Upf1p, Nmd2p, and Upf3p regulate the decapping and exonucleolytic degradation of both nonsense-containing mRNAs and wild-type mRNAs

Abstract

In Saccharomyces cerevisiae, rapid degradation of nonsense-containing mRNAs requires the decapping enzyme Dcp1p, the 5'-to-3' exoribonuclease Xrn1p, and the three nonsense-mediated mRNA decay (NMD) factors, Upf1p, Nmd2p, and Upf3p. To identify specific functions for the NMD factors, we analyzed the mRNA decay phenotypes of yeast strains containing deletions of DCP1 or XRN1 and UPF1, NMD2, or UPF3. Our results indicate that Upf1p, Nmd2p, and Upf3p regulate decapping and exonucleolytic degradation of nonsense-containing mRNAs. In addition, we show that these factors also regulate the same processes in the degradation of wild-type mRNAs. The participation of the NMD factors in general mRNA degradation suggests that they may regulate an aspect of translation termination common to all transcripts.

FIG. 1

Analysis of the 5′ ends and cap status of nonsense-containing mRNAs that accumulate in yeast strains defective in nonsense-mediated mRNA decay. (A) Analysis of the 5′ ends of the CYH2 and MER2 pre-mRNAs by primer extension. Total RNA was isolated from yeast strains of the indicated genotypes. Radiolabeled primers (CYH2-IN4 or MER2-2) were annealed to aliquots (20 μg) of each RNA sample and extended by avian myeloblastosis virus reverse transcriptase. DNA sequencing reactions with the same primers (run in lanes G, A, T, and C) were used to determine the positions of the primer extension products. The major transcriptional start sites (positions noted are relative to the initiation codon) for both pre-mRNAs are indicated by arrows. The atypical extension products detected in RNA from xrn1Δ cells are denoted by asterisks. (B) Analysis of the 5′ cap status of the CYH2 pre-mRNA by anti-m⁷ G immunoprecipitation. Aliquots (10 μg) of total RNA isolated from the indicated yeast strains were immunoprecipitated using polycolonal anti-m⁷ G antibodies. RNA comprising the supernatant (S) (uncapped) and pellet (P) (capped) fractions, as well as an aliquot of the input RNA (I), were analyzed by Northern blotting, using a CYH2 probe. The positions of the CYH2 pre-mRNA and mRNA are indicated. Quantitation of this experiment is summarized in Table 2. WT, wild type.

FIG. 2

Inactivation of Rat1p inhibits accumulation of CYH2 pre-mRNA decay intermediates in xrn1Δ cells. rat1-1 and rat1-1 xrn1Δ cells were grown at 24°C and then shifted to 37°C for 1, 2, or 4 h. Total RNA was isolated from each culture at the indicated time points and used for primer extension analysis of the 5′ ends (A) or Northern analysis of the levels of the CYH2 pre-mRNA (B), as in Fig. 1.

FIG. 3

Formation of decay intermediates of wild-type mRNAs in xrn1Δ cells requires the decapping enzyme Dcp1p and the 5′-to-3′ exoribonuclease Rat1p. (A) Inactivation of Dcp1p inhibits the formation of mRNA decay intermediates in xrn1Δ cells. Total RNA was isolated from yeast strains of the indicated genotypes and the 5′ ends of wild-type ADH1, URA5, and CUP1 mRNAs were analyzed by primer extension, as in Fig. 1A. Radiolabeled primers (ADH1-1, URA5-1, and CUP1-1) were used for both reverse transcription and DNA sequencing reactions. The major transcriptional start sites (position noted is relative to the translation initiation codon) for each mRNA are indicated by arrows. mRNA decay intermediates that accumulate in xrn1Δ cells are marked by asterisks. WT, wild type. (B) Inactivation of Rat1p inhibits the formation of mRNA decay intermediates in xrn1Δ cells. rat1-1 xrn1Δ cells were grown at 24°C and then shifted to 37°C. Total RNA was isolated from cells at the indicated time points, and the 5′ ends of the URA5 mRNA were analyzed by primer extension as in panel A.

