Repression of yeast RNA polymerase III by stress leads to ubiquitylation and proteasomal degradation of its largest subunit, C160

https://doi.org/10.1016/j.bbagrm.2018.10.007Get rights and content

Highlights

  • Repression of RNA polymerase III by stress leads to degradation of C160 subunit

  • C160 protein is ubiquitylated and degraded in proteasome

  • Blocking of C160 degradation is uncoupled from inhibition of transcription

Abstract

Respiratory growth and various stress conditions repress RNA polymerase III (Pol III) transcription in Saccharomyces cerevisiae. Here we report a degradation of the largest Pol III catalytic subunit, C160 as a consequence of Pol III transcription repression. We observed C160 degradation in response to transfer of yeast from fermentation to respiration conditions, as well as treatment with rapamycin or inhibition of nucleotide biosynthesis. We also detected ubiquitylated forms of C160 and demonstrated that C160 protein degradation is dependent on proteasome activity. A comparable time-course study of Pol III repression upon metabolic shift from fermentation to respiration shows that the transcription inhibition is correlated with Pol III dissociation from chromatin but that the degradation of C160 subunit is a downstream event. Despite blocking degradation of C160 by proteasome, Pol III-transcribed genes are under proper regulation. We postulate that the degradation of C160 is activated under stress conditions to reduce the amount of existing Pol III complex and prevent its de novo assembly.

Introduction

RNA synthesis in the eukaryote nucleus is carried out by the multisubunit RNA polymerases I, II, and III. Whereas Pol I and Pol II synthesize ribosomal and mainly messenger RNA, respectively, Pol III transcribes small RNAs, including transfer RNAs, 5S ribosomal RNA, and U6 small nuclear RNA. Yeast Saccharomyces cerevisiae Pol III comprises 17 subunits. The Pol III core, composed of ten subunits, is conserved relative to Pol I and Pol II. The largest catalytic Pol III subunit, C160, shows substantial homology to the largest subunits of Pol I and Pol II, A190 and Rpb1, respectively. Rbp1 contains a repetitive carboxyl terminal domain (CTD), unique to Pol II which, depending on its phosphorylation state, controls subsequent steps of transcription and processing of the primary transcript [1]. Specific features of a carboxyl-terminal extension of C160 that are specific for Pol III were revealed by structure analysis [2] but relevant functional studies are lacking for this subunit.

Over two decades ago Rbp1 was identified as a target for ubiquitylation-mediated degradation in response to DNA damage [3,4]. Blockage of transcription due to DNA damage promotes Rbp1 ubiquitylation in the nucleus and its proteasomal degradation associated with gene transcription [5,6]. Ubiquitylation of Rpb1 is reversible in that once DNA damage is repaired, the ubiquitin moiety is removed by the ubiquitin protease Ubp3, which in turn stabilizes Pol II [7]. Pol II proteolysis is an evolutionarily conserved, tightly regulated, multistep pathway [8]. The S. cerevisiae Rsp5 protein was the first ubiquitin ligase implicated in Rpb1 degradation and is the only essential HECT family ubiquitin ligase in budding yeast [9]. Pol II ubiquitylation is signed by phosphorylation of CTD, the site of Rsp5 association [[10], [11], [12]]. NEDD4, the mammalian homologue of Rsp5, was also shown to bind to and ubiquitylate Pol II [13]. In addition, polyubiquitylation of Rbp1 for proteasomal degradation requires the Elc1–Cul3 ubiquitin ligase complex that acts in tandem with Rsp5 [14]. Degradation of Rpb1 in the presence of DNA damage requires the Cdc48 protein, a component of the ubiquitin-proteasome system that interacts with the chromatin remodeling complex INO80 that can disrupt contacts between ubiquitylated Rpb1 and chromatin [15]. Although originally identified as a response to DNA damage, Rpb1 degradation also occurs under a number of conditions that lead to Pol II stalling/arrest during transcript elongation [8]. There are at least two alternative pathways that regulate Rpb1 degradation and depend on the stress type. In response to rapamycin, which induces stress similar to that associated with nutrient limitation, chromatin-bound Rpb1 is degraded by a ubiquitin-independent mechanism involving the Rrd1 peptidyl prolyl isomerase [16].

Expression of the largest Pol I subunit, A190, both in yeast and mammals, is also controlled by ubiquitylation and proteasomal degradation [17,18]. In yeast, A190 ubiquitylation serves as a checkpoint for a cold-sensitive step during rRNA transcription. The A190 protein is stabilized via Ubp10-mediated deubiquitylation that is required to achieve optimal levels of ribosomes and cell growth [17]. In contrast to Rpb1, DNA damage and rapamycin treatment has no effect on A190 levels [17,19], indicating that Pol I degradation has a different regulation pathway than does Pol II.

We are interested in mechanisms that control Pol III biogenesis and activity. Pol III is specialized to carry out high-level transcription of short DNA templates and, like Pol I, is regulated in a global manner. Several mechanisms account for repression of Pol III-mediated transcription (referred by [20]). Previous studies explored negative regulation by the Maf1 protein, general and global repressor which binds directly the Pol III complex [21,22]. Pol III-Maf1 association is increased under unfavorable growth conditions and correlated with reduced Pol III occupancy at Pol III genes [23]. Stress conditions also down-regulate Pol III by phosphorylation of C53 subunit [23]. Another Pol III regulator that, depending on growth conditions, could act as an activator or repressor, is the general transcription factor TFIIIC [24]. Lastly, a novel mechanism that accounts for Pol III repression is the sumoylation of C53, and possibly other subunits, that leads to ubiquitylation of C160 catalytic subunit. SUMO specifically targets defective Pol III inactivated by mutations or decreased expression [25].

