Ribosomal protein and biogenesis factors affect multiple steps during movement of the Saccharomyces cerevisiae Ty1 retrotransposon
© Suresh et al. 2016
Received: 21 September 2015
Accepted: 30 November 2015
Published: 8 December 2015
A large number of Saccharomyces cerevisiae cellular factors modulate the movement of the retrovirus-like transposon Ty1. Surprisingly, a significant number of chromosomal genes required for Ty1 transposition encode components of the translational machinery, including ribosomal proteins, ribosomal biogenesis factors, protein trafficking proteins and protein or RNA modification enzymes.
To assess the mechanistic connection between Ty1 mobility and the translation machinery, we have determined the effect of these mutations on ribosome biogenesis and Ty1 transcriptional and post-transcriptional regulation. Lack of genes encoding ribosomal proteins or ribosome assembly factors causes reduced accumulation of the ribosomal subunit with which they are associated. In addition, these mutations cause decreased Ty1 + 1 programmed translational frameshifting, and reduced Gag protein accumulation despite at least normal levels of Ty1 mRNA. Several ribosome subunit mutations increase the level of both an internally initiated Ty1 transcript and its encoded truncated Gag-p22 protein, which inhibits transposition.
Together, our results suggest that this large class of cellular genes modulate Ty1 transposition through multiple pathways. The effects are largely post-transcriptional acting at a variety of levels that may include translation initiation, protein stability and subcellular protein localization.
KeywordsRetrotransposition Host factors Programmed frameshifting Ribosomal protein insufficiency Ribosome biogenesis
The Saccharomyces cerevisiae Ty (Transposons of yeast) retrotransposons are members of the LTR (long terminal repeat) group and are similar to retroviruses both structurally and functionally [1, 2]. Like retroviruses, Ty elements undergo reverse transcription that occurs within virus-like particles (VLPs) formed from structural and enzymatic proteins encoded by two genes, GAG and POL. Ty elements are valuable as models for human retroviruses; several groups have exploited yeast genetic tools to identify genes encoding Ty host factors that modulate transposition. Knowing how these factors affect Ty retrotransposition can provide clues as to what host processes affect retrovirus or retrotransposon replication and pathogenicity. Genome-wide forward genetic screens identified host factors that are required for (cofactor genes) or prevent (restriction genes) retrotransposition by Ty1 [3–7]. The most salient feature of the genes identified in these screens is the diversity of function of their encoded products, including roles in transcription, chromatin structure and modification, intracellular signaling, cytoplasmic protein synthesis, DNA repair, RNA processing and cell cycle regulation among others. Among the most statistically overrepresented host cofactor genes are those encoding cytoplasmic ribosomal proteins  suggesting that Ty transposition might depend on efficient biogenesis of ribosomes. Host factors for other plus stranded viruses in yeast have not been found to be as diverse. Prominent among these is the endogenous L-A virus of S. cerevisiae. It supports the replication of satellite dsRNA molecules, one of which encodes a peptide toxin lethal to uninfected cells . Maintenance of L-A and the satellites depends on availability of the large (60S) ribosomal subunit , implying a more global role of protein synthesis for positive stranded viruses. Because, unlike Ty1, L-A has no integrated DNA form, it does not share a dependence on genes such as those involved in transcription, chromatin recombination and DNA repair. Its dependence on 60S abundance may relate to the L-A mRNA not being polyadenylated since polyA tails facilitate 60S joining during translation initiation . Thus, reduced 60S availability could reduce L-A mRNA translation relative to bulk poly(A)+ mRNA (reviewed in ). Ty1 expresses an abundant, poly(A)+ mRNA and depends on both 40S and 60S availability so its dependence on the translation machinery may have a different origin. Also, only three Ty1 cofactor genes were also identified as L-A host factors—SKI1/KEM1/XRNI, SKI2 and SKI8—and their Ty1 phenotype is opposite to their effect of L-A virus; these factors are required for Ty1 mobility but restrict L-A propagation. Therefore, Ty1 and L-A occupy distinct genetic niches with respect to their dependence on host proteins.
Ty elements, as well as many viruses and virus-like elements including L-A, employ an unusual translational control mechanism—programmed translational frameshifting . The Ty and L-A frameshift mechanisms are distinct. Ty elements employ +1 frameshifting, in which translation shifts one base in the downstream or 3′ direction, while L-A uses -1 frameshifting, shifting one base in the opposite direction. The Ty1-encoded enzymatic (Pol) protein is encoded as a fusion to the upstream-encoded Gag structural protein by +1 frameshifting at a 7 nt RNA signal . A similar or identical signal is used in all but the Ty5 element. The frequency of Ty1 frameshifting is approximately 40 % measured in a reporter gene construct containing only the frameshift signal . In the intact Ty1 element the Gag-Pol protein is expressed at 3 % the amount of the Gag protein, suggesting a further ~10-fold reduction in expression of Gag-Pol, which may result from either a translational effect during elongation through the POL gene or reduced stability of Gag-Pol relative to Gag protein; changes to this ratio blocked retrotransposition . Altered Gag to Gag-Pol stoichiometry also reduces transposition of many other viruses [15–20]. Because retrotransposition frequency requires a specific level of programmed frameshifting, that process could explain the dependence of retrotransposition on efficient ribosome biogenesis.