FIG. 4

In xrn1Δ cells, single or multiple deletions of UPF1, NMD2, or UPF3 differentially affect the levels of decapped nonsense-containing transcripts. (A) Analysis of the levels of CYH2 pre-mRNAs by anti-m⁷ G immunoprecipitation. Total RNA was isolated from yeast strains of the indicated genotypes, capped mRNAs were immunoprecipitated as in Fig. 1B, and each sample was analyzed by Northern blotting, using a CYH2 probe. Lanes I, S, and P designate input, supernatant, and pellet, respectively. (B) Analysis of the levels of CYH2 pre-mRNA decay intermediates. Primer extension analysis of the CYH2 pre-mRNA was performed on total RNA from each yeast strain as in Fig. 1A. The major transcriptional start sites of the CYH2 pre-mRNA and the 5′ ends of its decay intermediates are indicated by arrows and asterisks, respectively. Total RNA from the upf3Δ strain was used as a control. (C) Northern analysis of the steady-state levels of CYH2 pre-mRNA. Total RNA was isolated from yeast strains of the indicated genotypes and analyzed by Northern hybridization. The SCR1 RNA (20), which is transcribed by RNA polymerase III, was used as an internal control. WT, wild type. In panels A and C, the CYH2 DNA probe used was the same as in Fig. 1B. Quantitation of this experiment is summarized in Table 2.

FIG. 5

Single or multiple deletions of UPF1, NMD2, or UPF3 in xrn1Δ cells differentially affect the levels of wild-type (WT) mRNAs. Total RNA was isolated from yeast strains of the indicated genotypes and analyzed by Northern blotting, using the SCR1 RNA as an internal control. Quantitation of the results for the URA5, TCM1, and PGK1 mRNAs is summarized in Table 3.

FIG. 6

Effects of single or mutiple deletions of UPF1, NMD2, UPF3, and XRN1 on the accumulation of capped and decapped wild-type mRNAs. (A and B) Analysis of the levels of capped and decapped wild-type (WT) mRNAs by anti-m⁷ G immunoprecipitation. Total RNA was isolated from yeast strains of the indicated genotypes, and anticap immunoprecipitation was carried out as in Fig. 1B. DNA probes specific for URA5, TCM1, and PGK1 were used for Northern analysis of the respective RNA fractions. Lanes I, S, and P represent input, supernant, and pellet, respectively. Quantitation of this experiment is summarized in Table 3. (C) Analysis of the levels of URA5 mRNA decay intermediates. Primer extension analysis of the URA5 mRNA was performed on total RNA from each indicated yeast strain, as in Fig. 3A. Total RNA isolated from the upf3Δ strain was used as a control. The major transcriptional start sites of the URA5 mRNA and the 5′ ends of its decay intermediates are indicated by arrows and asterisks, respectively.

FIG. 7

In xrn1Δ cells, overexpression of the UPF1 gene decreases total mRNA levels as well as the levels of mRNA decay intermediates. (A) Northern analysis of total mRNA. xrn1Δ upf1Δ, xrn1Δ nmd2Δ, and xrn1Δ upf3Δ strains were transformed with a single-copy (S.C.) or a high-copy-number (H.C.) plasmid harboring the UPF1 gene. Total RNAs isolated from the resulting yeast strains, as well as the xrn1Δupf1Δ strain, were analyzed by Northern hybridization, using probes for the CYH2, URA5, GCN4, and CUP1 mRNAs. The SCR1 RNA was used as an internal control. (B) Analysis of mRNA decay intermediates. Primer extension analysis of the CYH2 pre-mRNA and the URA5 mRNA was performed on each of the RNAs used in panel A. Radiolabeled primers CYH2-IN4 and URA5-1 were used for both reverse transcription and DNA sequencing reactions. The major transcriptional start sites and the 5′ ends of decay intermediates are indicated by arrows and asterisks, respectively. The yeast strains labeled 1 to 7 correspond to those in panel A.

FIG. 8

Deletion of UPF1, NMD2, or UPF3 in dcp1Δ cells differentially affects the levels of the CYH2 pre-mRNA and mRNA. (A) Analysis of 5′ cap status. Total RNA was isolated from yeast strains of the indicated genotypes and anti-m⁷ G immunoprecipitation was performed as in Fig. 1B. Lanes I, S, and P represent input, supernatant, and pellet samples, respectively. (B) Northern analysis of total mRNA. Total RNA from yeast strains of the indicated genotypes was isolated and analyzed by Northern hybridization, using the SCR1 RNA as an internal control. WT, wild type. In both panels A and B, the CYH2 probe used was the same as in Fig. 1B. Quantitation of this experiment is summarized in Table 4.