BUD22 Affects Ty1 Retrotransposition and Ribosome Biogenesis in Saccharomyces cerevisiae
Arun Dakshinamurthy, Katherine M. Nyswaner, Philip J. Farabaugh, David J. Garfinkel


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 bud22Δ 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 bud22Δ 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.

THE Saccharomyces cerevisiae retrotransposon Ty1 is structurally and functionally related to retroviruses (Voytas and Boeke 2002). Ty1 elements are flanked by long terminal repeats (LTRs) and are transcribed from end to end, resulting in an mRNA that serves as a template for both translation and reverse transcription. The 45-kDa Gag protein is the major capsid protein of Ty1 virus-like particles (VLPs) (Adams et al. 1987). Most Gag-p45 is derived from proteolytic cleavage of Gag-p49, which is the primary translation product of the GAG gene. A +1 frameshift signal near the 3′ end of GAG results in the production of a 199-kDa Gag-Pol fusion protein (Clare et al. 1988; Belcourt and Farabaugh 1990), which contains the Gag structural protein, and the enzymes protease (PR), integrase (IN), and reverse transcriptase (RT) (Adams et al. 1987; Muller et al. 1987; Eichinger and Boeke 1988; Youngren et al. 1988; Garfinkel et al. 1991). The efficiency of frameshifting helps determine the optimal ratio of Gag and Gag-Pol proteins (estimated at ∼30:1) that is required for PR function and retrotransposition (Xu and Boeke 1990; Kawakami et al. 1993). PR processes both the Gag and Gag-Pol translation products during maturation of Ty1 VLPs (Youngren et al. 1988;Garfinkel et al. 1991). Cleavage at the Gag-PR cleavage site present in Gag-p49 or the Gag-Pol-p199 precursor yields the mature Gag-p45 protein. Gag-Pol-p199 is hydrolyzed first at the Gag-PR cleavage site, and the p160-Pol processing intermediate is cleaved to release PR, IN, and RT (Garfinkel et al. 1991; Merkulov et al. 1996, 2001).

Programmed +1 frameshifting occurs in a 38- to 44-bp overlap between the Ty1 GAG and POL open reading frames (Clare et al. 1988; Belcourt and Farabaugh 1990). Extensive deletion and missense mutagenesis analyses have defined the heptamer CUU-AGG-C as necessary and sufficient for +1 frameshifting. Overlapping leucine codons, CUU in the 0 frame and UUA in the +1 frame, are recognized by a single isoacceptor, leucyl-tRNA (UAG), which can recognize all six of the leucine codons by virtue of an unmodified uracil in its wobble position. The AGG codon in the heptamer is recognized by the low-abundance arginyl-tRNA (CCU) produced from the HSX1 gene (Kawakami et al. 1993). When ribosomes encountering the AGG codon pause because of the low availability of arginyl-tRNA, the peptidyl-leucyl-tRNA in the P site of the ribosome (on the CUU codon) slips forward +1 during the pause onto the UUA codon, and translation continues in the +1 reading frame (Belcourt and Farabaugh 1990).

BUD22 was discovered in a systematic screen of homozygous diploid yeast deletion mutants with an altered budding pattern (Ni and Snyder 2001). Several genome-wide analyses suggest that Bud22p may be involved in RNA processing and ribosome biogenesis. Affinity capture–mass spectrometry and two-hybrid studies implicate Bud22p in rRNA synthesis (Krogan et al. 2006). Bud22p associates with Ssf1p (Fatica et al. 2002), which is involved in the prevention of premature processing of an early pre-60S ribosomal particle and is also present in the nucleus and nucleolus (Ghaemmaghami et al. 2003; Malmstrom et al. 2007). Finally, BUD22 is one of >200 genes enriched for PAC and RRPE sequence motifs that are present in the promoters of genes comprising the ribosomal and rRNA transcriptional regulon (Wade et al. 2006). However, investigation of BUD22's role in RNA processing and ribosome biogenesis has not been explored directly.

Here, we report the results of a systematic screen of the S. cerevisiae deletion collection for genes that are required for the movement of endogenous Ty1 elements. In addition, the level of Gag-p49 and -p45 was monitored in a subset of the cofactor mutants. The secondary screen identified BUD22 as a Ty1 cofactor required for the accumulation of mature Gag-p45 and +1 frameshifting. BUD22 also plays a role in the formation of 18S and 5.8S rRNAs and the 40S subunit.