In addition to cellular cofactor and restriction genes that affect Ty1 transposition, a protein expressed from subgenomic internally initiated Ty1i transcripts (Gag-p22) containing the C-terminal half of Gag is a self-encoded restriction factorthat inhibits transposition and controls Ty1 copy number . Gag-p22 antagonizes VLP function by interfering with assembly of VLPs and assembly foci , called T-bodies  or retrosomes . Well-known Ty1 cofactors such as SPT3 and XRN1, which are implicated in full-length transcription , and RNA turnover and VLP function [26–28], respectively, influence the level of Ty1i RNA . However, additional cellular genes that modulate Ty1i/Gag-p22 expression remain to be discovered, and in fact, may be present in Ty1 cofactor or restriction gene collections. A clue to what types of factors might influence this effect is the fact that formation of retrosomes requires co-translational insertion of the Ty1 Gag protein into the endoplasmic reticulum (ER) . Interfering with ER insertion blocks formation of retrosomes and the Gag protein produced is more rapidly degraded. This suggests that some Ty1 cofactor genes might encode factors required for Gag ER insertion.
To gain a more thorough understanding of the relationship between ribosome biogenesis and Ty1 transposition, we analyzed the effect on Ty1 transposition of chromosomal deletions that remove structural proteins of the 40S and 60S ribosomal subunits as well as proteins involved in ribosomal processing or protein synthesis. We show that translation-associated cofactor deletion mutants affect Ty1 transposition through a combination of mechanisms. Most of the mutants tested show reduced accumulation of the corresponding ribosomal subunit, significantly decreased +1 programmed translational frameshifting at the Ty1 site, and reduced expression of Gag protein despite expressing at least normal amounts of Ty1 mRNA. Interestingly, several ribosome subunit mutants also express more Ty1i RNA relative to Ty1 mRNA and significant amounts of Gag-p22 and its C-terminally processed product, Gag-p18, consistent with the idea that producing more of the transpositional inhibitor Gag-p22 contributes to the Ty1 defects in these mutants . Together, our results suggest that multiple post-transcriptional processes are required for optimal Ty1 transposition.
Media and yeast strains
Yeast genetic techniques and media were used as described previously [29, 30]. Strains from the haploid MATα deletion collection  were obtained from Invitrogen (Carlsbad, CA). The mutant strains, constructed in BY4742 (MATα his3-∆1 leu2-∆0 lys2-∆0 ura3-∆0)  were transformed with pJC573, a URA3-based integrating plasmid carrying an active Ty1 element tagged with a modified indicator gene his3-AI, which cannot recombine with the his3-∆1 allele present in BY4742 to generate a functional HIS3 gene . The centromere-based Ty1 overexpression plasmid pGTy1his3-AI  was also introduced into BY4742 and an isogenic rpl1BΔ mutant.
Frequency of Ty1his3-AI mobility
Mobility of Ty elements in each mutant strain was determined essentially as described [5, 33]. Strains were streaked for single colonies on SC –Ura plates at 20 °C and a single colony suspended in SC –Ura liquid and ~103 cells inoculated into each of six tubes and incubated at 20 °C to saturation. Aliquots were plated on SC –Ura and SC –His –Ura and incubated at 30 °C. The frequency of Ty1his3-AI was calculated by dividing the average number of His+ Ura+ cells per milliliter by the average number of Ura+ cells per milliliter. Mobility of cells expressing a GAL1-promoted Ty1his3-AI plasmid (pGTy1his3-AI) was determined as described by Saha et al. .
Ty1 frameshifting efficiency
Ty1 programmed +1 frameshifting efficiency was measured as described . Briefly, the assay employs two reporter plasmids that include a translational fusion of the first 30 codons of the yeast HIS4 gene to the Escherichia coli lacZ gene, which encodes β-galactosidase. In the plasmid pMB38-9merWT, a short linker connecting the two genes includes the Ty1 heptameric frameshifting site fused to lacZ in the +1 reading frame. In a second plasmid, pMB38-9merFF, a single nucleotide deletion in the heptamer places the lacZ gene in the 0 reading frame so its expression does not require frameshifting. The two plasmids are transformed separately into the recipient strain. Frameshifting efficiency is calculated as the ratio of expression from pMB38-9merWT to that of pMB38-9merFF.
Sucrose gradient analysis of yeast ribosomes was performed essentially as described . Briefly, 200 ml of each strain were grown in YPED medium to mid-exponential phase and harvested after addition of 10 mg cycloheximide. After washing, cells were lysed with glass beads and 40 A260 units of supernatant was layered on a 10 to 50 % sucrose gradient and centrifuged in an SW40 rotor for 4 h at 41,000 rpm. Fractions were collected and continuously analyzed for absorption at 260 nm using an ISCO Foxy Jr fraction collector.
The steady-state level of Ty1 mRNA was determined essentially as described . Total cell RNA was isolated by the acid-phenol method  and 5 μg was separated by electrophoresis in 1 % agarose-glyoxal-DMSO gels and blotted to Brightstar-Plus positively charged nylon membranes (Life Sciences). For poly(A)+ RNA purification, total RNA was prepared using the MasturePure yeast RNA purification kit (Epicentre Biotechnologies, Madison, WI). Poly(A)+ RNA was isolated from 250 μg total RNA using the NucleoTrap mRNA purification kit (Clontech, Mountain View, CA). A DNA probe obtained as a 1.6 kb PvuII-ClaI fragment of the Ty1 POL gene and as a 1.4 kb EcoRI-XbaI fragment of the PYK1 gene were labeled by random priming using α-[32P]dATP using the Deca Prime II kit (Life Sciences). In vitro transcription of Ty1 GAG (nt 1266-1601) was performed using a MAXIscript kit (Life Technologies, Carlsbad, CA) and α-[32P]UTP (3,000 Ci/mmol; Perkin Elmer, Waltham, MA). Hybridization was visualized by autoradiography or by image analysis using a STORM 840 phosphor imager (GE Healthcare). The experimental results shown in the figure are representative of three experiments performed.
