rates. Using the latter assay, we confirmed some of the proposed secondary structure of the yeast core domain. The experiments indicate that both regions contribute to U1 snRNP activity but that mutations in a single region do not have a substantial effect on growth rate because U1 snRNP activity is not rate-limiting for growth.