Different nuclease requirements for exosome-mediated degradation of normal and nonstop mRNAs

Abstract

Two general pathways of mRNA decay have been characterized in yeast. In one pathway, the mRNA is degraded by the cytoplasmic form of the exosome. The exosome has both 3' to 5' exoribonuclease and endoribonuclease activity, and the available evidence suggests that the exonuclease activity is required for the degradation of mRNAs. We confirm here that this is true for normal mRNAs, but that aberrant mRNAs that lack a stop codon can be efficiently degraded in the absence of the exonuclease activity of the exosome. Specifically, we show that the endo- and exonuclease activities of the exosome are both capable of rapidly degrading nonstop mRNAs and ribozyme-cleaved mRNAs. Additionally, the endonuclease activity of the exosome is not required for endonucleolytic cleavage in no-go decay. In vitro, the endonuclease domain of the exosome is active only under nonphysiological conditions, but our findings show that the in vivo activity is sufficient for the rapid degradation of nonstop mRNAs. Thus, whereas normal mRNAs are degraded by two exonucleases (Xrn1p and Rrp44p), several endonucleases contribute to the decay of many aberrant mRNAs, including transcripts subject to nonstop and no-go decay. Our findings suggest that the nuclease requirements for general and nonstop mRNA decay are different, and describe a molecular function of the core exosome that is not disrupted by inactivating its exonuclease activity.

Fig. 1.

Mutations that disrupt the endo- or exoribonuclease activity of Rrp44p do not affect expression of nonstop reporters. (A) Strains containing the rrp44-endo⁻ or rrp44-exo⁻ mutations were transformed with a his3-nonstop reporter. Each of the indicated strains were serially diluted and spotted onto media lacking histidine to assay suppression of the his3-nonstop allele. (B and C) Strains containing the rrp44-endo⁻, rrp44-exo⁻, or rrp44-CR3 mutations (B) or completely lacking the exonucleolytic RNB domain of Rrp44p (C) were transformed with a PGK1pG-nonstop reporter under the control of a galactose-inducible promoter. Expression of the reporter was repressed by the addition of glucose. Total RNA was isolated and PGK1pG-nonstop mRNA levels were analyzed by Northern blot analysis. Plotted is the mRNA remaining at each time point after correcting for loading differences using a probe specific for the RNA subunit of the signal recognition particle (SCR1).

Fig. 2.

Either the endo- or exoribonuclease activity of Rrp44p can efficiently degrade nonstop mRNAs. (A) Mutation of the CR3 region of Rrp44p affects expression of a his3-nonstop reporter. A strain containing the rrp44-CR3 mutation was transformed with a his3-nonstop reporter. Each of the indicated strains were serially diluted and spotted onto media lacking histidine. (B) Protein expression of the Rrp44p-CR3 mutant was analyzed using Western blot analysis with antibodies specific for Protein A, and Pgk1p as a loading control. (C) The nonstop mRNA decay defect seen in the rrp44-CR3 strain can be complemented by wild-type RRP44 or by the rrp44-endo⁻ or rrp44-exo⁻ allele, but not by the double-mutant rrp44-endo⁻exo⁻.

Fig. 3.

The exoribonuclease activity of Rrp44p is required for exosome-mediated decay of normal mRNAs. (A) Strains containing either the rrp44-endo⁻ or rrp44-exo⁻ mutation in combination with dcp1-2 were serially diluted and grown at the indicated temperatures. (B) The stability of GAL7 mRNA in the dcp1-2 rrp44-endo⁻ or dcp1-2 rrp44-exo⁻ mutants was examined by growing cells at 23 °C in media containing galactose. The 5′-to-3′ decay pathway was inactivated by incubating cells for 1 h at 37 °C. Following the addition of glucose, RNA was isolated at the times indicated, and GAL7 mRNA levels were analyzed by Northern blot analysis. Plotted is the mRNA remaining at each time point after correcting for loading differences using a probe specific for the RNA subunit of the signal recognition particle (SCR1).

Fig. 4.

Mutations that disrupt the endo- or exoribonuclease activity of Rrp44p do not affect expression of a hammerhead ribozyme cleavage product. Strains containing the rrp44-endo⁻, rrp44-exo⁻, or rrp44-CR3 mutations were transformed with a reporter containing a hammerhead ribozyme inserted into the HIS3 gene. Each of the indicated strains were serially diluted and spotted onto media lacking histidine to assay suppression of the his3-ribozyme allele.

Fig. 5.

Endoribonuclease-mediated nonstop mRNA decay is distinct from no-go decay. A wild-type strain, a ski7Δ strain, and a ski7Δ rrp44-endo⁻ double-mutant strain were transformed with a PGK1 or PGK1-SL reporters under the control of a galactose-inducible promoter. Total RNA was isolated from cultures grown in galactose, and PGK1 mRNA levels were analyzed by Northern blot analysis. The RNA subunit of the signal recognition particle (SCR1) served as a loading control.

Fig. 6.

The endoribonuclease activity of Rrp44p is not redundant with the C-terminal domain of Ski7p in nonstop mRNA decay. The indicated strains were transformed with a his3-nonstop reporter and analyzed as in Fig. 1A.