Strains, media, and genetic methods:

Yeast genetic techniques and media were used as described previously (Sherman et al. 1986; Guthrie and Fink 1991). Strains were grown at 20° for all experiments (Paquin and Williamson 1984) unless otherwise mentioned. The haploid MATα deletion collection (Giaever et al. 2002) was obtained from Invitrogen (Carlsbad, CA). A total of 4739 deletion mutants derived from BY4742 (MATα his31 leu20 lys20 ura30) (Brachmann et al. 1998) were transformed with pJC573, a URA3-based integrating plasmid carrying an active Ty1 element tagged with a modified indicator gene his3-AId1 (designated his3-AI) (Curcio and Garfinkel 1991; Bryk et al. 2001; Scholes et al. 2001;Nyswaner et al. 2008), which cannot undergo recombination with the his31 allele present in BY4742 to generate a functional HIS3 gene. Strain DG2122 was constructed by introducing pJC573 into BY4742 (Nyswaner et al. 2008). Strain DG2984 was constructed by introducing pBDG606 (pGTy1-H3his3AI/Cen-URA3) into BY4742. Strain JC242 was isolated after galactose induction of plasmid pGTy1his3AI in strain GRF167 (MATα, ura3-167, his3200, GAL) and subsequent loss of the pGTy1his3-AI plasmid (Curcio and Garfinkel 1991). JC242 contains a genetically tagged chromosomal element (Ty1-242his3-AI) that can generate Ty1HIS3 mobility events. Strains ADY1, ADY2, and ADY3 were generated by disrupting the BUD22 gene in strains DG2122, JC242, and DG2984, respectively. One-step gene disruptions were performed using KanMX4-targeting fragments (Wach et al. 1994), amplified from the deletion mutants using the gene-specific flanking primers A and D (http://www-sequence.stanford.edu/group/yeast_deletion_project/Deletion_primers_PCR_sizes.txt). Mutant identity was verified by PCR using A and D primers and phenotypic analyses. The plasmids pMB38-9merWT and pMB38-9merFrameFusion contain the frameshift heptamer fused to the Escherichia coli lacZ gene in the +1 reading or 0 reading frame, respectively (Belcourt and Farabaugh 1990).

Ty1 mobility:

The Ty1his3-AI mobility assay used for systematic screening has been previously described (Nyswaner et al. 2008). Ty1 host cofactor mutants that consistently displayed a lower level of Ty1his3-AI mobility in at least three of the four transformants were saved for further analysis. A threefold decrease in Ty1his3-AI mobility could be reproducibly detected using this qualitative assay. The efficiency of Ty1his3-AI mobility was determined as described previously (Curcio and Garfinkel 1991; Nyswaner et al. 2008).

Detecting Ty1 insertions at the SUF16 locus:

Spontaneous Ty1 insertions upstream of the SUF16 (glycine-tRNA) locus on chromosome III were detected as described previously (Nyswaner et al. 2008). Briefly, PCR was performed with oligonucleotide primers specific to Ty1 POL (TYB OUT: 5′-GAACATTGCTGATGTGATGACA-3′) and SNR33 (SNR33 OUT: 5′-TTTTAGAGTGACACCATCGTAC-3′). Control reactions using primers specific to the CPR7 locus (CPR7A: 5′-GTTTGTGATTTATCTCTGGACTGCT-3′ and CPR7D: 5′-AGTTCGTCTCTCCTTCATATTCTCA-3′) ensured that the DNA samples were PCR competent.

Northern analysis:

Total RNA was isolated as previously described (Martin-Marcos et al. 2007; Schmitt et al. 1990) from strains grown to late log phase in 10 ml SC −Ura medium. Hybridization analysis to detect Ty1 mRNA was performed as described previously (Lee and Culbertson 1995; Lee et al. 1998), and the signals were quantified using a Typhoon Trio phosphorimager and ImageQuant 1.2 software (GE Healthcare, Piscataway, NJ). To measure steady-state levels of pre-rRNA and rRNA in wild-type and bud22Δ strains, total RNA was prepared and hybridized as described previously (Martin-Marcos et al. 2007). Briefly, cells were grown in YEPD broth to an OD600 of 1. RNA samples (5 μg) were separated by electrophoresis using 1.2% agarose–formaldehyde gels. The RNAs were blotted to positively charged nylon membranes (GE Healthcare), immobilized by UV crosslinking using a Stratalinker 1800 (Stratagene, La Jolla, CA), and detected by hybridization with 20-base oligonucleotides labeled at their 5′ ends with [γ-32P]ATP (6000 Ci/mmol) (Perkin-Elmer, Waltham, MA) using T4 polynucleotide kinase (KINASEMAX; Ambion, Austin, TX). The oligonucleotides used as probes are oligonucleotides 2, 5′-AGCCATTCGCAGTTTCACTG-3′; 3, 5′-TTAAGCGCAGGCCCGGCTGG-3′; and 6, 5′-GGCCAGCAATTTCAAGTTA-3′.

Western analysis:

Total cell protein was isolated from strains as described previously (Atkin et al. 1995). Total protein (50 μg/lane) and isolated VLPs were analyzed by Western blotting using polyclonal antisera B2 for IN, B8 for RT, and anti-VLP (a generous gift from Alan and Susan Kingsman) for Gag as previously described (Adams et al. 1987; Garfinkel et al. 1991). Cross-reactivity was detected by chemiluminescence with secondary ECL HRP-linked anti-rabbit antibodies (GE Healthcare). Protein concentrations were determined by the Bradford dye-binding assay (Bio-Rad, Hercules CA).