Western blot analyses
Three-milliliter SC-Ura liquid cultures were grown at 20 °C until saturated, which occurred between 24 and 48 h for different mutants. Strains were grown under similar conditions but split into different groups according to growth rate, and each group contained a wild type control. Total cell protein was prepared as previously described . Protein isolated by trichloroacetic acid (TCA) extraction  from wild type and the rpl1bΔ mutant either expressing pGTy1 or not was also subjected to immunoblotting. Galactose-induction of cells containing pGTy1 was performed as previously described . Protein concentration was determined using Coomassie Plus (Bradford) Assay Reagent (Thermo scientific, Waltham, MA). Protein samples were separated on a 10 % SDS-PAGE gel, and then transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in 5 % powdered milk–Tris buffered saline (100 mM Tris–HCl, 150 mM NaCl pH 7.5) with 0.1 % Tween 20 (TBST) and then incubated with primary antibody for 1 h at room temperature. Rabbit antisera directed against Ty1 VLPs (used to detect Gag; gift of Alan J. Kingsman), recombinant p18 (used to detect Gag and p22/p18) , and the control protein Hts1p (Gift of Thomas L. Mason) were used at 1:7,000, 1:5000 and 1:40,000 dilutions, respectively. Blots were washed three times for 10 min each in TBST. Primary antibody was detected with ECL anti-rabbit IgG, Horseradish peroxide linked whole antibody from donkey (GE healthcare, Pittsburgh, PA) at a 1:4,000 dilution in TBST for 1 h. Blots were washed three times for 10 min each in TBST, visualized by ECL Western Blotting Detection Reagents (GE healthcare, Pittsburgh, PA) and exposed to X-ray film. The experimental results shown are representative of two or three experiments performed.
Identifying Ty1 host cofactor and restriction genes involved in protein synthesis
We have previously described screens to identify Ty1 cellular cofactor  and restriction genes  using a Ty1 mobility assay. The assay employs a plasmid (pJC573) bearing a modified Ty1 element, Ty1his3-AI . HIS3 is inserted downstream of the POL gene opposite to the direction of Ty1 transcription and is transcribed from its own promoter; the gene is interrupted by the artificial intron (AI), which is oriented in the direction of Ty1 transcription. The Ty1 RNA expressed from this construct is spliced before undergoing reverse transcription, removing the disruption of the HIS3 gene and resulting after its reintegration into the genome in complementation of the chromosomal his3 deletion (His+). Most His+ cells result from transposition of the element. A minority of retromobility events can occur by homologous recombination with an endogenous Ty1 transposon .
Among 457 Ty1 cofactor genes isolated in various systematic screens of the viable deletion mutants [3–7], 71 encode ribosomal proteins, ribosome biogenesis factors and translation factors including 33 ribosomal proteins genes: RPL1B, RPL4A, RPL6A, RPL7A, RPL14A, RPL15B, RPL16B, RPL18A, RPL19A, RPL19B, RPL20B, RPL21A, RPL21B, RPL27A, RPL31A, RPL33B, RPL34A, RPL37A, RPL39, RPL40A, RPL41B, RPL43A, RPP1A, RPP2B, RPS0B, RPS9B, RPS10A, RPS11A, RPS19A, RPS19B, RPS25A, RPS27B and RPS30A. On the other hand, of the 91 identified Ty1 restriction genes only three are translation related [3, 5]. ASC1 is an integral ribosomal protein of the 40S ribosomal subunit and is the yeast homolog of the mammalian Receptor of Activated C Kinase 1 (RACK1) protein . ARC1 is a cofactor for aminoacyl-tRNA synthetases  and TRM7 encodes a tRNA 2′-O-ribose methyltransferase .
Quantitative Ty1 mobility is reduced in ribosomal protein gene Ty1 cofactor mutants
Ty1 mobility ± SEM (× 10−7)b
Fold reduced from WT
40 ± 2.0
4.7 ± 1.1
1.9 ± 0.56
2.6 ± 0.78
4.9 ± 1.3
6.5 ± 1.6
0.19 ± 0.14
8.1 ± 1.9
3.0 ± 1.0
5.6 ± 0.81
4.7 ± 0.79
4.8 ± 0.66
1.4 ± 0.27
7.8 ± 1.4
2.8 ± 0.56
4.0 ± 0.79
60S nuclear export
0.67 ± 0.67
Most translation associated Ty1 cofactor mutants impair ribosome biogenesis or function
Deficits in ribosomal proteins generally results in impaired ribosome biogenesis (reviewed in ). These defects include blocks to ribosome biogenesis events including rRNA processing, binding of other ribosomal proteins to the pre-ribosome and transport to the cytoplasm. We therefore expected that the translation-associated Ty1 cofactor mutants would show effects on ribosome biogenesis. Many of the mutants showed slowed growth rates, consistent with reduced ribosome availability, however since most ribosomal protein gene deletion mutants grow at a normal rate  this is a poor test of their effect on biogenesis. Therefore, we directly assessed the effect of a subset of the cofactor mutants by analyzing polysomes from the wild type control and 11 of the translation-associated cofactor gene deletions using sucrose density centrifugation .
Similarly, lack of most large subunit protein genes tested showed evidence of reduced 60S accumulation or activity. Deletions of 60S ribosomal protein genes (RPL1B and RPL27A) or a 60S subunit assembly factor gene involved in 3′-end processing of 5.8S rRNA (RRP6) all resulted in reduced amounts of 60S subunits and all three showed evidence of “halfmers”, which are secondary peaks indicating complexes with masses slightly greater than a 70S or polysome peak. Halfmers are caused by the presence of mRNA-bound 43S pre-initiation complexes to which 60S subunits have failed to assemble in addition to one or more 80S ribosomes on an mRNA . These peaks are direct evidence of slowed 60S subunit recruitment.
