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. Author manuscript; available in PMC: 2014 Jul 11.
Published in final edited form as: Mol Cell. 2013 Jun 6;51(1):105–115. doi: 10.1016/j.molcel.2013.05.005

RNA Polymerase I Stability Couples Cellular Growth to Metal Availability

Yueh-Jung Lee 1, Chrissie Young Lee 1,2, Agnieszka Grzechnik 1,3, Fernando Gonzales-Zubiate 1,4, Ajay A Vashisht 5, Albert Lee 1,6, James Wohlschlegel 5, Guillaume Chanfreau 1,7
PMCID: PMC3713077  NIHMSID: NIHMS477710  PMID: 23747013

Summary

Zinc is an essential cofactor of all major eukaryotic RNA polymerases. How the activity of these enzymes is coordinated or regulated according to cellular zinc levels is largely unknown. Here we show that the stability of RNA Polymerase I (RNAPI) is tightly coupled to zinc availability in vivo. In zinc deficiency, RNAPI is specifically degraded by proteolysis in the vacuole in a pathway dependent on the exportin Xpo1p and deubiquitination of the RNAPI large subunit Rpa190p by Ubp2p and Ubp4p. RNAPII is unaffected, which allows for expression of genes required in zinc deficiency. RNAPI export to the vacuole is required for survival during zinc starvation, suggesting that degradation of zinc-binding subunits might provide a last resort zinc reservoir. These results reveal a hierarchy of cellular transcriptional activities during zinc starvation, and show that degradation of the most active cellular transcriptional machinery couples cellular growth and proliferation to zinc availability.

Introduction

Ribosome biogenesis is a highly energetically costly process (Warner, 1999) that must be tightly regulated according to cellular demands, nutrient abundance and stress conditions. One of the most frequent strategies to regulate ribosome biogenesis is to control the transcription of ribosomal DNA (rDNA) by RNA polymerase I (RNAPI). Negative regulation of RNAPI activity is frequently observed when cells are exposed to nutrient deprivation or suboptimal growth conditions (Grummt, 2003; Lempiainen and Shore, 2009; Warner, 1999). In general, RNAPI-mediated transcription is controlled by two major mechanisms: epigenetic control of rDNA chromatin can modulate the number of active rDNA transcription units, while regulation of RNAPI transcription factors directly controls RNAPI activity, typically by limiting the number of initiation-competent RNAPI complexes (Grummt, 2003; Moss, 2004). The latter strategy is most frequently utilized to quickly respond to environmental cues, for example by modulating the association of RNAPI with one of its key transcription factors such as Rrn3p, the yeast orthologue of mammalian TIF-IA (Grummt, 2003; Moss, 2004). Such a mechanism is used in nutrient starvation conditions, during which the target of rapamycin (TOR) signal transduction pathway regulates RNAPI activity by reducing the number of RNAPI complexes associated with Rrn3p/TIF-IA (Claypool et al., 2003; Mayer and Grummt, 2006; Powers and Walter, 1999; Philippi et al., 2010). RNAPI activity can also be regulated at the elongation stage (Stefanovsky et al., 2006), and by stresses that affect the integrity of cellular components. For example, induction of DNA damage activates the ATM signal transduction pathway in mammalian cells and inhibits RNAPI activity (Kruhlak et al., 2007). Exposure of mammalian cells to oxidative stress triggers the export of transcription factor TIF-IA to the cytoplasm (Mayer et al., 2005), resulting in RNAPI downregulation through a reduction of the number of RNAPI core complexes associated with TIF-IA in the nucleus. The fact that RNAPI activity is regulated during the cell cycle and in conditions of cellular damage underscores the importance of coupling RNAPI activity to cellular activity and integrity. Recently, the stability of the large subunit of RNAPI was shown to be modulated by ubiquitination (Richardson et al., 2012). Deubiquitination of Rpa190p by the Ubp10p ubiquitin protease was shown to stabilize RNAPI and to enhance rRNA synthesis (Richardson et al., 2012). This study showed that rRNA production can be controlled not only by modulating the level of transcriptionally competent RNAPI complexes, but also by changing the stability of the large subunit of RNAPI.

Zinc is an essential component of all three major eukaryotic RNA polymerases, including RNAPI (Cramer et al., 2001; Donaldson and Friesen, 2000; Naryshkina et al., 2003; Treich et al., 1991), and is also found in a large number of cellular proteins. Despite a thorough characterization of the response of eukaryotic cells to zinc deficiency (North et al., 2012), it is unclear how the activity of eukaryotic RNA polymerases is influenced by zinc levels, which is critical for the activity of these enzymes. In this study, we report the unexpected finding that RNAPI complexes are downregulated during zinc deficiency via export to the vacuole and proteolytic degradation, while the levels of most RNA Polymerase II and III subunits remain unchanged. Given the importance of RNAPI activity in controlling cellular growth and proliferation (Drygin et al., 2010), degradation of RNAPI provides an unprecedented mechanism to limit cellular growth control in conditions of zinc scarcity.

