Identification and characterization of yeast mutants that overcome an experimentally introduced block to splicing at the 3' splice site.
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.
Lin
J
J
Department of Microbiology and Molecular Genetics, Loma Linda University, California 92350, USA.
Rossi
J J
JJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018628:Gene Dosage; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010641:Phenotype; D017433:Protein Structure, Secondary; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D016337:RNA, Catalytic / Q000737:chemistry; D012331:RNA, Fungal / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013489:Suppression, Genetic; D013696:Temperature; D001616:beta-Galactosidase / Q000235:genetics
D016337:RNA, Catalytic; D012331:RNA, Fungal; D000255:Adenosine Triphosphate; D012316:RNA Nucleotidyltransferases; D001616:beta-Galactosidase; D020365:RNA Helicases