Four mutants displayed profiles resembling the wild type; the deletion of these genes does not appear to grossly alter the rate of assembly of either subunit. These genes encode a 40S ribosomal protein (RPS25A) two 60S proteins (RPL15B, RPL41B) and a ribosome nuclear export factor (KAP123). Previous work showed that lack of the 60S Ty1 cofactor gene RPP2B also does not alter subunit abundance . Of the five encoded proteins, only Rpl15 is essential for viability; the proteins encoded by the other four can be eliminated without affecting viability although only a strain lacking Rpl41 grows at a normal rate . These five proteins must affect Ty1 mobility without altering ribosome biogenesis.
Most ribosomal protein gene deletions cause a significant reduction in Ty1 frameshift activity
An obvious reason for the connection between translation and Ty1 retrotransposition could be the Ty1 + 1 programmed frameshifting event responsible for expression of the Gag-Pol fusion protein. The stoichiometry of Gag to Gag-Pol sensitively controls transposition efficiency and even slight changes in the ratio of Gag to Gag-Pol proteins can block retrotransposition . For Ty1, increasing frameshifting blocks transposition by causing incomplete proteolytic processing of the Gag-Pol polyprotein leading to formation of defective VLPs . Reducing Ty1 frameshifting also blocks transposition  although the mechanism of this blockage is not known.
Frameshift activity in mutant strains was determined using a well characterized β-galactosidase reporter construct . The construct has the first 33 codons of the HIS4 gene fused to the β-galactosidase gene through a minimal Ty1 frameshift site with expression of the enzyme requiring frameshifting. The percent frameshift activity is expressed as the ratio of the frameshift activity to that of a frame fusion control in which the genes are in one open reading frame so expression does not require frameshifting. The use of the frame fusion control eliminates other transcriptional, post-transcriptional and translational effects on the activity of the enzyme.
Comparison of the frameshift and transposition phenotypes of each of the mutants showed no significant correlation (Pearson’s correlation coefficient, r = 0.07). If we exclude the mutants that had no effect on frameshifting or the polysome profile the correlation is better (r = 0.27) but still weak. This statistical analysis suggests that the magnitude of the effect on transposition does not correlate well with the magnitude of the reduction of frameshifting, suggesting that effects beyond frameshift efficiency explain the reduction in Ty1 mobility. Clearly, for translation-associated cofactor mutants that do not alter either frameshifting or the polysome profile the effect on transposition must be from another cause, possibly extraribosomal .
Post-transcriptional regulation reduces Ty1 Gag protein accumulation in most Ty1 translation-associated cofactor mutants
Previously, we reported that the 40S assembly factor mutant bud22∆ accumulated lower amounts of Gag and higher amounts of Gag-p49 or Gag† compared to Gag-p45 . We confirmed the bud22∆ phenotype (Fig. 3) and found that three of the mutants tested here showed a similar phenotype (rps10A∆, rps19B∆ and rps25A). Three other mutants, rps0B∆, rps9B∆ and rrp6, accumulated similarly reduced amounts of Gag but do not show reduced processing of Gag-p49 to Gag-p45. All of these five genes encode 40S ribosomal proteins or 40S biogenesis proteins implying a link between 40S availability and the Bud22 phenotype and suggesting that the phenotype results specifically from a reduction in 40S availability.
The phenotypes of the remaining 60S ribosomal protein mutants are quite different than those of the 40S mutants. First, none of the 60S ribosomal protein mutants show an obvious deficit in p45 processing; the amount of the processed Gag-p45 is consistently greater than that of unprocessed Gag-p49/Gag†. Second, the 60S mutants, other than rpl1B∆ and rpl39, clearly accumulate higher amounts of Gag than the 40S mutants although most express less than wild type (>50 % of wild type level). The co-occurrence of these two effects suggests that Gag processing or other posttranslational modifications  may explicitly depend on a sufficient supply of Gag protein. Reduced numbers of Gag proteins relative to Ty1 genomic RNA in the 40S mutants could result in increased production of defective VLPs. Combined with a reduction in the frequency of programmed translational frameshifting in these mutants, which reduces the ratio of Gag to Gag-Pol to far less than 50:1, many of these VLPs might then actually lack the protease activity of Ty1 Pol protein, blocking processing of the Gag and presumably Pol proteins as well. For the 60S mutants, the increased amounts of Gag and Gag-Pol per genomic RNA might ensure the presence of protease in more of the VLPs.
The steady state level of full-length Ty1 mRNA cannot explain observed differences in Gag accumulation
Ty1i RNA increases relative to Ty1 mRNA in several ribosomal subunit mutants
Ty1i mRNA is translated to produce an N-terminally truncated Gag-p22, which is likely the primary translational product, and a C-terminally processed Gag-p18 protein; the presence of these proteins leads to defective VLP assembly and function, and reduced Ty1 mobility . We performed immunoblotting with an antibody raised against recombinant p18 on the same set of wild type and mutant strains and found evidence for p22/p18 in all of the mutants (Fig. 5c). As expected, the spt3∆ mutant accumulated large amounts of p22; because little Ty1-encoded proteins are expressed in this strain, no p18 was detected. In the four Ty1 ribosome-associated mutants there was evidence of p22 and/or p18. The amount of p18 was correlated with the amount of full-length Gag protein, which indicated the extent of translation of full-length Ty1 proteins. The rpl27A∆ mutant had both the highest amount of both Gag-p49/p45/Gag† and Gag-p22. The increasingly lower amounts of Gag-p49/45 in the rpl21A∆, rps0B∆ and rps39∆ mutants correlated with increasingly lower amounts of Gag-p18 and an increased ratio of Gag-p22 to Gag-p18. These results confirm that the mutants tested express substantial amounts of Ty1i mRNA and the inhibitory Gag-p22/p18 proteins and suggest that the reduced Ty1 mobility in these strains in part results from this inhibitory mechanism.