Detecting unincorporated Ty1 cDNA:

Southern analysis of Ty1 cDNA was performed as described previously (Lee et al. 1998; Nyswaner et al. 2008). The intensity of the 2-kb cDNA band was determined by phosphorimage analysis and normalized to three conserved Ty1-chromosomal junction fragments.

Ty1 frameshifting efficiency:

In vitro assays of β-galactosidase activity were performed as described previously (Rose and Botstein 1983). Six transformants of pMB38-9merWT and pMB38-9merFrameFusion were analyzed for wild-type and mutant strains. The frameshifting efficiency was obtained from the ratio of β-galactosidase activity produced by cells containing pMB38-9merWT that requires a +1 frameshift to express lacZ to the activity produced by the pMB38-9merFrameFusion plasmid in which the upstream and downstream sequences are fused in frame (Belcourt and Farabaugh 1990).

Ty1-VLP purification:

VLPs were prepared by differential centrifugation and sucrose gradient sedimentation as previously described (Eichinger and Boeke 1988; Moore and Garfinkel 2009).

Ty1-VLP integrity:

This qualitative assay was modified from one previously described (McCarthy et al. 1998; Roth 2000). Briefly, an aliquot of a VLP preparation (60 μg) was mixed with VLP disruption buffer (250 mm Tris-HCl, pH 7.4, 15 mm KCl, 10 mm HEPES-KOH, pH 7.4, and 5 mm EDTA) in a total volume of 100 μl. The VLPs were incubated at 37° for 30 min and layered on a 2-ml 30% sucrose cushion, also prepared in VLP disruption buffer. Samples were centrifuged for 3 h, at 36,000 rpm, at 30° in a Beckman TL-100 centrifuge using a swinging-bucket rotor (Beckman Coulter, Brea, CA). A 200-μl aliquot from the top of the tube was removed and saved as the supernatant (S) fraction. The remaining solution from the tube was drained and the pellet (P) was dissolved in 200 μl buffer A (15 mm KCl, 10 mm HEPES-KOH, pH 7.4, and 5 mm EDTA). The presence of Ty1-Gag proteins at the top or bottom of the 30% sucrose cushion was determined by Western analysis.

Estimating the levels of 18S and 25S rRNA:

Total RNA was extracted from individual cultures of wild-type and mutant strains that were grown in YEPD to late log phase. Samples (10 μg) were 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 flurorescence imaging using a Typhoon Trio phosphorimager and ImageQuant 1.2 software. Band intensities were determined using the object average for background correction. The rRNA signals were robust and well separated, with standard deviations <0.1% of the object volume.

Polysome analysis and total ribosomal subunit quantification:

Polysomes were prepared from cells grown in SC −Ura and fractionated on 7–50% sucrose gradients as described previously (Baim et al. 1985). The gradients were centrifuged at 35,000 rpm in a Beckman SW41 rotor (Beckman Coulter) at 4° for 2 hr 45 min and analyzed with an ISCO UV-6 continuous gradient collector (Teledyne Isco, Lincoln, NE) with the UV detector set at 254 nm.

Ty1 mRNA in polyribosomal fractions:

The polyribosomal RNA was extracted and the Ty1 mRNA was assayed using established procedures (Ballinger and Pardue 1983; Zaret and Sherman 1984). Fractions (1 ml) of the sucrose gradient were collected and precipitated overnight at −20° with 3 ml of 95% ethanol. The precipitate was sedimented by centrifugation for 10 min at 10,000 × g. The precipitate was resuspended in 300 μl of buffer containing 20 mm Tris (pH 7.4), 2.5 mm EDTA, 100 mm NaCl, and 1% sodium dodecyl sulfate. The solution was extracted twice with phenol, once with phenol–choloroform–isoamyl alcohol (25:24:1), and once with chloroform–isoamyl alcohol (24:1) and then precipitated with 2.5 ml of 95% ethanol at −20°. The precipitated RNA was collected, suspended in 100 μl of sterile water, and lyophilized. Each sample of RNA was suspended in 10 μl of sterile water and run on a 1.2% agarose gel containing 4% formaldehyde. RNA was transferred to nylon membranes in 10× SSC and sequentially hybridized with 32P-labeled Ty1 and PYK1 probes.


Identifying Ty1 host cofactor genes involved in Gag accumulation and processing:

The efficiency of Ty1 movement was estimated using a qualitative papillation assay for histidine prototrophs after integrating a plasmid-borne (pJC573) Ty1his3-AI (artificial intron) element (Bryk et al. 2001; Scholes et al. 2001) upstream of the HIS4 gene in 4739 MATα haploid deletion mutants (Nyswaner et al. 2008). His+ cells usually arise by retrotransposition of Ty1HIS3 to a new location following removal of the AI from the mRNA by splicing and reverse transcription. However, His+ events can also occur by homologous recombination between the Ty1HIS3 cDNA and a chromosomal element. Therefore, “Ty1 mobility” is used to describe both classes of Ty1HIS3 events. Four independent transformants of each mutant were analyzed for Ty1his3-AI mobility alongside the isogenic wild-type strain DG2122 that also contains pJC573. An increase or a decrease of about threefold in His+ papillae could be reproducibly detected using this assay and at least three of four transformants for a given mutant had to show a decrease in mobility to be studied further. Mutations that increase Ty1his3-AI mobility have been published previously (Nyswaner et al. 2008), and mutations that decrease mobility are presented here (supporting information, Table S1).

