Yeast RNA viruses as indicators of exosome activity: human exosome hCsl4p participates in RNA degradation in Saccharomyces cerevisiae'

Abstract

The exosome is an evolutionarily conserved 10-mer complex involved in RNA metabolism, located in both the nucleus and the cytoplasm. The cytoplasmic exosome plays an important role in mRNA turnover through its 3′!5′ exonucleolytic activity. The superkiller (SKI) phenotype of yeast was originally identified as an increase of killer toxin production due to elevated levels of the L-A double-stranded RNA (dsRNA) Totivirus and its satellite toxin-encoding M dsRNA. Most SKI genes were later shown to be either components of the exosome or modulators of its activity. Variations in the amount of Totivirus are, thus, good indicators of yeast exosome activity, and can be used to analyse its components. Furthermore, if exosome proteins of higher eukaryotes were functional in S. cerevisiae, these viruses would provide a simple tool to analyse their function. In this work, we have found that hCSL4, the human orthologue of SKI4 in the yeast exosome, rescues the null phenotype of the deletion mutant. hCsl4p shares with Ski4p conserved S1 RNA-binding domains, but lacks the N-terminal third of Ski4p. Nevertheless, it interacts with the Dis3p exonuclease of yeast exosome, and partially complements the superkiller phenotype of ski4-1 mutation. The elimination of the N-terminal third of Ski4p does not affect its activity, indicating that it is dispensable for RNA degradation. We have also identified the point mutation G152E in hCSL4, equivalent to the ski4-1 mutation G253E, which impairs the activity of the protein, thus validating our approach of using yeast RNA virus to analyse the exosome of higher eukaryotes.

Variation and Distribution of L-A Helper Totiviruses in Saccharomyces sensu stricto Yeasts Producing Different Killer Toxins

“…Total nucleic acids were obtained from 1 mL stationary-phase cultures by breaking cells with glass beads followed by phenol extraction and ethanol precipitation as described [ 62 ]. Total RNAs were separated on native agarose gels, denatured by treatment with formamide and formaldehyde and transferred to neutral nylon membranes [ 63 ].…”

Relationships and Evolution of Double-Stranded RNA Totiviruses of Yeasts Inferred from Analysis of L-A-2 and L-BC Variants in Wine Yeast Strain Populations

“…Total RNAs were obtained from 1-ml stationary-phase cultures, separated on agarose gels, and denatured in the gels before transfer to neutral nylon membranes (GE Healthcare). The details of nucleic acid preparation and Northern hybridization conditions have been provided elsewhere (43,44). 32 P-labeled-specific probes were made by T3 or T7 runoff transcription from plasmids that had been linearized with appropriate restriction enzymes.…”

L-A-lus, a New Variant of the L-A Totivirus Found in Wine Yeasts with Klus Killer Toxin-Encoding Mlus Double-Stranded RNA: Possible Role of Killer Toxin-Encoding Satellite RNAs in the Evolution of Their Helper Viruses

“…Table 1 summarizes the strains used in this study. Most were constructed for this work, and others are laboratory strains that have already been described (26,33). ski2⌬, ski1⌬, and mak10⌬ deletion strains, all of them derivatives from strain BY4741, are from the EUROFAN collection (kindly supplied by J. L. Revuelta).…”

“…Strains were grown in YPAD medium or in complete minimal medium deprived of the appropriate amino acid for 2 to 3 days at 28°C. Cells from 1 ml of culture were broken with glass beads by vortex mixing, and nucleic acids were extracted from cell lysates as described previously (33). Total RNAs were separated on 1.3% agarose gels, denatured in the gel, and transferred to neutral nylon membranes (GE Healthcare).…”