P-body components are required for Ty1 retrotransposition posttranscriptionally. (A) Schematic representation of a Ty1 element showing the positions of mRNAs, hybridization probes, precursor (p49) and mature (p45) Gag proteins, and mature Pol proteins PR, IN, and RT. (B) Northern analysis of P-body component mutants using a ³²P-labeled Gag 5′-end riboprobe. Below the blot is the amount of Ty1 mRNA relative to the 18S and 25S rRNAs after normalization to the wild-type (wt) transcript level. An spt3Δ mutant (DG2247) served as a negative control. SD, standard deviation from at least three independent experiments. (C) Northern analysis of Ty1 transcripts detected in a kem1Δ mutant using a radiolabeled RT#2 probe. Note that a 5′-truncated Ty1 transcript is detected in the kem1Δ mutant. Below the blot are the levels of full-length and 5′-truncated mRNAs relative to the level of full-length mRNA from the wild type. (D) Levels of unintegrated Ty1 cDNA are reduced in P-body component mutants. The graph summarizes the relative percentage of unintegrated Ty1 cDNA, as determined by Southern blot analysis. The decrease in Ty1 cDNA levels detected in the P-body mutants is expressed relative to the wild-type level. The error bars represent standard deviations from three independent cultures analyzed per strain. The level of unintegrated cDNA decreased at least 85% in the mutants compared with the wild-type level (P < 0.0001). (E) Levels of p49 and p45 in the P-body mutants. Total protein extracts from the wild type and P-body mutants, extracted from equivalent cell densities, were subjected to Western blot analysis using an anti-Ty1 VLP serum. Protein extracts from an spt3Δ mutant and a Ty1-less S. paradoxus strain (DG1768) were included as negative controls. The p45/p49 ratio was determined by densitometry. Standard deviations were calculated from three independent experiments (P < 0.005).

Localization of Ty1 mRNA and Gag in discrete cytoplasmic foci requires optimal conditions for retrotransposition. Cells were grown in SC-glucose (2%) medium at 20°C, unless otherwise noted, and visualized by differential interference contrast (DIC) microscopy. Ty1 mRNA and Gag were detected by FISH-IF, and DNA was stained with DAPI. Representative images are shown. (A) Detection of Ty1 mRNA and Gag under conditions optimal and suboptimal for retrotransposition. Conditions optimal for retrotransposition are present when the wild type is grown at 20°C. Conditions suboptimal for retrotransposition are present when the wild type is grown at 30°C. An spt3Δ mutant (DG2247) and a Ty1-less S. paradoxus strain (DG1768) grown at 20°C served as negative controls. Ty1 mRNA and Gag were detected as described in the legend to Fig. 3B. Merged Ty1 mRNA and Gag staining is shown. Mean percentages of cells containing cytoplasmic Ty1 mRNA foci, Gag foci, and their respective colocalization ± the SEM are included. (B) Northern blot analysis of Ty1 mRNA of wild-type strain BY4742 grown at 20°C and 30°C. Equivalent amounts of total RNA were hybridized with a ³²P-labeled riboprobe corresponding to the 5′ end of Gag (see Fig. 2A). The signal detected for Ty1 mRNA was normalized to rRNA levels. Below the blot is the relative Ty1 mRNA level.

Gag colocalizes with VLP clusters. (A) Comparison of mRNA/Gag foci by FISH-IF in cells expressing endogenous levels of Ty1 (BY4742) versus cells overexpressing a Ty1 element from pGTy1 (DG2027). Cells containing pGTy1 were induced with galactose for 16 h. Ty1 mRNA and Gag were detected as described in the legend to Fig. 3B. Merged mRNA and Gag staining, DNA staining with DAPI, and corresponding DIC images are shown. The mean percentages of cells containing cytoplasmic Ty1 mRNA foci, Gag foci, and the propensity of Ty1 mRNA and Gag foci to colocalize ± the SEM are indicated. (B) Localization of Gag by IEM in cells expressing endogenous levels of Ty1 (BY4742) and cells overexpressing a pGTy1 plasmid (DG2027 and DG1781) after 16 h of galactose induction. A Ty1-less S. paradoxus strain (DG1768) served as a negative control. Gag was detected with anti-Ty1 VLP serum and visualized with 10-nm gold particles conjugated to anti-rabbit IgG. Enlargements of sections containing Gag are shown, and representative VLPs are indicated with arrows. A black bar shows the scale of each image. N, nucleus.

P-body components are required for accumulation of IN and RT. Levels of IN, RT, and Gag in P-body component mutants. Total protein extracts from the wild type (wt) and P-body component mutants overexpressing pGTy1 for 16 h, extracted from equivalent cell densities, were subjected to Western blot analysis using anti-IN, anti-RT, and anti-Ty1 VLP antibodies. Comparable levels of protein were loaded, as judged from Coomassie blue staining. Protein molecular size standards (kilodaltons) are indicated.

Colocalization of Ty1 mRNA and antisense RNAs in wild-type (wt) and kem1Δ mutant cells. Ty1 antisense RNAs were detected as described in the legend to Fig. 9A, and Ty1 mRNA was detected using a probe labeled with Alexa Fluor 594 corresponding to the RT region. Merged Ty1 antisense RNA and mRNA staining is shown, and colocalized foci and aggregates are indicated by arrows.

