TORC1-dependent sumoylation of Rpc82 promotes RNA polymerase III assembly and activity

Abstract

Maintaining cellular homeostasis under changing nutrient conditions is essential for the growth and development of all organisms. The mechanisms that maintain homeostasis upon loss of nutrient supply are not well understood. By mapping the SUMO proteome in Saccharomyces cerevisiae, we discovered a specific set of differentially sumoylated proteins mainly involved in transcription. RNA polymerase III (RNAPIII) components, including Rpc53, Rpc82, and Ret1, are particularly prominent nutrient-dependent SUMO targets. Nitrogen starvation, as well as direct inhibition of the master nutrient response regulator target of rapamycin complex 1 (TORC1), results in rapid desumoylation of these proteins, which is reflected by loss of SUMO at tRNA genes. TORC1-dependent sumoylation of Rpc82 in particular is required for robust tRNA transcription. Mechanistically, sumoylation of Rpc82 is important for assembly of the RNAPIII holoenzyme and recruitment of Rpc82 to tRNA genes. In conclusion, our data show that TORC1-dependent sumoylation of Rpc82 bolsters the transcriptional capacity of RNAPIII under optimal growth conditions.

Keywords: RNA polymerase III; Sumo; TORC1; tRNA; transcription.

Fig. 1.

Inhibition of TORC1 alters the sumoylated protein landscape. (A) Nitrogen starvation alters the profile of protein sumoylation. Sumoylated proteins were purified under denaturing conditions from cells expressing HIS₆-FLAG–tagged SUMO (6HF-SMT3, which encodes yeast SUMO) and analyzed by Western blotting using SUMO antibodies. (B) Differential sumoylation of proteins in response to nitrogen starvation. Sumoylation of Cyc8, Tup1, and Rap1 was increased, whereas sumoylation of RNAPIII components Rpc53, Rpc82, and Ret1 was decreased upon nitrogen starvation. (C) GO analysis showing that proteins that are desumoylated following nitrogen stress and are primarily involved in mRNA translation. The size of the nodes indicates the relative number of proteins associated with that specific GO term; the width of the edges indicates the relative number of proteins in common between the GO terms. (D) Differential sumoylation of proteins in response to inhibition of TORC1. Sumoylation of RNAPIII components Rpc53, Rpc82, and Ret1 was decreased upon rapamycin treatment. (E) Validation of the MS data. SUMO pull-downs were performed under denaturing conditions using cell lysates of cells grown under nitrogen-rich or nitrogen-starved conditions (Starv.), and the levels of copurifying Rpc82-HA, Rpc53-HA, and Ret1-HA were analyzed by Western blotting with anti-HA antibodies. Rpc82-HA signals (Lower graphs) were quantified using ImageJ. Error bars indicate the deviation from the average of two experiments. (F) Validation of MS data. Cells were treated with rapamycin (Rapa), and cell lysates were analyzed as in E.

Fig. S1.

Nitrogen starvation and rapamycin treatment remodel the Sumo proteome. (A) GO analysis of the proteins that are more highly sumoylated in response to nitrogen stress reveals overrepresentation of transcriptional regulators. The size of the nodes indicates the relative number of proteins associated with that specific GO term; the width of the edges indicates the relative number of proteins in common between the GO terms. (B) Rapamycin treatment induces increased sumoylation of the bulk of cellular proteins. Cells expressing untagged SUMO (SMT3) or His₆-FLAG–tagged SUMO (HF-SMT3) were grown to log phase and treated with either DMSO or 100 nM rapamycin for 30 min, and global protein sumoylation was analyzed as described in Fig. 1A.

Fig. 2.

SUMO is enriched at tRNA genes and regulates tRNA transcription. (A) Snapshots of SUMO ChIP-seq experiments (Upper, gray) and sRNA-seq experiments (Lower, yellow) in WT and ubc9-1 cells grown at permissive temperature and treated with either DMSO or rapamycin. “Class II genes” indicates genes that are transcribed by RNAPII, whereas “class III genes” indicates genes transcribed by RNAPIII. (B) Meta-analysis of SUMO levels at tRNA genes. Rapa, rapamycin; TSS, TSS. (C and D) Global expression levels of tRNAs (C) and other short RNAs (D) in WT and ubc9-1 cells grown at permissive temperature and treated with either DMSO or 100 nM of rapamycin (Rapa) for 30 min.