The number and diversity of genes identified as host factors for Ty1 retrotransposition reflects the complexity of the Ty1 lifecycle . Many host restriction and cofactor genes encode proteins involved in basic processes of cellular information transfer with those involved in protein synthesis being significantly overrepresented among the Ty1 cofactor genes, which are required for optimal level of transposition . Similarly, mutations targeting 60S ribosomal proteins are also required for propagation of the yeast L-A double-stranded RNA virus ; L-A shares several features with retroviruses and retrotransposons (reviewed in ) so this shared mode of control may reflect similar mechanisms of propagation. Quantitative assays of Ty1 mobility (Table 1) validate the requirement for 13 ribosomal proteins genes and three ribosome biogenesis genes (RRP6, RRP8 and KAP123). As expected, most but not all mutants lacking 40S or 60S structural proteins or biogenesis factors are deficient in the corresponding subunit. It has long been recognized that biogenesis of the two subunits diverges early in biogenesis with a 90S pre-ribosome containing immature forms of both subunits dividing into a pre-40S and pre-60S complexes (reviewed in ). No comprehensive test of the effect of ribosomal protein depletion on ribosome biogenesis has been performed but most of the proteins have been tested and in all cases the lack of a ribosomal protein blocks maturation and accumulation of the corresponding subunit [49, 64].
Summary of experimental results
Ty1 moblility relative to WT
Frameshifting relative to WT
Gag relative to WT
Ty1 mRNA relative to WT
60S nuclear export
The phenotype of the rpl1B∆ and rpl39∆ mutants is quite distinctive. The lack of accumulation of Gag in these mutants despite the presence of Ty1 mRNA strongly suggests a post-transcriptional block. The striking inability to detect Gag protein in the rpl39∆ mutant is consistent with it having the lowest Ty1 mobility frequency of any of the mutants, 210-fold less than wild type. The rpl1B∆ mutant with a similar Gag accumulation phenotype, however, supports Ty1 mobility slightly more than the average of the mutants tested. Because the rpl1B∆ mutation causes a relatively small decrease in mobility we suspected that the low level of Gag detected in the rpl1B∆ mutant results from its being sequestered and not easily extracted. A harsher method of extraction detected no more Gag protein but overexpressing the Ty1 mRNA in this mutant background using a Gal-driven element resulted in significantly more Gag detected but also restored near normal Ty1 mobility. We cannot exclude that Gag is sequestered in the rpl1B∆ mutant and we are unable to explain why, despite their similar Gag phenotype, the rpl1B∆ and rpl39 mobility phenotypes are so different. The location of the Rpl39 protein in the ribosomal subunit provides a clue to the origin of the difference. RPL39 being a single copy gene, the deletion mutant accumulates 60S subunits lacking the protein. Rpl39 is located at the opening of the peptide exit tunnel (see Fig. 6e) and interacts with the hydrophobic signal anchor sequence of a nascent protein during co-translational insertion into the endoplasmic reticulum (ER) ; this interaction appears to be important for targeting proteins to the ER . Doh et al.  demonstrated that VLP assembly sites are nucleated by targeting of ribosomes translating Ty1 mRNAs to the ER by contranslational insertion of Ty1 proteins into the ER. The formation of cytoplasmic foci , called T-bodies  or retrosomes  may be necessary for efficient formation of VLPs and therefore for maximal Ty1 mobility. Ty1 Gag is synthesized but is less stable when targeting to the ER is blocked. This suggests that a block to ER targeting caused by the absence of Rpl39 inhibits VLP assembly and Gag accumulation, resulting in a severe transposition defect.
The overall conclusion of this work is that failure in ribosome biogenesis results in reduced Ty1 mobility with distinct phenotypes for mutants deficient in 40S and 60S subunit proteins. The effect shows no clear connection to a particular step in biogenesis or the position of the protein within either subunit. The effect is largely translational involving both decreased programmed translational frameshifting, reduced efficiency of translation and possibly increased instability of newly synthesized Ty1 Gag protein. A connection has been made to co-translational insertion of Ty1 Gag protein into the endoplasmic reticulum both by the severe phenotype of a mutant lacking the Rpl39 protein, which plays a role in targeting co-translational ER insertion and our demonstration of the accumulation of the Ty1i protein and Gag-p22/p18 in several of the translation-associated Ty1 cofactor mutant strains. Experiments are continuing to determine whether the connection between ribosome subunit sufficiency and Ty1 mobility is through the disruption of this newly discovered step in the Ty1 transposition process. Future studies will address how the individual pathways identified here modulate Ty1 gene expression and function, and whether similar processes also affect other retroelements.
group specific antigen
a Large double-stranded RNA virus of Saccharomyces cerevisiae
large ribosomal subunit
long terminal repeat
POLymerase; the retroviral polyprotein processed to produce the protease, reverse transcriptase, RNase H and integrase enzymes
small ribosomal subunit
transposon of yeast
We thank Stephen Hajduk for sharing equipment, and Jessica M. Tucker, Agniva Saha and Yuri Nishida for helpful discussions. Thanks to Thomas L. Mason for the gift of anti-Hts1 antibody. This work was supported by NIH grants GM095622 (D.J.G) and GM029480 (P.J.F.).
