BUD22 affects Ty1 retrotransposition and ribosome biogenesis in Saccharomyces cerevisiae

Abstract

A variety of cellular factors affect the movement of the retrovirus-like transposon Ty1. To identify genes involved in Ty1 virus-like particle (VLP) function, the level of the major capsid protein (Gag-p45) and its proteolytic precursor (Gag-p49p) was monitored in a subset of Ty1 cofactor mutants. Twenty-nine of 87 mutants contained alterations in the level of Gag; however, only bud22Delta showed a striking defect in Gag processing. BUD22 affected the +1 translational frameshifting event required to express the Pol proteins protease, integrase, and reverse transcriptase. Therefore, it is possible that the bud22Delta mutant may not produce enough functional Ty1 protease to completely process Gag-p49 to p45. Furthermore, BUD22 is required for 18S rRNA processing and 40S subunit biogenesis and influences polysome density. Together our results suggest that BUD22 is involved in a step in ribosome biogenesis that not only affects general translation, but also may alter the frameshifting efficiency of ribosomes, an event central to Ty1 retrotransposition.

Figure 1.—

Strategy used for identifying Ty1 cofactor mutants that affect the level or processing of the Ty1 Gag protein. A total of 4739 haploid MATα deletion mutants were screened to identify cellular genes that restrict (Nyswaner et al. 2008) or are required for endogenous Ty1 retrotransposition by monitoring the movement of a Ty1 element containing the his3-AI marker. Of the 168 Ty1 cofactor mutants (Table S1), 87 candidates were analyzed for Gag production (Figure S1), and a subset of these were analyzed for Ty1 mRNA level (Figure S2). Cofactor mutants obtained in screens for Ty1 (Griffith et al. 2003) and Ty3 (Aye et al. 2004; Irwin et al. 2005) are designated by asterisks (*, Ty3; **, Ty1 and Ty3 modulator).

Figure 2.—

Gag protein and Ty1 mRNA levels in the bud22Δ mutant. On the top is a scheme for Ty1 Gag processing. Most Gag-p45 protein is derived from PR-mediated cleavage of the Gag-p49 precursor near the C terminus (reviewed by Voytas and Boeke 2002). A minor amount of Gag-p45 is derived by cleavage of the Gag-Pol-p199 precursor protein (Garfinkel et al. 1991; Kawakami et al. 1993) that is produced by +1 frameshifting (+1Fs). Below is a Western blot showing the level and processing of Ty1-Gag in the wild type (WT; DG2122) and a bud22Δ mutant rederived in DG2122 (ADY1). Total protein was isolated from both wild-type and mutant strains, separated by SDS–polyacrylamide gel electrophoresis, and subjected to Western analysis using a polyclonal antiserum raised against purified Ty1-VLPs. The unprocessed (p49) and processed (p45) forms of Gag are indicated. The bottom panels show a Northern analysis of total RNA isolated from wild-type and bud22Δ strains. Total RNA was isolated from cells grown to mid-log phase and subjected to Northern analysis using a ³²P-labeled probe from the Ty1 reverse transcriptase (RT) domain. Ty1 mRNA was quantified by phosphorimage analysis and normalized to the total amount of 25S and 18S rRNA signals (bottom panel; also see Figure S2). The numbers below the lanes indicate the fold change in Ty1 mRNA level relative to the wild-type strain.

Figure 3.—

Spontaneous Ty1 insertions upstream of the glycine tRNA gene SUF16. (A) Schematic representation of the SUF16 locus on chromosome III. The arrows indicate the orientation of the PCR primers, SNR33 OUT and TYB OUT, which are derived from the SNR33 gene and Ty1, respectively. The insertion patterns for various strains reflect the hotspots for Ty1 integration upstream of SUF16 (Ji et al. 1993). (B) PCR analysis of DNA from five independent cultures of DG2122 (wild type) and ADY1 (bud22Δ). Control reactions using primers specific for the CPR7 gene are shown below. The PCR products were separated by electrophoresis on a 1.2% agarose gel, stained with ethidium bromide, and visualized by fluorescence imaging.