Ty1 integration events upstream of the SUF16 tRNA^Gly gene are reduced in P-body component mutants. (A) Schematic representation depicting the region where Ty1 elements integrate upstream of SUF16 on chromosome III. LTRs are represented by hatched triangles. PCR using TyB Out and SNR33 Out primers (black horizontal arrows) yields products derived from the junctions of de novo Ty1 integration upstream of SUF16. For example, the vertical black arrow represents the location of a new Ty1 integration, which would generate a PCR product of 780 bp. PCR products range between 780 bp and ∼1,400 bp, with a periodicity of ∼80 bp (21). (B) Ty1 integration assay for wild-type (wt) strain DG2122 and isogenic P-body component mutants. An isogenic spt3Δ mutant, DG2277, served as a negative control. Three independent colonies were analyzed for each strain, and PCR products were resolved on 1.5% agarose gels, stained with ethidium bromide, and visualized by fluorescence imaging. Below are control PCR using primers specific for the CPR7 gene. Molecular size standards (kilobases) are alongside the gels.

Localization of Ty1 mRNA and Gag in discrete cytoplasmic foci requires P-body components. Cells were grown in SC-glucose (2%) medium at 20°C and visualized by differential interference contrast (DIC) microscopy. Ty1 mRNA and Gag were detected by FISH-IF, and DNA was stained with DAPI. Representative images are shown. (A) Ty1 mRNA localizes in discrete cytoplasmic foci in wild-type (wt) BY4742 cells. Ty1 mRNA was detected using a probe corresponding to the RT region labeled with either DIG or Alexa Fluor 594. Merged Ty1 mRNA and DAPI staining is shown. Mean percentages of cells containing cytoplasmic Ty1 mRNA foci ± the standard error of the mean (SEM) calculated from more than six independent experiments are included. (B) P-body components are required for the formation of discrete Ty1 mRNA/Gag foci. Ty1 mRNA and Gag for the wild-type strain and isogenic P-body component mutants were detected using a DIG-labeled probe corresponding to the GAG sequence and an anti-Ty1 VLP serum, respectively. Merged Ty1 mRNA and Gag staining is shown. (C) Localization of ASH1 mRNA and Gag as a control for FISH-IF specificity. ASH1 mRNA was detected using ASH1-433 and -884 oligonucleotides labeled with DIG. Gag was detected as described for panel B. Merged ASH1 mRNA, Gag, and DAPI staining is shown.

Ty1 mRNA/Gag foci are distinct from P-bodies. (A) Ty1 mRNA/Gag foci disassemble when P-bodies are activated. Wild-type (wt) cells were grown at 20°C in SC medium in the presence of glucose (+Glu) and then deprived of glucose (−Glu) for 25 min, which activates the formation of P-bodies. Ty1 mRNA and Gag were detected as described in the legend to Fig. 3B. (B) Levels of Ty1 mRNA in cells deprived of glucose. Northern blot analysis of Ty1 mRNA for the wild-type strain (BY4742) and isogenic HA-tagged derivatives grown in the presence of glucose (+Glu) and then deprived of glucose (−Glu) for 25 min. Equivalent amounts of total RNA were hybridized with a ³²P-labeled riboprobe corresponding to the 5′ end of GAG (see Fig. 2A). The signal detected for Ty1 mRNA was normalized to rRNA levels. Below the blot is the level of Ty1 mRNA without Glu relative to that of Ty1 mRNA with Glu.

P-body components are required for the formation of retrotransposition-competent VLPs. (A) P-body components are required for colocalization of Ty1 mRNA/Gag in foci after galactose induction of a pGTy1 plasmid. Cells overexpressing pGTy1 (DG2027, MAC350, MAC587, and MAC590) were induced for 16 h. Ty1 mRNA and Gag were detected as described in the legend to Fig. 3B. Merged mRNA/Gag and Gag/DAPI staining, DNA staining with DAPI, and corresponding DIC images are shown. The percentages of cells containing cytoplasmic Ty1 mRNA and Gag foci and the propensity of Ty1 mRNA/Gag foci or aggregates to colocalize and Gag/DAPI overlap are indicated. (B) Localization of VLPs by TEM in P-body component mutants overexpressing a pGTy1 plasmid after 16 h of galactose induction. Representative cells containing VLP clusters outlined in blue (right and middle panels) and dispersed VLPs indicated by yellow arrows (right panel) are shown. The black bars represent 500 nm.

Localization and levels of Ty1 antisense RNAs in wild-type (wt) cells and P-body component mutants. (A) Localization of Ty1 antisense RNAs in cytoplasmic puncta or aggregates in wild-type (BY4742) cells and isogenic P-body mutants by FISH-IF. Ty1 antisense RNAs were detected with a DIG-labeled probe corresponding to the GAG sequence (Gag340 probe). Merged antisense RNA and DAPI staining is shown. Ty1-less S. paradoxus strain DG1768 served as a negative control. (B) P-body component mutants have elevated levels of Ty1 antisense RNAs. Northern analysis of Ty1 antisense RNAs in the wild type and isogenic P-body component mutants. Equivalent amounts of total RNA were hybridized with a ³²P-labeled riboprobe corresponding to the 5′ end of Ty1 (Gag 5′-end riboprobe). The hybridization signals for Ty1 antisense RNAs were normalized to the level of 18S and 25S rRNA, as determined by phosphorimaging. An isogenic spt3Δ mutant (DG2247) was included to verify that Ty1 mRNA synthesis and antisense RNA synthesis were independent. RNA molecular size standards (kilobases) are indicated. SD, standard deviation.