Here we report a decrease in steady state levels of the largest subunit of Pol III, C160, under various conditions that repress Pol III gene transcription. In response to stress, C160 protein is ubiquitylated and degraded by proteasomes, similar to the degradation of the largest subunits of Pol I and Pol II. Dynamics of C160 degradation has been analyzed in the context of down-regulation of Pol III activity and association of Pol III with chromatin.

Section snippets

Growth conditions

Yeast were grown in: rich media YPD (2% glucose, 2% peptone, 1% yeast extract), or YPGly (2% glycerol, 2% peptone, 1% yeast extract), minimal media, SC + aa, SC-ura, or SC-trp (2% glucose, 0.67% yeast nitrogen base, supplemented with 20 μg/ml of all the amino acids required for growth, or all except for uracil or tryptophan, respectively). 200 μg/ml geneticin, 200 ng/ml rapamycin (RAP) or 100 μg/ml cycloheximide (CHX) were added to YPD, as required. For nucleotide depletion strains grown on

C160 subunit has limited stability that varies depending on Pol III transcription activity

To evaluate C160 protein stability, yeast cells encoding C160 tagged with the HA epitope were treated with cycloheximide (CHX) in a time-course experiment, and cell lysates were analyzed by immunoblotting. The blots were probed with anti-HA antibodies followed by antibodies directed against other Pol III subunits and the Pol III repressor, Maf1. The level of actin was used to normalize sample loading. The blot showed that incubation with CHX for 120 min resulted in continuing C160 degradation

Discussion

In this work we present evidence that inhibition of yeast Pol III by drugs or stress conditions leads to degradation of the Pol III catalytic subunit C160 by proteasomal system. Furthermore, we showed for the first time that C160 is ubiquitylated. Moreover, a comparable time-course study showed that inhibition of Pol III transcription upon shift to glycerol medium is correlated with Pol III dissociation from chromatin but the degradation of C160 subunit is a downstream event.

We sought to

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Acknowledgements

We thank Teresa Zoladek and Damian Graczyk for critical reading of the manuscript. This work was supported by the National Science Centre [UMO-2012/04/A/NZ1/00052] and the Foundation for Polish Science [MISTRZ 7/2014].

References (52)

  • L.A. Richardson et al.

    A conserved deubiquitinating enzyme controls cell growth by regulating RNA polymerase I stability

    Cell Rep.

    (2012)
  • K. Peltonen et al.

    A targeting modality for destruction of RNA polymerase I that possesses anticancer activity

    Cancer Cell

    (2014)
  • D. Graczyk et al.

    Regulation of tRNA synthesis by the general transcription factors of RNA polymerase III - TFIIIB and TFIIIC, and by the MAF1 protein

    Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms.

    (2018)
  • R. Upadhya et al.

    Maf1 is an essential mediator of diverse signals that repress RNA polymerase III transcription

    Mol. Cell

    (2002)
  • J. Towpik et al.

    Derepression of RNA polymerase III transcription by phosphorylation and nuclear export of its negative regulator, Maf1

    J. Biol. Chem.

    (2008)
  • P. Kaiser et al.

    Is this protein ubiquitinated?

  • M. Boguta

    Maf1, a general negative regulator of RNA polymerase III in yeast

    Biochim. Biophys. Acta

    (2013)
  • D. Oficjalska-Pham et al.

    General repression of RNA polymerase III transcription is triggered by protein phosphatase type 2A-mediated dephosphorylation of Maf1

    Mol. Cell

    (2006)
  • M. Escobar-Henriques et al.

    Proteome analysis and morphological studies reveal multiple effects of the immunosuppressive drug mycophenolic acid specifically resulting from Guanylic nucleotide depletion

    J. Biol. Chem.

    (2001)
  • F. Gómez-Herreros et al.

    Balanced production of ribosome components is required for proper G1/S transition in Saccharomyces cerevisiae

    J. Biol. Chem.

    (2013)
  • I. Karkusiewicz et al.

    Maf1 protein, repressor of RNA polymerase III, indirectly affects tRNA processing

    J. Biol. Chem.

    (2011)
  • T. Mayor et al.

    Quantitative profiling of ubiquitylated proteins reveals proteasome substrates and the substrate repertoire influenced by the Rpn10 receptor pathway

    Mol. Cell. Proteomics

    (2007)
  • V. Iesmantavicius et al.

    Convergence of ubiquitylation and phosphorylation signaling in rapamycin-treated yeast cells

    Mol. Cell. Proteomics

    (2014)
  • T. Wei et al.

    Small-molecule targeting of RNA polymerase I activates a conserved transcription elongation checkpoint

    Cell Rep.

    (2018)
  • N.A. Hoffmann et al.

    Molecular structures of unbound and transcribing RNA polymerase III

    Nature

    (2015)
  • D.B. Bregman et al.

    UV-induced ubiquitination of RNA polymerase II: a novel modification deficient in Cockayne syndrome cells

    Proc. Natl. Acad. Sci.

    (1996)
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