To identify mutations required for the synthesis and processing of Ty1 Gag, a secondary screen based on Western blotting was performed with an antiserum raised against purified Ty1-VLPs and total cell extracts from 87 of 168 cofactor mutants (Table S1). A candidate gene approach was used to select Ty1 cofactors that may affect the level or processing of Ty1 Gag post-transcriptionally. The mutants were defective in a variety of cellular functions, including bud site selection, RNA transactions, and protein trafficking. Each set of Ty1 cofactor mutants was analyzed in parallel with the wild-type strain DG2122 and an isogenic spt3Δ mutant that produces a low level of Ty1-Gag due to a defect in Ty1 transcription (Winston et al. 1984). Although there was some variability in the level of Ty1-Gag p49 and p45 detected from DG2122, several expression patterns of Gag proteins were observed (Figure 1 and Figure S1). Fifty-eight mutants showed essentially no change in Gag levels when compared with the wild type. However, 29 mutants contained an alteration in the level or processing of Gag; 26 contained a lower level of Gag, 2 contained more Gag, and 1 mutant exhibited an apparent defect in PR-mediated cleavage of Gag-p49 to mature Gag-p45. To determine if the alteration in Gag level was caused by a change in Ty1 mRNA abundance, Northern blotting was performed using a 32P-labeled probe from the RT domain of Ty1 and total RNA isolated from the 22 of 29 cofactor mutants that had an alteration in Gag level or processing (Figure 1 and Figure S1). Twelve mutants showed more than a twofold decrease in Ty1 mRNA level and were not analyzed further (Figure S2). Of the remaining 10 mutants that showed less than a twofold decrease in Ty1 mRNA, 7 contained a lower level of Gag (dhh1Δ, pho88Δ, pop2Δ, pub1Δ, ski8Δ, tgs1Δ, and ynl228wΔ), 2 contained a higher level of Gag (clc1Δ and vps16Δ), and bud22Δ contained mostly unprocessed Gag-p49. The post-transcriptional defect in Gag processing was evident in the original bud22Δ isolate from the deletion collection (Figure S1 and Figure S2) as well as when the bud22Δ mutation was rederived (ADY1) in DG2122 (Figure 2). Since BUD22 affects the formation of mature Gag-p45, which is an indicator of VLP assembly and the synthesis of Pol proteins, and BUD22's cellular function was not well understood, we chose this gene for further analyses.

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.

Ty1his3-AI mobility decreases in a bud22Δ mutant:

We determined the efficiency of Ty1his3-AI mobility in the wild-type strain DG2122, which contains a functional Ty1his3-AI element carried on pJC573 (Bryk et al. 2001; Scholes et al. 2001; Nyswaner et al. 2008), and the isogenic bud22Δ mutant ADY1. Deletion of BUD22 resulted in a fivefold decrease in the rate of His+ colony formation (Table 1). In addition, mobility of a chromosomal Ty1his3-AI element decreased sixfold in an independent strain background (JC242) lacking BUD22 (ADY2). Also, we examined GAL1-driven Ty1his3-AI mobility and observed an eightfold reduction in a bud22Δ mutant (ADY3) when compared with a wild-type strain (DG2984). Therefore, BUD22 is required for mobility of native Ty1 elements as well as when Ty1his3-AI is overexpressed from a pGTy1his3-AI plasmid.

View this table:

Ty1his3-AI mobility in bud22Δ mutants

BUD22 is required for de novo Ty1 retrotransposition events:

Ty1 elements preferentially integrate in specific regions upstream of genes transcribed by RNA polymerase III (Devine and Boeke 1996). Transposition into the region upstream of the SUF16 (tRNA glycine) locus on chromosome III (Ji et al. 1993) can be monitored by a qualitative PCR assay that detects spontaneous insertion events in a growing culture of cells (Lee et al. 2000). Genomic DNA was isolated from five independent cultures of DG2122 (wild type) and ADY1 (bud22Δ) strains that were grown to late log phase. The DNA samples were subjected to PCR using primers specific to Ty1 (TYB OUT) and SNR33 (SNR33 OUT), which is a single-copy gene adjacent to SUF16 (Figure 3A). The PCR products were resolved on a 1.2% agarose gel containing ethidium bromide and analyzed by fluorescence imaging. Exceptionally intense bands in certain cultures suggest that a Ty1 insertion occurred early in cell growth, creating a “jackpot” event. The results show a decreased level of Ty1 insertions upstream of the SUF16 locus in the bud22Δ mutant compared with the wild-type strain (Figure 3B). Although the frequency of insertions was lower in the mutant, the targeting pattern resembled that of 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.

