Desumoylation of RNA polymerase III lies at the core of the Sumo stress response in yeast

Abstract

Post-translational modification by small ubiquitin-like modifier (Sumo) regulates many cellular processes, including the adaptive response to various types of stress, referred to as the Sumo stress response (SSR). However, it remains unclear whether the SSR involves a common set of core proteins regardless of the type of stress or whether each particular type of stress induces a stress-specific SSR that targets a unique, largely nonoverlapping set of Sumo substrates. In this study, we used MS and a Gene Ontology approach to identify differentially sumoylated proteins during heat stress, hyperosmotic stress, oxidative stress, nitrogen starvation, and DNA alkylation in Saccharomyces cerevisiae cells. Our results indicate that each stress triggers a specific SSR signature centered on proteins involved in transcription, translation, and chromatin regulation. Strikingly, whereas the various stress-specific SSRs were largely nonoverlapping, all types of stress tested here resulted in desumoylation of subunits of RNA polymerase III, which correlated with a decrease in tRNA synthesis. We conclude that desumoylation and subsequent inhibition of RNA polymerase III constitutes the core of all stress-specific SSRs in yeast.

Keywords: Nutrient starvation; RNA polymerase III; Saccharomyces cerevisiae; gene regulation; mass spectrometry (MS); post-translational modification (PTM); small ubiquitin-like modifier (SUMO); stress response; transcription regulation; transfer RNA (tRNA).

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

© 2019 Nguéa P et al.

Figures

Figure 1.

Characterization of Sumo proteomes in five stress conditions by MS.A, experimental workflow for Ni–NTA enrichment of sumoylated proteins in diverse stress conditions. Exponentially growing cells expressing HIS6-FLAG-SMT3 were exposed to different cellular stressors as indicated on the scheme. The cells were lysed under denaturing conditions, and sumoylated proteins were enriched with Ni–NTA affinity matrix. Sumo substrates in each condition were identified by MS. B and C, heat maps showing specific signatures of differentially sumoylated proteins under different stress conditions. All identified Sumo targets are shown in B, whereas C shows only transcription machineries subunits. Relative changes in the sumoylation status of identified targets after each stress are displayed, where green denotes an increase in sumoylation, and red denotes a decrease. Values from two biological replicates per stress are depicted. The asterisk to the right of each heat map highlights proteins displaying a SSR that is consistent across all five stresses. D and E, Circos plot representation of overlaps between the different stresses for proteins enriched in Sumo pulldowns. The outer arcs (multiple colors) on each plot represent a given stress, whereas the inner arcs (blue) represent proteins depleted (D) or enriched (E) from Sumo pulldowns in the given stress. Lines connect the same proteins shared by multiple stresses. F and G, heat map of differentially enriched biological processes (F) or cellular components (G) based on GO identifiers generated using Metascape (52). The GO terms were assigned from the list of proteins identified in Sumo pulldowns in stressed versus unstressed samples. The respective −log10 (p values) are visualized with a color scale ranging from 0 for no representation to +20 for overrepresentation. HS, heat stress; Os, osmotic stress; Ox, oxidative stress; DA, DNA alkylation; -N, nitrogen starvation.

Figure 2.

Identification of sumo-modified complexes: heat stress and osmotic stress. Networks generated for differentially sumoylated substrates following heat stress (A) and osmotic stress (B). Nodes represent proteins, and edges represent protein–protein associations. Desumoylated proteins are circled in red, whereas hypersumoylated proteins are circled in blue. Enriched complexes are depicted with different colors (see legend). Black nodes represent proteins not associated with enriched complexes. Networks were generated using the STRING database (55).

Figure 3.

Identification of sumo-modified complexes: oxidative stress, DNA alkylation, and nitrogen starvation. Networks were generated for differentially sumoylated substrates following oxidative stress (A), DNA alkylation (B), and nitrogen starvation (C). Nodes represent proteins and edges represent protein–protein associations. Desumoylated proteins are circled in red, whereas more sumoylated proteins are circled in blue. Enriched complexes are depicted with different colors (see legend). Black nodes represent proteins not associated with enriched complexes. Networks were generated using the STRING database (55).

Figure 4.

Desumoylation of Ret1 and Rpc82 upon stress rewire their binding to class III genes and affect RNAPIII transcriptional activity.A and B, validation of MS data. Sumo pulldowns were performed under denaturing conditions using cell lysates of 6HF-SMT3 cells grown in the indicated conditions. The levels of copurifying Rpc82-HA and Ret1-MYC were analyzed by Western blotting (WB) using anti-HA (A) and anti-MYC (B) antibodies, respectively. The lower graphs display the quantification of Rpc82-HA (A) and Ret1-MYC (B) signals. Error bars indicate the deviation from the average of three different experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. n.s., nonsignificant (Student's t test); UT, untagged; NT, no treatment; HS, heat stress; Os, osmotic stress; Ox, oxidative stress; DA, DNA alkylation; -N, nitrogen starvation. C–E, stress reduces the recruitment of RNAPIII holoenzyme and general transcription factor TFIIIB at class III genes. The occupancy of RNAPIII and TFIIIB subunits were analyzed by ChIP-qPCR. Yeasts were stressed as indicated in Fig. 1, and the relative levels of Rpc82-HA (C), Ret1-MYC (D), and Brf1-GFP (E) at tDNALeu and SCR1 were determined by ChIP-qPCR using anti-HA, -MYC, and -GFP antibodies, respectively. F, schematic representation of the regions where primers used for ChIP anneal. G, schematic representation or Norther blotting probes. H, precursor tRNAs (pre-tRNA) do not accumulate under stress. Pre-tRNA levels were analyzed by Northern blotting. Northern blots are representative of three independent biological replicates of experiments. WT cells were grown at 30 °C to exponential phase and then stressed as specified in Fig. 1. Total RNA was isolated and analyzed by Northern hybridization with oligonucleotide probes specific to precursor and mature tRNALeu(CAA) (top panel) and tRNATrp(CCA) (middle panel). Loading control is provided by ethidium bromide–stained 5.8S rRNA (bottom panel). I, ratios of precursor over mature tRNAs. Errors bars, S.E. of three different experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.