Article Figures & Data
Figures
- Figure 1. Hmt1 occupancy at tRNA genes is decreased under stress conditions.
(A–C) Hmt1 occupancy across tRNA genes was measured in yeast cells before and after nutrient deprivation (A), treatment with CPZ (B), or treatment with tunicamycin (C). qPCR results for products of ChIP are displayed in bar graphs. Percentage of input is calculated as ΔCT, with error bars representing the SEM of three biological samples (n = 3). P-value as calculated by t test: *<0.05; **<0.01; and ***<0.001.
- Figure 2. Under optimal growth conditions, Hmt1 methylates Rpc31 and loss of this modification adversely affects biogenesis of pre-tRNAs.
(A) The amino acid sequence of yeast Rpc31 with methylated arginines at positions 5 and 9 denoted in bold lettering. (B) Immunoblotting showing the relative levels of Rpc31-MORF to endogenous Rpc31 was analyzed using lysates made from WT and hmt1Δ cells. Double asterisks denote MORF-tagged Rpc31 and a single asterisk denotes endogenous Rpc31. The level of Pgk1 is used as a loading control for the relative total protein levels loaded. (C) In vitro methylation of Rpc31 from the yeast MORF collection, after purification from WT and hmt1Δ cells, using recombinant Hmt1 and [methyl-3H]-SAM. The full protein complement in each reaction was resolved on a 4–12% SDS–PAGE; methylation was visualized by fluorography (arrow) and protein levels by Ponceau S staining. Recombinant GST-tagged Rps2 served as a positive control (highlighted by asterisk). (D) Biochemical purification of TAP-tagged Rpc82 from hmt1Δ cells. TAP was performed from hmt1Δ cells expressing TAP-tagged Rpc82 (top panel). The purified proteins were resolved on a 4–12% SDS–PAGE and the gel was silver stained to determine the protein composition (bottom panel). The TAP-purified Rpc82 and associated proteins were subjected to an in vitro methylation assay using recombinant Hmt1 and [methyl-3H]-SAM. The full protein complement in each reaction was resolved on a 4–12% gel by SDS–PAGE. Methylation of Rpc31 was visualized by fluorography and protein levels by Ponceau S staining. Recombinant GST-tagged Rps2 served as a control. (E) In vitro methylation of GST-tagged WT Rpc31, Rpc31R5K, Rpc31R9K, and Rpc31R5,9K, after purification from Escherichia coli, using recombinant Hmt1 and [methyl-3H]-SAM. Visualization of methylation and protein as in Part (B). Recombinant GST-tagged Rps2 served as a positive control. (F) Fold change in levels of pre-tRNAs in hmt1Δ or Rpc31R5,9A versus WT cells under optimal growth condition in either SC + glucose (for WT versus hmt1Δ cells) or YPD (for WT versus Rpc31R5,9A cells), as assessed by hybridization of probes to intronic regions. Bars show abundance of pre-tRNAs tL(CAA), tL(UAG), tP(UGG), and tY(GUA) in hmt1Δ (left panel) or Rpc31R5,9A (right panel) relative to those in WT cells. In each case, the signal was normalized based on the levels of U4 snRNA. Error bars represent the SEM of three biological replicates (n = 3). P-value as calculated by t test: *<0.05 and **<0.01.
- Figure 3. In the context of stress, methylation of Rpc31 is required for robust repression of pre-tRNA biogenesis.
(A, B) RNA hybridization was carried out for four pre-tRNAs in WT versus hmt1Δ cells (A) or WT versus Rpc31R5,9A cells (B) grown in YPD before and after treatment with CPZ. Ratios of signal intensities for each pre-tRNA were individually normalized against three internal controls: U4, U3, and U5. The normalized signals were plotted on a bar graph to compare against signal obtained from the untreated WT cells, which is set to 100%. Error bars represent the SEM of three biological replicates (n = 3). P-value as calculated by t test: *<0.05, **<0.01, and ***<0.001. (C) Fold decrease in expression of four candidate pre-tRNAs in WT, hmt1Δ, or Rpc31R5,9A cells after treatment with CPZ, as assessed by hybridization of probes to intronic regions. Signal was normalized to levels of the U4 snRNA. Analysis of variance (ANOVA) revealed significant variation among the strains(P-value = 1.4 × 10−6). Post hoc Tukey’s Honest significant differences method revealed a significant difference between WT and hmt1Δ (after adjustment for the multiple comparisons, the adjusted P-value is 0.0014), WT and Rpc31R5,9A (adjusted P-value is 2.8 × 10−6). n = at least three per pre-tRNA.
- Figure S1. RNA hybridization data from WT, hmt1Δ, and Rpc31R5,9A cells before and after CPZ treatment.
Total RNAs were extracted from WT, hmt1Δ, and Rpc31R5,9A cells before and after CPZ treatment. The media used to grow the cell in each panel are stated. 10 μg of RNA from each biological sample is loaded and resolve by an 8% urea-TBE gel. Resolved RNAs were transferred onto nylon membrane and subjected to RNA hybridization using radiolabeled oligonucleotide probes corresponding to pre-tRNAs of tL(CAA), tL(UAG), tP(UGG), and tY(GUA). (A, B) As a control for loading, the blots were also probed for U3, U4, and U5. Sample hybridization data from WT versus hmt1Δ (A) or versus Rpc31R5,9A (B) before and after CPZ treatment.