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- Voytas DF, Boeke JD. Yeast retrotransposon revealed. Nature. 1992;358:717.View ArticlePubMedGoogle Scholar
- Curcio MJ, Lutz S, Lesage P. The Ty1 LTR-retrotransposon of budding yeast, Saccharomyces cerevisiae. Microbiol Spectr. 2015;3:1–35.PubMedGoogle Scholar
- Scholes DT, Banerjee M, Bowen B, Curcio MJ. Multiple regulators of Ty1 transposition in Saccharomyces cerevisiae have conserved roles in genome maintenance. Genetics. 2001;159:1449–65.PubMed CentralPubMedGoogle Scholar
- Griffith JL, Coleman LE, Raymond AS, Goodson SG, Pittard WS, Tsui C, et al. Functional genomics reveals relationships between the retrovirus-like Ty1 element and its host Saccharomyces cerevisiae. Genetics. 2003;164:867–79.PubMed CentralPubMedGoogle Scholar
- Nyswaner KM, Checkley MA, Yi M, Stephens RM, Garfinkel DJ. Chromatin-associated genes protect the yeast genome from Ty1 insertional mutagenesis. Genetics. 2008;178:197–214.PubMed CentralView ArticlePubMedGoogle Scholar
- Dakshinamurthy A, Nyswaner KM, Farabaugh PJ, Garfinkel DJ. BUD22 affects Ty1 retrotransposition and ribosome biogenesis in Saccharomyces cerevisiae. Genetics. 2010;185:1193–205.PubMed CentralView ArticlePubMedGoogle Scholar
- Risler JK, Kenny AE, Palumbo RJ, Gamache ER, Curcio MJ. Host co-factors of the retrovirus-like transposon Ty1. Mob DNA. 2012;3:12.PubMed CentralView ArticlePubMedGoogle Scholar
- Wickner RB. Double-stranded RNA viruses of Saccharomyces cerevisiae. Microbiol Rev. 1996;60:250–65.PubMed CentralPubMedGoogle Scholar
- Ohtake Y, Wickner RB. Yeast virus propagation depends critically on free 60S ribosomal subunit concentration. Mol Cell Biol. 1995;15:2772–81.PubMed CentralView ArticlePubMedGoogle Scholar
- Searfoss A, Dever TE, Wickner R. Linking the 3′ poly(A) tail to the subunit joining step of translation initiation: relations of Pab1p, eukaryotic translation initiation factor 5b (Fun12p), and Ski2p-Slh1p. Mol Cell Biol. 2001;21:4900–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Wickner RB, Fujimura T, Esteban R. Viruses and prions of Saccharomyces cerevisiae. Adv Virus Res. 2013;86:1–36.PubMed CentralView ArticlePubMedGoogle Scholar
- Farabaugh P. Post-transcriptional regulation of transposition by Ty retrotransposons of Saccharomyces cerevisiae. J Biol Chem. 1995;270:10361–4.View ArticlePubMedGoogle Scholar
- Belcourt MF, Farabaugh PJ. Ribosomal frameshifting in the yeast retrotransposon Ty: tRNAs induce slippage on a 7 nucleotide minimal site. Cell. 1990;62:339–52.View ArticlePubMedGoogle Scholar
- Kawakami K, Pande S, Faiola B, Moore D, Boeke J, Farabaugh P, et al. A rare tRNA-Arg(CCU) that regulates Ty1 element ribosomal frameshifting is essential for Ty1 retrotransposition in Saccharomyces cerevisiae. Genetics. 1993;135:309–20.PubMed CentralPubMedGoogle Scholar
- Felsenstein K, Goff S. Expression of the gag–pol fusion protein of Moloney murine leukemia virus without gag protein does not induce virion formation or proteolytic processing. J Virol. 1988;62:2179–82.PubMed CentralPubMedGoogle Scholar
- Hung M, Patel P, Davis S, Green SR. Importance of ribosomal frameshifting for human immunodeficiency virus type 1 particle assembly and replication. J Virol. 1998;72:4819–24.PubMed CentralPubMedGoogle Scholar
- Xu H, Boeke JD. Host genes that influence transposition in yeast: the abundance of a rare tRNA regulates Ty1 transposition frequency. Proc Natl Acad Sci U S A. 1990;87:8360–4.PubMed CentralView ArticlePubMedGoogle Scholar
- Dinman JD, Wickner RB. Ribosomal frameshifting efficiency and gag/gag–pol ratio are critical for yeast M1 double–stranded RNA virus propagation. J Virol. 1992;66:3669–76.PubMed CentralPubMedGoogle Scholar
- Park J, Morrow CD. Overexpression of the gag–pol precursor from human immunodeficiency virus type 1 proviral genomes results in efficient poteolytic processing in the absence of virion production. J Virol. 1992;65:5111–7.Google Scholar
- Karacostas V, Wolffe EJ, Nagashima K, Gonda MA, Moss B. Overexpression of the HIV-1 gag-pol polyprotein results in intracellular activation of HIV-1 protease and inhibition of assembly and budding of virus-like particles. Virology. 1993;193:661–71.View ArticlePubMedGoogle Scholar
- Saha A, Mitchell JA, Nishida Y, Hildreth JE, Ariberre JA, Gilbert WV, et al. A trans-dominant form of Gag restricts Ty1 retrotransposition and mediates copy number control. J Virol. 2015;89:3922–38.PubMed CentralView ArticlePubMedGoogle Scholar
- Doh JH, Lutz S, Curcio MJ. Co-translational localization of an LTR-retrotransposon RNA to the endoplasmic reticulum nucleates virus-like particle assembly sites. PLoS Genet. 2014;10:e1004219.PubMed CentralView ArticlePubMedGoogle Scholar
- Malagon F, Jensen TH. The T body, a new cytoplasmic RNA granule in Saccharomyces cerevisiae. Mol Cell Biol. 2008;28:6022–32.PubMed CentralView ArticlePubMedGoogle Scholar
- Sandmeyer SB, Clemens KA. Function of a retrotransposon nucleocapsid protein. RNA Biol. 2010;7:642–54.PubMed CentralView ArticlePubMedGoogle Scholar