The level of unincorporated Ty1 cDNA decreases in a bud22Δ mutant:

Genomic DNA samples from wild-type (DG2122) and bud22Δ (ADY1) strains grown in triplicate were analyzed by Southern blot hybridization using a 32P-labeled DNA probe derived from the RT region of Ty1. PvuII digestion of linear unincorporated cDNA present in total DNA generates a characteristic 2-kb fragment (Figure 4A) containing sequences from a conserved internal PvuII restriction site in Ty1 (nt 3944) to the end of the element (nt 5918). The RT probe hybridizes with unincorporated Ty1 cDNA as well as with genomic Ty1 elements, thereby generating a variety of Ty1-chromosomal junction fragments (Figure 4B). The level of unincorporated Ty1 cDNA was quantified relative to the level of three different Ty1-genomic junction fragments in wild-type and bud22Δ strains. The bud22Δ mutant had 2.5-fold less Ty1 cDNA relative to the wild-type strain. Therefore, the lower level of Ty1 transposition is correlated with a decrease in Ty1 cDNA in the mutant.

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.

Processing of Gag-p49 is defective in a bud22Δ mutant overexpressing a Ty1 element:

Strains DG2984 (wild type, WT) and ADY3 (bud22Δ) containing a pGTy1his3-AI plasmid were grown in SC −Ura medium containing glucose and transferred to SC −Ura containing galactose to induce GAL1-promoted Ty1 expression (Figure 5). Total protein was extracted from cells at various times up to 8 hr after galactose addition and assayed by Western analysis using anti-VLP antiserum (Figure 5A). Gag-p49 remained mostly unprocessed even after 8 hr of galactose induction in the bud22Δ mutant, whereas processing of Gag-p49 to -p45 occurred in wild-type cells.

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.

To examine VLP proteins more closely, wild-type and bud22Δ strains were induced with galactose and Ty1-VLPs were isolated by sucrose gradient sedimentation (Eichinger and Boeke 1988). Fractions enriched for VLPs were pooled and analyzed by Western blotting for the presence of Gag, IN, and RT (Figure 5B). Anti-VLP antiserum detected both the Gag-p49 precursor protein and mature Gag-p45 in VLPs from the wild type (DG2984). However, the Gag-p49 precursor was the major capsid protein in VLPs isolated from the bud22Δ mutant. Different amounts of VLP-enriched fractions from both wild-type and mutant cells were analyzed to estimate the relative abundance of VLP proteins. In general, the level of Gag, IN, and RT was decreased in VLP preparations from the bud22Δ mutant when compared with the wild type. In contrast to Gag, IN and RT were processed at wild-type levels in bud22Δ VLPs. Reverse transcriptase activity using exogenously added primer and template also reflected the lower yield of VLPs from the bud22Δ mutant (data not shown). Together, these results suggest the production of VLPs is reduced in the bud22Δ mutant; however, the VLPs isolated from the mutant show a significant Gag-processing defect.

To determine if the VLPs composed mainly of the Gag-p49 capsid precursor were less stable (Figure S3), samples were treated with 0.25 m Tris–HCl for 30 min at 37° and then pelleted through a 30% sucrose cushion. This treatment has been shown to destabilize Ty1 VLPs (Roth 2000), as monitored by the amount of capsid protein in the VLP pellet vs. the supernatant. However, the sedimentation profile of VLPs from the wild type or the bud22Δ mutant remained unchanged after treatment with Tris-HCl, suggesting that VLPs from the bud22Δ mutant are stable.

rRNA ratios are altered in bud22Δ and several other mutants involved in ribosomal biogenesis:

During the course of these studies, we noted that the ratio of 25S:18S rRNA was consistently altered in ADY1 (bud22Δ) (Figure 2 and Figure S2). To check if this aberration was unique to cells lacking Bud22p, we estimated the relative amounts of 25S and18S rRNAs in bud22Δ and 41 other Ty1 host cofactor mutants (Table S1) defective in RNA processing or protein synthesis to the wild-type strain DG2122 by agarose gel electrophoresis and fluorescence imaging (Figure 6). Most of the mutants had 25S:18S ratios within 50% of that observed in the wild-type strain and were not analyzed further. However, with the exception of rps0BΔ, bud22Δ showed the highest 25S:18S ratio among the Ty1 cofactor mutants screened qualitatively for discordant rRNA levels. Rps0Bp is a component of the 40S ribosomal subunit, and cells lacking this protein have reduced amounts of 40S ribosomal subunits (Ford et al. 1999). Interestingly, the rps0BΔ mutant also exhibited defects in Ty1his3-AI mobility (data not shown, Table S1) and Gag processing similar to that of bud22Δ (Figure S4).

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.

