Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
Repression of yeast RNA polymerase III by stress leads to ubiquitylation and proteasomal degradation of its largest subunit, C160
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].
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