- Grant PA, Duggan L, Cote J, Roberts SM, Brownell JE, Candau R, et al. Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. Genes Dev. 1997;11:1640–50.View ArticlePubMedGoogle Scholar
- Berretta J, Pinskaya M, Morillon A. A cryptic unstable transcript mediates transcriptional trans-silencing of the Ty1 retrotransposon in S. cerevisiae. Genes Dev. 2008;22:615–26.PubMed CentralView ArticlePubMedGoogle Scholar
- Checkley MA, Nagashima K, Lockett SJ, Nyswaner KM, Garfinkel DJ. P-body components are required for Ty1 retrotransposition during assembly of retrotransposition-competent virus-like particles. Mol Cell Biol. 2010;30:382–98.PubMed CentralView ArticlePubMedGoogle Scholar
- Dutko JA, Kenny AE, Gamache ER, Curcio MJ. 5′ to 3′ mRNA decay factors colocalize with Ty1 gag and human APOBEC3G and promote Ty1 retrotransposition. J Virol. 2010;84:5052–66.PubMed CentralView ArticlePubMedGoogle Scholar
- Sherman F, Fink GR, Hicks JB. Methods in yeast genetics. Cold Spring Harbor: Cold Spring Harbor Laboratoy; 1986.Google Scholar
- Guthrie C, Fink GR. Guide to yeast genetics and molecular biology. In: Abelson JN, Simon MI, editors. Methods in Enzymology. San Diego, California: Academic; 1991.Google Scholar
- Giaever G, Chu AM, Ni L, Connelly C, Riles L, Veronneau S, et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature. 2002;418:387–91.View ArticlePubMedGoogle Scholar
- Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast. 1998;14:115–32.View ArticlePubMedGoogle Scholar
- Curcio MJ, Garfinkel DJ. Single-step selection for Ty1 element retrotransposition. Proc Natl Acad Sci U S A. 1991;88:936–40.PubMed CentralView ArticlePubMedGoogle Scholar
- Deshmukh M, Tsay YF, Paulovich AG, Woolford Jr JL. Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits. Mol Cell Biol. 1993;13:2835–45.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee BS, Lichtenstein CP, Faiola B, Rinckel LA, Wysock W, Curcio MJ, et al. Posttranslational inhibition of Ty1 retrotransposition by nucleotide excision repair/transcription factor TFIIH subunits Ssl2p and Rad3p. Genetics. 1998;148:1743–61.PubMed CentralPubMedGoogle Scholar
- Schmitt ME, Brown TA, Trumpower BL. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 1990;18:3091–2.PubMed CentralView ArticlePubMedGoogle Scholar
- Braiterman LT, Monokian GM, Eichinger DJ, Merbs SL, Gabriel A, Boeke JD. In-frame linker insertion mutagenesis of yeast transposon Ty1: phenotypic analysis. Gene. 1994;139:19–26.View ArticlePubMedGoogle Scholar
- Lawler Jr JF, Merkulov GV, Boeke JD. A nucleocapsid functionality contained within the amino terminus of the Ty1 protease that is distinct and separable from proteolytic activity. J Virol. 2002;76:346–54.PubMed CentralView ArticlePubMedGoogle Scholar
- Sharon G, Burkett TJ, Garfinkel DJ. Efficient homologous recombination of Ty1 element cDNA when integration is blocked. Mol Cell Biol. 1994;14:6540–51.PubMed CentralView ArticlePubMedGoogle Scholar
- Gerbasi VR, Weaver CM, Hill S, Friedman DB, Link AJ. Yeast Asc1p and mammalian RACK1 are functionally orthologous core 40S ribosomal proteins that repress gene expression. Mol Cell Biol. 2004;24:8276–87.PubMed CentralView ArticlePubMedGoogle Scholar
- Simos G, Segref A, Fasiolo F, Hellmuth K, Shevchenko A, Mann M, et al. The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases. EMBO J. 1996;15:5437–48.PubMed CentralPubMedGoogle Scholar
- Pintard L, Lecointe F, Bujnicki JM, Bonnerot C, Grosjean H, Lapeyre B. Trm7p catalyses the formation of two 2′-O-methylriboses in yeast tRNA anticodon loop. EMBO J. 2002;21:1811–20.PubMed CentralView ArticlePubMedGoogle Scholar
- Briggs MW, Burkard KT, Butler JS. Rrp6p, the yeast homologue of the human PM-Scl 100-kDa autoantigen, is essential for efficient 5.8 S rRNA 3′ end formation. J Biol Chem. 1998;273:13255–63.View ArticlePubMedGoogle Scholar
- Bousquet-Antonelli C, Vanrobays E, Gelugne JP, Caizergues-Ferrer M, Henry Y. Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2. RNA. 2000;6:826–43.PubMed CentralView ArticlePubMedGoogle Scholar
- Peifer C, Sharma S, Watzinger P, Lamberth S, Kotter P, Entian KD. Yeast Rrp8p, a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA. Nucleic Acids Res. 2013;41:1151–63.PubMed CentralView ArticlePubMedGoogle Scholar
- Sydorskyy Y, Dilworth DJ, Yi EC, Goodlett DR, Wozniak RW, Aitchison JD. Intersection of the Kap123p-mediated nuclear import and ribosome export pathways. Mol Cell Biol. 2003;23:2042–54.PubMed CentralView ArticlePubMedGoogle Scholar
- Askree SH, Yehuda T, Smolikov S, Gurevich R, Hawk J, Coker C, et al. A genome-wide screen for Saccharomyces cerevisiae deletion mutants that affect telomere length. Proc Natl Acad Sci U S A. 2004;101:8658–63.PubMed CentralView ArticlePubMedGoogle Scholar
- Hamasaki-Katagiri N, Tabor CW, Tabor H. Spermidine biosynthesis in Saccharomyces cerevisae: polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase). Gene. 1997;187:35–43.View ArticlePubMedGoogle Scholar
- de la Cruz J, Karbstein K, Woolford Jr JL. Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo. Annu Rev Biochem. 2015;84:93–129.View ArticlePubMedGoogle Scholar