BUD22 and RPS0B are required for optimal +1 Ty1 frameshifting:

As with certain retroviral POL genes, programmed ribosomal frameshifting is used to express Ty1 POL (Clare et al. 1988; Belcourt and Farabaugh 1990). The efficiency of Ty1 frameshifting was estimated using the pMB38-9merWT and pMB38-9merFrameFusion reporter plasmids. Both plasmids carry a HIS4A∷lacZ fusion gene; however, pMB38-9merWT contains the minimal heptameric sequence required for +1 frameshifting inserted between the yeast HIS4A gene and the bacterial lacZ reporter. In the pMB38-9merFrameFusion plasmid the HIS4A and lacZ genes are fused into a single open reading frame such that expression of β-galactosidase does not require frameshifting.

β-Galactosidase activity was determined from six independent transformants containing each plasmid and the Ty1 frameshifting efficiencies were calculated by determining the ratio of β-galactosidase activity produced from a construct (pMB38-9mer) requiring a +1 frameshift to express lacZ to that of a construct (pMB38-9merFrameFusion) in which the upstream and downstream genes are in frame (Table 2). A frameshifting efficiency of 13.5% was obtained in the wild-type (DG2122) strain, but decreased to 4.4% in the bud22Δ mutant (ADY1). Similar results were also obtained with the rps0BΔ mutant. Two Ty1 cofactor mutants (rps10AΔ and rps19BΔ) with 25S:18S rRNA ratios elevated to almost the same extent as bud22Δ were analyzed for +1 Ty1 frameshifting as additional controls for specificity. In contrast to bud22Δ and rps0BΔ, Ty1 frameshifting remained at wild-type levels in the rps10AΔ and rps19BΔ mutants, although there may be a general defect in synthesizing β-galactosidase. These results suggest that the bud22Δ and rps0BΔ mutations specifically reduce Ty1 transposition and +1 frameshifting.

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The efficiency of Ty1 frameshifting

The bud22Δ mutant's sensitivity to aminoglycoside antibiotics suggests a defect in ribosome function:

The growth of isogenic bud22Δ (ADY1) and wild-type (DG2122) strains was assessed in the presence of the protein synthesis inhibitors, paromomycin and neomycin (both aminoglycosides), sparsomycin (kindly provided by John Beutler, National Cancer Institute, Frederick, MD) and cycloheximide (Figure 7). The strains were grown overnight in YEPD liquid medium at 30° and serially diluted onto YEPD plates with or without the antibiotics. Although the bud22Δ mutation did not alter the sensitivity to sparsomycin or cycloheximide, the mutation conferred a marked increase in sensitivity to paromomycin and neomycin. Sensitivity to aminoglycoside drugs has been previously reported to be a manifestation of defects in the 40S subunit (Lee et al. 1992; Kressler et al. 1997).

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°.

Bud22p affects rRNA processing:

To investigate possible defects in the rRNA maturation pathway caused by the bud22Δ mutation, rRNA processing was examined by Northern blotting. We detected various rRNA processing intermediates in total RNA isolated from bud22Δ (ADY1) and wild-type (DG2122) cells grown to mid-log phase (Figure 8A). In the bud22Δ mutant, the 35S rRNA accumulated and 32S was depleted, indicating a possible defect in cleavage at sites A0 and A1 (Figure 8B). Nevertheless, the overall level of 25S was unaffected (data not shown). In addition, 20S pre-rRNA, which is exported to the cytoplasm as part of the 43S preribosomal particle and subsequently processed at site D, accumulated to a higher level in the mutant, while the level of 18S rRNA was reduced. The bud22Δ mutant showed a marked accumulation of 7S pre-RNA, which is a precursor to 5.8S rRNA. These results suggest that Bud22p may not only be required for the initial cleavage events, but could directly or indirectly affect events downstream of 20S and 7S pre-rRNAs. Also, the lack of complete processing of 20S pre-rRNA to 18S rRNA may reflect a defect in export or processing of 20S rRNA.

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.

BUD22 is involved in biogenesis of the 40S subunit:

Because the production of 18S rRNA is modulated by BUD22, we determined if the quantity of 40S ribosomal subunit is depleted in the bud22Δ mutant ADY1. Wild-type (DG2122) cells had a typical polysome profile consisting of the 40S and 60S subunits, 80S monoribosomes, and seven to eight polysomes (Figure 9A), as detected by sucrose gradient centrifugation analysis. In contrast, the bud22Δ mutant had a reduced 40S peak and a dramatically enlarged 60S peak, which is often observed when there is a defect in 18S processing and accumulation of free large ribosomal subunits (Liu and Thiele 2001). The distribution of PYK1 and Ty1 mRNA transcripts in the fractionated polyribosomes was also determined by Northern analysis. These results show that the bulk of PYK1 and Ty1 mRNA transcripts was associated with polysomes containing six to eight ribosomes in the wild-type cells, whereas these transcripts were distributed among a broader range of polysomes in the bud22Δ mutant.