- Figure 4. Increased RNA Pol III occupancy at tRNA genes is observed in hmt1Δ and Rpc31R5,9A cells under stress.
(A, B) The in vivo occupancy across the four tRNA genes for Rpc82 (A) and Rpc160 (B) was determined by ChIP. qPCR results for products of ChIP performed on WT, hmt1Δ, orRpc31R5,9A cells before and after treatment of CPZ are displayed as bar graphs. Percentage of input is calculated by ΔCT. The error bars representing SEM of three biological samples (n = 3). P-value as calculated by t test: *<0.05; **<0.01, and ***<0.001. (C) Fold decrease in Rpc82 and Rpc160 occupancy for four candidate tRNA genes in WT, hmt1Δ, or Rpc31R5,9A cells after treatment with CPZ. qPCR was performed for products of ChIP on WT, hmt1Δ, or Rpc31R5,9A cells before and after treatment with CPZ. Percentage of input is calculated as ΔCT. ANOVA on these values yielded significant variation among the three strains (P-value = 0.0015). Post hoc Tukey test revealed significant differences between WT and hmt1Δ cells (adjusted P-value is 5.4 × 10−4), and between WT and Rpc31R5,9A cells (adjusted P-value is 9.8 × 10−4). n = 3 per tRNA gene.
- Figure 5. During stress, methylation of Rpc31 is required for the association of Pol III with its negative regulator Maf1.
(A) Rpc31 levels in yeast lysates generated from WT or hmt1Δ cells before and after treatment with CPZ, assessed by CoIP with an α-Rpc31 antibody. The levels of Myc-tagged Maf1-7SA were probed using an α-Myc antibody and the results are displayed in a bar graph. Error bars represent the SEM of three biological replicates (n = 3). P-value as calculated by t test: *<0.05 and **<0.01. (B) Rpc31 and Myc-tagged Maf1-7SA levels in yeast lysates generated from WT or Rpc31R5,9A cells before and after treatment of CPZ, assessed using CoIP as in (A) (including the number of replicates). P-value as calculated by t test: *<0.05 and **<0.01. (C) Rpc160 and Rpc34 levels in complexes immunoprecipitated with α-Rpc31 antibody. The samples were probed with α-Rpc160, α-Rpc34, and α-Pgk1 (as a negative control). (D) Hmt1 physically interacts with Rpc31-containing complex. CoIP of Rpc31 was carried out using cell lysates generated from WT or hmt1Δ cells and resolved on a 4–12% SDS–PAGE followed by immunoblotting using α-Hmt1, α-Rpc31, and α-Rpc34 antibodies to determine the levels of Hmt1, Rpc31, and Rpc34 present in the co-immunoprecipitates. The level of Pgk1 was used as a negative control in the immunoprecipitation experiment.
Source data are available for this figure.
Source Data for Figure 5[LSA-2018-00261_Sdata5A,B.tif][LSA-2018-00261_Sdata5C,D.tif]
- Figure 6. Decreased Maf1 occupancy at tRNA genes is observed in hmt1Δ and Rpc31R5,9A cells under stress.
(A) Maf1 occupancy across the four tRNA genes was determined by ChIP. qPCR results for products of ChIP performed on WT, hmt1Δ, or Rpc31R5,9A cells before and after treatment of CPZ are displayed as bar graphs. Percentage of input is calculated by ΔCT. The error bars representing SEM of three biological samples (n = 3). P-value as calculated by t test: *<0.05; **<0.01, and ***<0.001. (B) Fold increase in Maf1 occupancy of four candidate tRNA genes in WT, hmt1Δ, or Rpc31R5,9A cells after treatment with CPZ. qPCR was performed on products of ChIP in WT, hmt1Δ, or Rpc31R5,9A cells before and after treatment with CPZ. Percentage of input was calculated as ΔCT. ANOVA on these values revealed significant variation among the three strains (P-value = 4.6 × 10−6). Post hoc Tukey test revealed significant difference between WT and hmt1Δ (adjusted P-value is 8.6 × 10−4), and between WT and Rpc31R5,9A (adjusted P-value is 3.7 × 10−6). n = 3 per tRNA gene.
- Figure 7. Mammalian PRMT1 methylates human Rpc31 homolog RPC32β, but not RPC32α.
(A) Sequence alignment of N-terminal amino acid sequences of yeast Rpc31 with its homologs in Caenorhabditis elegans, Drosophila melanogaster, and various mammalian species. Arginines on yeast Rpc31 that are colored red represent the identified methylated arginine and are conserved in RPC32β. (B) WT RPC32α and RPC32β were purified from E. coli and then subjected to in vitro methylation by rat PRMT1 and [methyl-3H]-SAM. In parallel, methylarginine substitutionmutants of RPC32β (R4K, R8K and R4, 8K) were also tested. The arrow on the fluorograph denotes methylated RPC32β. RBP16 served as a positive control for the in vitro methylation. The protein loading levels for each sample areshownby Ponceau S staining of the same membrane before fluorography, with the arrow denoting the substrate tested.
Supplementary Materials
Table S1 Yeast strains used in this study.
Table S2 Plasmids used in this study.