- Steffen KK, McCormick MA, Pham KM, MacKay VL, Delaney JR, Murakami CJ, et al. Ribosome deficiency protects against ER stress in Saccharomyces cerevisiae. Genetics. 2012;191:107–18.PubMed CentralView ArticlePubMedGoogle Scholar
- Sagliocco FA, Moore PA, Brown AJ. Polysome analysis. Methods Mol Biol. 1996;53:297–311.PubMedGoogle Scholar
- Helser TL, Baan RA, Dahlberg AE. Characterization of a 40S ribosomal subunit complex in polyribosomes of Saccharomyces cerevisiae treated with cycloheximide. Mol Cell Biol. 1981;1:51–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Cardenas D, Revuelta-Cervantes J, Jimenez-Diaz A, Camargo H, Remacha M, Ballesta JP. P1 and P2 protein heterodimer binding to the P0 protein of Saccharomyces cerevisiae is relatively non-specific and a source of ribosomal heterogeneity. Nucleic Acids Res. 2012;40:4520–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Warner JR, McIntosh KB. How common are extraribosomal functions of ribosomal proteins? Mol Cell. 2009;34:3–11.PubMed CentralView ArticlePubMedGoogle Scholar
- Adams SE, Mellor J, Gull K, Sim RB, Tuite MF, Kingsman SM, et al. The functions and relationships of TyVLP proteins in yeast reflect those of mammalian retroviral proteins. Cell. 1987;49:111–9.View ArticlePubMedGoogle Scholar
- Merkulov GV, Swiderek KM, Brachmann CB, Boeke JD. A critical proteolytic cleavage site near the C terminus of the yeast retrotransposon Ty1 Gag protein. J Virol. 1996;70:5548–56.PubMed CentralPubMedGoogle Scholar
- Ohashi A, Gibson J, Gregor I, Schatz G. Import of proteins into mitochondria. The precursor of cytochrome c1 is processed in two steps, one of them heme-dependent. J Biol Chem. 1982;257:13042–7.PubMedGoogle Scholar
- Servant G, Pennetier C, Lesage P. Remodeling yeast gene transcription by activating the Ty1 long terminal repeat retrotransposon under severe adenine deficiency. Mol Cell Biol. 2008;28:5543–54.PubMed CentralView ArticlePubMedGoogle Scholar
- Joo YJ, Kim JH, Kang UB, Yu MH, Kim J. Gcn4p-mediated transcriptional repression of ribosomal protein genes under amino-acid starvation. EMBO J. 2011;30:859–72.PubMed CentralView ArticlePubMedGoogle Scholar
- Deminoff SJ, Santangelo GM. Rap1p requires Gcr1p and Gcr2p homodimers to activate ribosomal protein and glycolytic genes, respectively. Genetics. 2001;158:133–43.PubMed CentralPubMedGoogle Scholar
- Paquin CE, Williamson VM. Temperature effects on the rate of ty transposition. Science. 1984;226:53–5.View ArticlePubMedGoogle Scholar
- Winston F, Durbin KJ, Fink GR. The SPT3 gene is required for normal transcription of Ty elements in S. cerevisiae. Cell. 1984;39:675–82.View ArticlePubMedGoogle Scholar
- Tschochner H, Hurt E. Pre-ribosomes on the road from the nucleolus to the cytoplasm. Trends Cell Biol. 2003;13:255–63.View ArticlePubMedGoogle Scholar
- Woolford Jr JL, Baserga SJ. Ribosome biogenesis in the yeast Saccharomyces cerevisiae. Genetics. 2013;195:643–81.PubMed CentralView ArticlePubMedGoogle Scholar
- Gale Jr M, Tan SL, Katze MG. Translational control of viral gene expression in eukaryotes. Microbiol Mol Biol Rev. 2000;64:239–80.PubMed CentralView ArticlePubMedGoogle Scholar
- Huang Q, Purzycka KJ, Lusvarghi S, Li D, Legrice SF, Boeke JD. Retrotransposon Ty1 RNA contains a 5′-terminal long-range pseudoknot required for efficient reverse transcription. RNA. 2013;19:320–32.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang Y, Wolfle T, Rospert S. Interaction of nascent chains with the ribosomal tunnel proteins Rpl4, Rpl17, and Rpl39 of Saccharomyces cerevisiae. J Biol Chem. 2013;288:33697–707.PubMed CentralView ArticlePubMedGoogle Scholar
- Lin PJ, Jongsma CG, Liao S, Johnson AE. Transmembrane segments of nascent polytopic membrane proteins control cytosol/ER targeting during membrane integration. J Cell Biol. 2011;195:41–54.PubMed CentralView ArticlePubMedGoogle Scholar
- Svidritskiy E, Brilot AF, Koh CS, Grigorieff N, Korostelev AA. Structures of yeast 80S ribosome-tRNA complexes in the rotated and nonrotated conformations. Structure. 2014;22:1210–8.PubMed CentralView ArticlePubMedGoogle Scholar
- Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. The protein data bank. Nucleic Acids Res. 2000;28:235–42.PubMed CentralView ArticlePubMedGoogle Scholar
- Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996;14:33–8.View ArticlePubMedGoogle Scholar
- Ohtake Y, Wickner RB. KRB1, a suppressor of mak7-1 (a mutant RPL4A), is RPL4B, a second ribosomal protein L4 gene, on a fragment of Saccharomyces chromosome XII. Genetics. 1995;140:129–37.PubMed CentralPubMedGoogle Scholar
- Sachs AB, Davis RW. The poly(A)–binding protein is required for poly(A) shortening and 60S ribosomal subunit dependent translation intiation. Cell. 1989;58:857–67.View ArticlePubMedGoogle Scholar
- Leger-Silvestre I, Caffrey JM, Dawaliby R, Alvarez-Arias DA, Gas N, Bertolone SJ, et al. Specific role for yeast homologs of the Diamond Blackfan Anemia-associated Rps19 Protein in Ribosome Synthesis. J Biol Chem. 2005;280:38177–85.View ArticlePubMedGoogle Scholar