Abstract
An experimentally introduced secondary structure in exon 2 adjacent to the 3' splice site of a yeast ACT-Escherichia coli lacZ fusion gene abolishes splicing in vivo and inhibits beta-galactosidase production. We have devised a genetic screen to isolate both cis and trans-acting mutants that restore beta-galactosidase activity. Two cis-acting mutants potentially destabilize the stem in the region close to the 3' splice site. One trans-acting mutant, designated rss1-1, partially restores beta-galactosidase activity by both increasing the splicing efficiency and stabilizing the precursor and lariat intermediate. The trans-acting suppression activity of rss1-1 is specific for a particular structure because another artificially introduced secondary structure, which also blocks splicing, is not suppressed by this mutant allele. We have cloned the gene encoding the trans-acting mutant protein. The RSS1 gene is located on Saccharomyces cerevisiae chromosome V and is a single copy, essential gene. The predicted RSS1 protein has marked similarity to members of the putative ATP-dependent RNA helicase family. At the nonpermissive temperature, the rss1-1 mutant allele decreases the steady-state levels of several endogenous messenger RNAs and increases the ratio of pre-mRNA to mRNA of specific messages. RSS1 is likely to play an interesting role in RNA processing.
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