Rat1p and Xrn1p Are Functionally Interchangeable Exoribonucleases That Are Restricted to and Required in the Nucleus and Cytoplasm, Respectively

Abstract

XRN1 encodes an abundant cytoplasmic exoribonuclease, Xrn1p, responsible for mRNA turnover in yeast. A screen for bypass suppressors of the inviability of xrn1 ski2 double mutants identified dominant alleles of RAT1, encoding an exoribonuclease homologous with Xrn1p. These RAT1 alleles restored XRN1-like functions, including cytoplasmic RNA turnover, wild-type sensitivity to the microtubule-destabilizing drug benomyl, and sporulation. The mutations were localized to a region of the RAT1 gene encoding a putative bipartite nuclear localization sequence (NLS). Fusions to green fluorescent protein were used to demonstrate that wild-type Rat1p is localized to the nucleus and that the mutant alleles result in mislocalization of Rat1p to the cytoplasm. Conversely, targeting Xrn1p to the nucleus by the addition of the simian virus 40 large-T-antigen NLS resulted in complementation of the temperature sensitivity of a rat1-1 strain. These results indicate that Xrn1p and Rat1p are functionally interchangeable exoribonucleases that function in and are restricted to the cytoplasm and nucleus, respectively. It is likely that the higher eukaryotic homologs of these proteins will function similarly in the cytoplasm and nucleus.In the yeast Saccharomyces cerevisiae, the 5Ј33Ј exoribonuclease I, Xrn1p, encoded by the XRN1 gene, is the primary cytoplasmic RNase responsible for mRNA degradation (30). Xrn1p acts at the last step in mRNA turnover, after deadenylation and decapping (reviewed in reference 4). Consequently, xrn1 mutants accumulate many different mRNA species as deadenylated and decapped RNAs, displaying the highest mRNA levels relative to the wild type for short-lived transcripts (9, 18). Mutants deficient in XRN1 also accumulate decapped intermediates of the deadenylation-independent nonsense-mediated decay pathway (34). Significantly, the decapped RNAs that accumulate in xrn1 mutants persist in polysomes (6, 15), suggesting that in the absence of Xrn1p exonuclease, transcripts targeted for degradation by decapping continue to be translated. Xrn1p degrades other classes of RNAs as well, including internal transcribed spacer 1 (ITS1) (39), a byproduct of rRNA processing. Thus, Xrn1p has broad substrate specificity.Xrn1p was first purified from yeast extracts in a search for RNA turnover factors based on its exoribonuclease activity (38). Subsequently, the enzyme has been purified from biochemical fractionations as a DNA strand exchange activity referred to as Sep1p (13, 28) and as a DNA G4 tetraplexspecific nuclease referred to as Kem1p (32). The gene has also been identified in various genetic screens. It was identified as KEM1, mutation of which enhances the karyogamy defect in kar1 mutants (26). kem1 mutants were shown to be hypersensitive to benomyl, a microtubule-destabilizing drug, and it has subsequently been suggested that Xrn1p may physically interact with microtubules (19). XRN1 was also identified as RAR5 in a screen for mutants that destabilized plasmids containing defective ARS elements (27). In addition, x...

Activation of 5′-3′ exoribonuclease Xrn1 by cofactor Dcs1 is essential for mitochondrial function in yeast

“…1B), showing that this defect is related to the enzyme's exoribonuclease activity. We were also able to rescue this growth defect by expressing the nuclear 5′-3′ exoribonuclease Rat1 in the cytoplasm in the absence of its nuclear localization signal, Rat1ΔNLS (20) (Fig. 1B).…”

A genome-wide RNAi screen identifies genes regulating the formation of P bodies in C. elegans and their functions in NMD and RNAi

“…One of the factors identified is T25G3.3, encoding the C. elegans NMD3 homolog. NMD3 directly interacts with UPF1 and mutations in NMD3 cause synthetic lethal with an Xrn1 mutant (He and Jacobson, 1995;Johnson, 1997). Mutant alleles of NMD3 also show altered rRNA stability.…”

The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo

“…[ 35 S]-methionine-labeled extracts were prepared by growing cells to mid-log phase in 7+5 mL of appropriate synthetic selective medium lacking methionine+ The cells were concentrated to 1 mL, 80 mCi of [ 35 S]-methionine (Expre 35 S 35 S Protein Labeling Mix, DuPont NEN) was added, and the cells were incubated for an additional 30 min at 30 8C with shaking+ Cells were collected, washed once with ice-cold extraction buffer (20 mM Tris-HCl (pH 7+5), 20 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM PMSF, 0+5 mg/mL leupeptin, 0+7 mg/mL pepstatin A), resuspended in 0+25 mL of ice-cold extraction buffer, and disrupted by vortexing with glass beads+ The extract was removed, the beads washed once with 0+25 mL of extraction buffer, and the extract and wash were combined+ The sample was clarified by centrifugation for 10 min at 15,000 ϫ g at 4 8C+ Clarified extract (0+2 mL) was mixed with an equal volume of extraction buffer supplemented to 500 mM (NH 4 ) 2 SO 4 and 0+2% NP-40, 50 mL of BSA-blocked Protein A agarose (Gibco BRL) was added, and the samples were incubated at 4 8C with rocking+ After 30 min, the beads were removed by centrifugation+ The supernatant was recovered and the appropriate primary antibody was added, followed 1 h later by the addition of BSA-blocked Protein A agarose beads+ After 2 h of rocking at 4 8C, the beads were collected by centrifugation and washed three times with extraction buffer containing 250 mM (NH 4 ) 2 SO 4 and 0+1% NP-40+ Proteins were eluted from the beads by heating at 100 8C in Laemmli buffer+ Extracts for immunoprecipitations to be visualized by Western blotting were prepared similarly except cells were not labeled with [ 35 S]-methionine and the extract buffer was composed differently (20 mM Tris-HCl (pH 7+5), 20 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0+5 mg/mL leupeptin, 0+7 mg/mL pepstatin A)+ For these reactions, immunoprecipitates were washed in extract buffer supplemented with 0+1% NP-40+ Western blot analysis was carried out as previously described (Johnson, 1997)+ In Western blots the only immunoreactive YEp351-SKI2 Johnson & Kolodner (1995) protein bands were those expected and no immunoreactive bands were observed in the absence of tagged protein (data not shown)+…”