Figure 4.—

Unincorporated Ty1 cDNA decreases in the bud22Δ mutant. (A) Schematic depicting the unincorporated 2-kb cDNA fragment of Ty1 produced by PvuII digestion of total DNA. The solid bar represents the PvuII/ClaI restriction fragment of Ty1 that was used to synthesize the probe used in the Southern hybridization. (B) Total DNA was prepared from three wild-type (DG2122) and bud22Δ (ADY1) mutant strains, digested with PvuII, and subjected to Southern analysis using a ³²P-labeled probe derived from the RT domain of POL. The 2-kb Ty1 cDNA (asterisks) as well as the three chromosomal junction fragments used for normalization of the cDNA fragment (solid circles at right) are indicated.

Figure 5.—

Expression of Ty1 proteins following induction of a pGTy1 plasmid. (A) Accumulation of Ty1 Gag during galactose induction. Total protein was isolated at the indicated time points from wild-type (DG2984) and bud22Δ (ADY3) mutant cells expressing pGTy1his3-AI and subjected to Western analysis (50 μg/lane) using anti-VLP antiserum. The Gag-p49 precursor and mature Gag-p45 proteins are indicated. (B) Western analysis of Ty1 VLPs. Equivalent amounts of pooled and concentrated VLPs were subjected to Western analysis using anti-VLP, anti-IN, and anti-RT antisera.

Figure 6.—

25S:18S rRNA ratios in Ty1 and Ty3 cofactor mutants. Total RNA was isolated from the wild type (DG2122) and a variety of Ty1 and Ty3 cofactor mutants including bud22Δ (ADY1) (Table S1), separated by electrophoresis on a 1.2% agarose gel, and stained with ethidium bromide. The relative amounts of 25S and 18S rRNAs were estimated by fluorescence imaging and plotted as a histogram. Shaded bars (Griffith et al. 2003) and solid bars (Irwin et al. 2005) represent mutants identified in previous studies. A stippled bar denotes the asc1Δ mutant, which was isolated as a Ty3 cofactor (Nyswaner et al. 2008) and a Ty1 restriction mutant (Nyswaner et al. 2008). A hatched bar indicates the bud22Δ mutant and the rest are Ty1 cofactors identified in this study.

Figure 7.—

Sensitivity of the bud22Δ mutant to protein synthesis inhibitors. DG2122 (wild type) and ADY1 (bud22Δ) cells were grown to mid-log phase and then serially diluted (10-fold) onto YEPD plates containing the following antibiotics: paromomycin (1 mg/ml), neomycin (0.1 mg/ml), sparsomycin (10 μg/ml), and cycloheximide (0.5 μg/ml). The plates were photographed after incubation for 2 days at 30°.

Figure 8.—

Defective processing of pre-rRNAs in a bud22Δ mutant. (A) Scheme of the pre-rRNA processing pathway. The 35S pre-rRNA contains the sequences for mature rRNAs (18S, 5.8S, and 25S) separated by two internal transcribed spaces (ITS1 and ITS2) and is flanked by two external transcribed spaces (5′ETS and 3′ETS). The rRNAs are shown as solid bars and the transcribed spaces are represented as lines. The processing sites are indicated above the diagram by the uppercase letters A–E (Martin-Marcos et al. 2007). The positions of oligonucleotides 2, 3, and 6 used as hybridization probes are indicated beneath the primary transcript. (B) Wild-type (DG2122) and bud22Δ (ADY1) mutant strains were cultured in YEPD medium to mid-logarithmic phase, and total RNA was extracted and subjected to Northern analysis. The blots were hybridized with oligomeric probes shown in A that were end labeled with [γ-³²P]ATP using T4 polynucleotide kinase.

Figure 9.—

Analysis of ribosomes from wild-type and bud22Δ mutant strains. (A) Ribosomal profiles and detection of Ty1 and PYK1 transcripts. Strains DG2122 (WT) and ADY1 (bud22Δ) were grown to mid-log phase in SC −Ura. Ribosomes and ribosomal subunits were isolated and separated on a 7–50% sucrose gradient (see materials and methods). The top and bottom of the gradient and the ribosomes and subunits are marked. The numbered peaks are polysomes containing the indicated numbers of ribosomes. RNA extracted from an equivalent volume of each gradient fraction was subjected to Northern analysis using ³²P-labeled probes specific to Ty1 mRNA and PYK1 and is shown below the ribosomal profiles. (B) Polysome profiles were analyzed under ribosome dissociating conditions to monitor 40S and 60S subunits (see materials and methods).