Fig. S2.

The Ubc9–SUMO pathway is required for efficient tRNA transcription. (A) Enrichment of SUMO at tRNA genes is dependent on TORC1 activity. WT cells and ubc9-1 mutants (both expressing 6HF-SMT3) were grown at permissive temperature to log phase and treated with either DMSO or 100 nM rapamycin (Rapa) for 30 min, and the relative level of SUMO at the indicated genes was determined by ChIP-qPCR. (B) Protein sumoylation is strongly reduced in ubc9-1 mutants. WT cells and ubc9-1 mutants (both expressing 6HF-SMT3) were grown at permissive temperature to log phase, after which global protein sumoylation was analyzed as described in Fig. 1A. (C) Enrichment of SUMO at tRNA genes is dependent on nitrogen availability. WT cells expressing either untagged SMT3 (CTRL) or 6HF-SMT3 were grown to log phase and either incubated in media supplemented with nitrogen and amino acids (Nitrogen-rich) or in media lacking these nitrogen sources (Nitrogen-poor), after which the relative level of SUMO at the indicated genes was determined by ChIP-qPCR. (D–F) The enrichment of the RNAPIII holoenzyme at class III genes is dependent on TORC1 activity. Indicated strains were grown to log phase, and the relative levels of 6HF-SUMO (D); GFP-Rpc82, GFP-Ret1, and GFP-Brf1 (E); and HA-Tfc4 (F) at tRNA^L, tRNA^W, and SCR1 were determined by ChIP-qPCR using antibodies against FLAG, HA, and GFP, respectively. Error bars, SEM of three independent experiments. (G) tRNA transcription is significantly reduced in ubc9-1 mutants. WT cells or ubc9-1 mutants were treated with DMSO of 100 nM of rapamycin for 30 min, after which RNA was purified and analyzed by qPCR using primer pairs against the indicated genes. Error bars, SEM of three independent experiments.

Fig. 3.

Sumoylation of Rpc82 promotes tRNA transcription. (A) Location of the putative SUMO sites in Rpc82. CC, coiled-coil domain; eWH, extended winged helix domain. Protein domain organization was derived from the structure of human RPC3 (31). (B) Rpc82-4KR is not sumoylated in vivo. Cells expressing RPC82-HA or rpc82-4KR-HA as well as HIS₆-FLAG–tagged SUMO were treated with DMSO or 100 nM rapamycin (Rapa) for 30 min, after which SUMO was purified and eluates were analyzed by Western blotting with HA antibodies. Dashed line indicates where the image was cropped to remove irrelevant data. (C) Rpc82 sumoylation is abolished in the ubc9-1 mutant. WT cells or ubc9-1 mutants (both expressing normal RPC82-HA and 6HF-SMT3) were grown to log phase at permissive temperature and treated with either DMSO or 100 nM rapamycin (Rapa) for 30 min. SUMO was then purified and eluates were analyzed as in B. (D) tRNA transcription is significantly reduced in rpc82-4KR mutants. WT cells or rpc82-4KR mutants were grown and treated as in B, after which RNA was purified and analyzed by qPCR using primer pairs against the indicated genes. Error bars, SEM of three independent experiments. (E) Deletion of MAF1 does not restore tRNA transcription in rpc82-4KR mutants. The indicated strains were grown to log phase and treated with either DMSO or 100 nM rapamycin (Rapa), after which RNA was purified and analyzed by qPCR as in D. Error bars, SEM of three independent experiments.

Fig. S3.

Mutating potential sumoylation sites in Rpc53 and Ret1 does not alter RNAPIII activity. (A) Mutations of putative SUMO sites do not abolish Rpc53 sumoylation. WT and ubc9-1 cells expressing RPC53-HA or rpc53-7KR-HA as well as HIS₆-FLAG-SMT3 were treated with DMSO or 100 nM of rapamycin for 30 min; SUMO was purified and eluates were analyzed by Western blotting with HA antibodies. (B) Mutations of putative SUMO sites do not fully abolish Ret1 sumoylation. WT and ubc9-1 cells expressing RET1-HA or ret1-3KR-HA as well as HIS₆-FLAG-SMT3 were treated with DMSO or 100 nM of rapamycin for 30 min, after which cell lysates were analyzed as in A. (C) tRNA synthesis is not affected in cells expressing rpc53-7KR. WT cells and rpc53-7KR mutants were grown and treated as in A, after which RNA was purified and analyzed by qPCR using primer pairs against the indicated genes. Error bars, SEM of three independent experiments. (D) Stimulation of tRNA synthesis in cells expressing ret1-3KR. WT cells and ret1-3KR mutants were grown and treated as in A, after which RNA was purified and analyzed by qPCR using primer pairs against the indicated genes. Error bars, SEM of three independent experiments.