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).

To quantify the deficiency of 40S subunits in the bud22Δ mutant, levels of 40S and 60S subunits were monitored under ribosome-dissociating conditions, which were achieved by omitting cycloheximide from the culture, and harvesting cells after a 20-min treatment with 1 mm NaN3 to produce polysome runoff (Liu and Thiele 2001). Analysis of sucrose gradients prepared without Mg2+ revealed a considerable increase in the 60S to 40S subunit ratio in bud22Δ cells (Figure 9B). The ratio of 25S to 18S rRNAs increased from 1.2 in the wild type to 2.1 in the bud22Δ mutant, and the relative concentration of 60S subunits increased from a mass fraction of 2.03 to 3.36 in the mutant. These results suggest that Bud22p is required for 40S subunit biogenesis and probably affects translation globally.


Of the 87 Ty1 cofactor mutants screened for the production of Gag proteins, bud22Δ was the only mutant that exhibited a Gag processing defect. The bud22Δ mutant shows at least a fivefold decrease in Ty1his3-AI mobility and a reduced level of de novo retrotransposition events upstream of SUF16. To understand how BUD22 modulates Ty1 retrotransposition, we examined the effect of a bud22Δ mutation on several additional steps in the process of Ty1 retrotransposition. Bud22p acts post-transcriptionally to enhance Ty1 transposition since cells lacking this protein contain essentially wild-type levels of Ty1 mRNA. The level of unincorporated Ty1 cDNA decreases in the bud22Δ mutant, suggesting that reverse transcription or cDNA stability is partially defective. GAL1-promoted production of Ty1 VLPs is reduced in the bud22Δ mutant, as monitored by the level of Gag, RT, and IN proteins and reverse transcriptase activity. However, the Gag protein present in total cell extracts or isolated VLPs is almost entirely unprocessed in the mutant. Thus, the reasons for a lower level of Ty1 transposition in the bud22Δ mutant may be attributed to inefficient Gag processing and an overall reduction in the synthesis of Ty1 proteins.

We further sought to understand how BUD22 affects the formation of Gag-p45, which is a key step in the process of retrotransposition. One possibility is that BUD22 is involved in optimizing Ty1 Gag and Gag-Pol stoichiometry through its effect on Ty1 frameshifting and the synthesis of PR. Previous work on factors affecting the efficiency of Ty1 frameshifting demonstrates the importance of maintaining the correct balance between Gag-Pol and Gag precursors for protein processing and retrotransposition (Xu and Boeke 1990; Kawakami et al. 1993). The efficiency of Ty1 frameshifting is ∼3-fold lower in the bud22Δ mutant, which may decrease the levels of the Gag-Pol precursor and mature PR. One idea generally consistent with our results is that the level of PR may be high enough to cleave Gag-Pol but is not sufficient to cleave Gag-p49 to Gag-p45 in the absence of Bud22p. Note that Gag-p49 is present at an ∼30-fold higher level than Gag-Pol (Garfinkel et al. 1991; Kawakami et al. 1993), thus requiring more cleavage events to generate Gag-p45. However, one would have still expected a reduction in Pol processing in the mutant if PR was limiting, which we did not observe by Western analysis of fractionated VLPs (Figure 5B). An additional factor to consider is that the level of Ty1 proteins and yield of VLPs decrease in the bud22Δ mutant. Therefore, BUD22 may also promote VLP assembly independently of +1 frameshifting by enhancing the synthesis of Ty1 proteins.

Differences in ribosomal frameshifting and the available information on the function of BUD22 led us to examine the relative amounts of 18S and 25S rRNAs in the mutant. We included 41 other Ty1 cofactor mutants that are defective in RNA processing or protein synthesis and listed them on the basis of their rRNA ratios. Among the mutants tested, bud22Δ shows an elevated 25S:18S ratio, second only to rps0BΔ. Rps0 proteins are required for the maturation of 40S ribosomal subunits by promoting the efficient processing of 20S rRNA precursor to 18S rRNA (Ford et al. 1999). Similar to bud22Δ's effect on cellular bud site selection (Ni and Snyder 2001), microscopic analysis of cells depleted of Rps0p also revealed an approximately 2:1 ratio of large unbudded cells relative to cells that had either a single large bud or multiple buds, showing that proteins involved in rRNA biogenesis can perturb budding patterns in yeasts (Ford et al. 1999). Furthermore, RPS0B was identified as a Ty1 cofactor that affects both Ty1 frameshifting and Gag processing.

Since bud22Δ and rps0BΔ mutants show similar defects in 18S rRNA processing, we determined if bud22Δ exhibits defects in 40S biogenesis using several different approaches. First, we show that the bud22Δ mutant is very sensitive to the aminoglycoside drugs paromomycin and neomycin. Sensitivity to paromomycin and neomycin has been previously demonstrated in fal1-1Δ, nsr1Δ, rps18aΔ, and rps18bΔ mutants that also manifest a 40S defect (Lee et al. 1992; Folley and Fox 1994; Kressler et al. 1997). Furthermore, increased drug sensitivity supports biophysical and structural data showing direct binding of these aminoglycosides to the bacterial small ribosomal subunit (Carter et al. 2000).

