Accumulation of poly(A)⁺ RNA in cytoplasmic bodies of rpb1 mutant cells. (A) RPB1, rpb1-67, and rpb1-1 cells grown in exponential phase were fixed and processed for poly(A)⁺ RNA-FISH using a Cy3-labeled and locked nucleic acid-modified dT₂₀ probe (THJ790 [62]). The chromatin-rich parts of yeast nuclei were visualized by DAPI staining and overlaid with the Cy3 signal as indicated. Bar = 2 μm. (B) Quantification of the fractions of RPB1 and rpb1 mutant (rpb1-1, rpb1-67, rpb1-70, rpb1-80, rpb1-488, rpb1-261, rpb1-1230, rpb1-1103, rpb1-320, and rpb1-260) cells harboring cytoplasmic poly(A)⁺ RNA bodies. (C) Triple poly(A)⁺ RNA, DAPI, and Nop1p (nucleolar marker) or Nsp1p (nuclear envelope marker) staining of fixed rpb1-67 cells carried out using the THJ790 RNA-FISH probe and the monoclonal Nop1p or Nsp1p antibody. The outskirts of the cell and the nuclear membrane are indicated by solid and dotted white borders, respectively. IF, immunofluorescence. Bar = 2 μm.

The Tya protein localizes to T bodies. (A) Triple Ty1 RNA, DAPI, and Tya protein staining of fixed haploid RPB1 and rpb1-67 cells as well as diploid rpb1-67/rpb1-67 cells. Ty1 RNA was visualized using the Ty1-INT probe, and Tya protein was visualized using a monoclonal anti-Tya antibody. The fractions of cells containing cytoplasmic Tya protein bodies are indicated in the upper-right corners of the “Tya” images. The propensities of Tya protein to overlap with Ty1 RNA, in cells harboring both signals, are indicated in the upper-right corners of the “Overlay” images. Thin and thick arrows point toward cells with Tya “speckles” and T bodies, respectively. n.a., not applicable. Bar = 2 μm. (B) Ty1 RNA primer extension analysis of total RNA harvested from WT, rpb1-67, and spt3Δ cells, carried out using a 5′-radiolabeled Ty1-GAG oligonucleotide. Size markers (in nucleotides) are indicated at left.

Enhanced T-body formation in transcription mutants. (A) Double poly(A)⁺ RNA/DAPI or Ty1 RNA/DAPI staining of the indicated fixed strains. Pie charts to the left of the images show quantifications of the numbers of T, T(A)⁺, and T(A)⁻ bodies, as described in the legend for Fig. 2C. Actual percentages of cells containing T(A)⁺ and total T bodies are indicated in the upper-right corners of the images. Bar = 2 μm. (B) Ty1 RNA Northern analysis of total RNA harvested from the indicated strains. ACT1 RNA was used as an internal standard. Ty1 and ACT1 RNAs were visualized using the 5′-radiolabeled Ty1-INT probe and a randomly labeled ACT1 probe, respectively. A.U., arbitrary units (Ty1/ACT1 ratios normalized to the WT). S.D., standard deviation of two independent experiments using two different strains of the same genotype.

Cytoplasmic poly(A)⁺ RNA bodies contain Ty1 RNA. (A) RPB1, rpb1-67, and rpb1-1 cells were fixed and processed for dual poly(A)⁺ and Ty1 RNA-FISH analysis using the Cy3-labeled dT₂₀ probe and the Cy5-labeled Ty1-INT probe. Cy3 and Cy5 signals were overlaid with each other and with DAPI stain as indicated. n.a., not applicable. Bar = 2 μm. (B) Ty1 and SED1 RNA Northern analysis of total or poly(A)⁺-purified RNA harvested from RPB1 or rpb1-67 cells. Ty1 and SED1 RNAs were visualized using the 5′-radiolabeled Ty1-INT probe and a randomly labeled SED1 probe, respectively. The poly(A)⁺ RNA recovery efficiencies for both RNAs are indicated below the images. (C) Quantification of the fractions of haploid WT, spt3Δ, rpb1-67, and rpb1-67 spt3Δ cells and diploid WT and rpb1-67/rpb1-67 cells harboring T bodies. Pie charts indicate the total numbers of T bodies and how they distribute into fractions with detectable [T(A)⁺] or undetectable [T(A)⁻] poly(A)⁺ RNA signals. Actual percentages are given below the pie charts. (D) Quantification of the fractions of WT, pap1-1, rpb1-67, and rpb1-67 pap1-1 cells harboring T bodies during exponential growth at 25°C or 30°C as indicated. Columns indicate the total numbers of T bodies and how they distribute into fractions with detectable [T(A)⁺] or undetectable [T(A)⁻] poly(A)⁺ RNA signals. Actual percentages of cells harboring T bodies are given above the columns. Temp, temperature. (E) Double Ty1 RNA and DAPI staining of fixed MEX67 and mex67-5 cells grown exponentially at 20°C or subjected to a 30-min temperature increase to 30°C. The fractions of cells containing T bodies and nucleus-retained Ty1 RNA are indicated. Bar = 2 μm.

T bodies are distinct from P bodies. (A) Triple poly(A)⁺ RNA, DAPI, and Dcp2-GFP staining of fixed WT, rpb1-67, and xrn1Δ cells grown at 25°C in glucose-rich medium (+Glucose) or for 10 min in glucose-deprived medium (−Glucose). Poly(A)⁺ RNA was labeled as described in the legend for Fig. 1A, and the Dcp2-GFP protein was visualized using a monoclonal GFP antibody. The fractions of cells containing T(A)⁺ bodies are indicated. Bar = 2 μm. (B) Triple Ty1 RNA, DAPI, and Dcp2-GFP staining of fixed WT, rpb1-67, and spt3Δ cells. Ty1 RNA was visualized as described in the legend for Fig. 2A. The fractions of cells containing T bodies are indicated. Bar = 2 μm.