Fig. 4.

Sumoylation of Rpc82 promotes its interaction with RNAPIII and TFIIIB. (A) Reduced binding of Rpc82-4KR to RNAPIII and TFIIIB. Cells were grown to log phase, and equal amounts of Rpc82-HA and Rpc82-4KR-HA were purified with HA antibodies in the presence of NEM to prevent desumoylation. Copurifying proteins were analyzed by quantitative MS. (B) Sumoylation of Rpc82 is important for the interaction with Brf1. Rpc82-HA and Rpc82-4KR-HA were purified with HA antibodies under native conditions in the presence of NEM, and the amount of copurifying Brf1-GFP was analyzed by Western blotting with GFP antibodies. GFP over HA ratios were determined by quantifying Western blot signals with ImageJ. Error bars indicate the deviation from the mean of two independent experiments. (C) Sumoylation of Rpc82 promotes the interaction with Ret1. Rpc82-HA and Rpc82-4KR-HA were purified from cells as described in B, and the amount of copurifying Ret1-GFP was analyzed by Western blotting with GFP antibodies. GFP over HA ratios were determined as in B. Error bars indicate the deviation from the mean of two independent experiments. (D and E) Sumoylation of Rpc82 promotes its recruitment to tRNA^L. RPC82-HA RET1-GFP cells and rpc82-4KR-HA RET1-GFP mutants were grown to log phase, and the relative level of Rpc82 or Rpc82-4KR and Ret1 at tRNA^L (D) or tRNA^W (E) was determined by ChIP-qPCR using antibodies against HA and GFP, respectively. Error bars, SEM of three independent experiments. (F) Rpc82 SUMO sites in the context of the structure of RNAPIII. K401 and K406 lie in a low-density flexible loop of Rpc82 WH3 domain. K591 lies in the coiled-coil domain of Rpc82 and faces outwards in a cavity formed by Rpc160, Rpc82, Rpc34, Rpc31, and Rpc25. K596 lies under a low-density (flexible) domain of Rpc31. (G) The putative positioning of SUMO density at K591 (purple) suggests that SUMO may stabilize the active conformation of RNAPIII. SUMO may either coordinate or stabilize the multidomain cavity formed by Rpc160, Rpc82, Rpc34, Rpc31, and Rpc25, potentially favoring the closed-clamp (active) conformation of RNAPIII. (H) rpc82-4KR cells are sensitive to heat stress. WT and rpc82-4KR cells were spotted on plates supplemented and were incubated at 30 °C or 37 °C until colonies appeared.

Fig. S4.

Rpc82 K596 is occluded by a flexible part of Rpc31. (A) Normal trafficking of Rpc82-KR. rpc82Δ cells transformed with GFP-RPC82 or GFP-rpc82-4KR were grown in dropout medium to log phase and further incubated 30 min with DAPI (100 ng/mL). Cells were gently harvested by centrifugation and washed once with PBS before being prepared for microscopy. (B) Rpc82-HA and Rpc82-4KR-HA were purified with HA antibodies and resolved by SDS/PAGE. The presence of ubiquitin and Rpc82 was detected by anti-ubiquitin (WB:Ub) and anti-HA (WB:HA) Western blots. (C) Normal stability of Rpc82-4KR. WT and rpc82-4KR cells were grown in YPD to log phase and treated with 0.2 mg/mL of cycloheximide (CHX). At indicated times, the presence of Rpc82-HA and Rpc82-4KR-HA was detected by anti-HA Western blot. As a loading control, Cdc11 protein levels were assessed by anti-Cdc11 Western blot. HA signals were determined by quantifying Western blot signals with ImageJ and were normalized to time point “0.” Error bars represent the mean of two independent experiments. (D) Cartoon showing that K596 is occluded by a low-density (flexible) loop of Rpc31.