Second, we analyzed the effects of Bud22p depletion on processing of pre-rRNAs. Consistent with the cellular deficiency of 40S subunits, analysis of steady-state levels of rRNAs revealed a partial block in 20S rRNA processing and moderately less 18S rRNA in the bud22Δ mutant. This pattern of rRNA processing is also observed in strains harboring conditional alleles in genes required for 40S biogenesis, including the RNA helicases ROK1, RRP3, and FAL1 (Venema and Tollervey 1995; O'Day; et al. 1996; Kressler et al. 1997); the nucleolar proteins NOP1, GAR1, SOF1, and NOP5 (Tollervey et al. 1991; Girard et al. 1992; Jansen et al. 1993; Wu et al. 1998); several snoRNAs (U3, U14, and snR30) (Li et al. 1990; Hughes and Ares 1991; Morrissey and Tollervey 1993); and the RNA methylase DIM1 (Lafontaine et al. 1995). In addition to the 20S rRNA processing deficiency, loss of BUD22 causes an accumulation of the 35S rRNA and 7S RNA precursors. Instead of cleavage at A2, 35S rRNA may be cleaved at site E to generate the 23S pre-rRNA species (Figure 8 and Figure S5). Subsequent cleavage at or near A2 would yield a 7S species that may no longer be susceptible to cleavage at site B1. The defect could be either in a processing enzyme responsible for the first cleavage in wild-type strains or due to a defective factor that leads the pre-rRNA to form an altered conformation in the processing complex. Loss of Bud22p also caused diminution of the 32S rRNA precursor, which is characteristic of typical A0 to A1 processing-site inhibition. Together, our results suggest that Bud22p affects multiple steps during rRNA maturation; however, its biochemical function remains to be determined.

Third, polysome analysis and total ribosomal subunit quantification reveal a subunit imbalance leading to an excess of free 60S over free 40S subunits in the bud22Δ mutant. Such profiles have been described for mutations in 40S ribosomal genes (Abovich et al. 1985; Folley and Fox 1994) and for genes implicated in 40S biogenesis (Finley et al. 1989; Lee et al. 1992). It has been previously shown that 40S subunits containing 20S rRNA are excluded from polysomes and presumably inactive in protein synthesis (Udem and Warner 1973). Cells depleted of Rps0 proteins have high levels of immature subunits containing the 20S precursor and may also be inactive in protein synthesis (Ford et al. 1999). If we assume that the immature subunits containing 20S rRNA are degraded at a faster rate than mature 40S subunits, the reduced rate of rRNA processing may explain the overall reduction in the level of 40S subunits in the bud22Δ mutant. Additionally, the decrease in the polysome density observed in a bud22Δ mutant suggests there may be a defect in the rate of translation initiation. This defect in translation may cause a reduction in the capacity to synthesize cellular and Ty1 proteins and may also help explain the growth defect of the mutant.

In conclusion, we have shown that BUD22 affects maturation of 18S rRNA and biogenesis of the 40S subunit. Previous genomic analyses also suggest a role for Bud22p in ribosome production or function (Fatica et al. 2002; Krogan et al. 2006; Wade et al. 2006). However, our work supports a more specific role for Bud22p and Rps0p in the biogenesis of the 40S ribosomal subunit and +1 frameshifting, since other Ty1 cofactors (Rps10Ap and Rps19Bp) that affect ribosome function do not affect +1 frameshifting. Although it remains to be determined how Bud22p and Rps0p enhance +1 frameshifting and the accumulation of mature Gag-p45, changes in ribosomal function may indirectly affect +1 frameshifting by altering the competition between continued in-frame decoding and +1 frameshifting at the frameshift site. There is abundant evidence that frameshifting can be strongly reduced by increasing the availability of tRNAs required to continue reading in the normal reading frame (Stahl et al. 2001); for example, frameshifting depends on the slow recognition of the AGG codon at the frameshift site by a low-abundance arginyl-tRNA (Belcourt and Farabaugh 1990). If the reduction in ribosome concentration was not accompanied by a comparable reduction in tRNA abundance, this tRNA might become too abundant relative to ribosome concentration to stimulate significant frameshifting. Alternatively, a defect in ribosome formation could generate a cellular signal that alters frameshifting by an unknown mechanism; this same cellular signal could affect multiple cellular processes including bud site selection. Clearly more functional analyses on the orphan BUD genes are needed to establish a link between budding and translational capacity.


We thank Mary-Ann Checkley, Amar Klar, Emiko Matsuda, Sharon Moore, Dwight Nissley, Gurjeet Singh, Karen Stafanisko, and Jeffrey Strathern for helpful discussions. This work is supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, and mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. government.


  • Received March 26, 2010.
  • Accepted May 21, 2010.


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