Results

Decrease of ribosomal RNA synthesis during zinc deficiency is due to RNA Polymerase I downregulation

In previous gene expression analysis of yeast cells shifted in a medium containing the non-permeable metal chelator bathophenanthroline-sulfonate (BPS; Lee et al., 2005), we observed a decrease in the amount of ribosomal RNAs (rRNAs; Fig.1A, bottom). Northern blot analysis of pre-rRNAs showed a progressive decrease in the amount of 35S, 27S, and 7S pre-rRNA intermediates in these conditions, suggesting a defect in transcription (Fig.1A, top). Pulse-chase analysis revealed that the transcription of rRNAs was severely reduced (Fig.1B) to levels comparable to those of a strain deleted for RNase III, which is deficient in rRNA transcription (Catala et al., 2008). To investigate the mechanism of this downregulation, we monitored the levels of RNA Polymerase I subunits using C-terminally Tandem-Affinity Purification (TAP)-tagged strains (Fig.2A). Western blot analysis performed from crude extracts prepared from these tagged strains showed that all RNAPI subunits tested were downregulated in cells grown with BPS. This effect was also detected for Rpa135p expressed from a plasmid-borne HA-tagged version or detected using an anti-Rpa135 monoclonal antibody (Fig.2A). We observed a downregulation of the Rpb6p subunit common to all three RNA Polymerases (Fig.2A) but most RNAPII-specific subunits did not exhibit any change (Fig.2A). The only exception was the large subunit Rpb1p, which appeared downregulated based on immunoblot analysis using the monoclonal antibody 8WG16 that recognizes the unphosphorylated C-terminal domain (CTD). Western blot analysis using the N200 antibody that recognizes the N-terminus of Rpb1p (Chen and Hahn, 2004) showed that a shorter fragment of Rpb1p accumulates during BPS treatment (Fig.2A), suggesting that the CTD is cleaved. In agreement with this hypothesis, the 8WG16 antibody detected a small fragment in high percentage polyacrylamide gels upon BPS treatment (Fig.2C). Additional experiments showed that the apparent Rpb1p CTD cleavage is the result of a proteolytic artifact occurring during preparation of crude extracts (Fig. S1).

Figure 1. Downregulation of ribosomal RNA synthesis in S. cerevisiae cells grown in the presence of the metal chelator BPS.

Figure 1

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A. Northern analysis and ethidium bromide staining of rRNAs. Top, the membrane was probed with an oligonucleotide hybridizing to the 35S pre-rRNA precursor. Cross-hybridization of the oligonucleotide is responsible for the faint signal observed at the positions of the 18S and 25S rRNAs. ScR1 was used as a loading control. Bottom, ethidium bromide staining. RNA profiles obtained with wild-type cells treated with BPS were compared to that of a rnt1Δ strain grown in normal medium, which is deficient in rRNA synthesis (Catala et al., 2008).

B. Pulse-chase analysis. Wild-type cells grown in normal medium or treated with BPS for 10 hours were analyzed by transcriptional pulse-chase analysis using 3H-uracil to monitor the kinetics of rRNAs production. A rnt1Δ strain grown in normal medium, which is deficient in rRNA synthesis was included for comparison. This analysis showed that the rate of rRNAs transcription was lower in wild-type cells shifted to BPS than in rnt1Δ cells.

Figure 2. Downregulation of RNA Polymerase I in Zinc deficiency.

Figure 2

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A. Western blot analysis of RNA polymerase subunits from crude protein extracts prepared from strains grown in the presence of BPS. PAP antibody was used for TAP-tagged proteins. The 8WG16 mAb (CTD) or N200 antibodies (NTD) were used to detect Rpb1p, and a monoclonal anti-Rpa135p antibody was used to detect Rpa135p.

B. Western blot analysis of RNA polymerase subunits during EDTA treatment. Legends as in A.

C. The CTD of Rpb1 is cleaved in extract from cells grown with BPS. Shown is a western blot analysis of Rpb1 using 8WG16 and a high percentage acrylamide gel (lower panel).

D. Downregulation of RNAPI subunits is due to zinc limitation. Shown are western blots of Rpa135p or Rpa190-TAP levels in low zinc medium (LZM) or low iron medium (LIM).

E. RNAPI downregulation is slower in cells pre-loaded with zinc. Shown is an Rpa135-GFP western analysis of wild-type cells pre-grown in minimal medium with (2mM) or without (0mM) zinc supplement, and shifted in a medium containing EDTA.

F. Rpa135p downregulation occurs faster in a strain genetically zinc deficient. Shown is an Rpa135-GFP western analysis in wild-type and zap1Δ strains.

G. Western blot analysis of the zinc-binding proteins Sad1p and Luc7p fused to GFP during zinc starvation.

BPS is generally used to chelate iron but can also chelate zinc (Zhao and Eide, 1996). We found that growing yeast cells in the presence of another divalent cation chelator, EDTA also resulted in RNAPI downregulation (Fig.2B and see below). By contrast, subunits of the two other major RNA Polymerases such as Rpb9 and Rb11p (RNAPII) and Rpc31, Rpc34, and Rpc82 (RNAPIII) were unaffected by growth in EDTA-containing medium (Fig.2B). Thus, the downregulation observed in cells grown in the presence of metal chelators such as BPS or EDTA is specific to RNAPI. These results raised the question of which metal deficiency was responsible for the effects observed. To answer this question, we prepared media containing low zinc (LZM) or low iron (LIM) by pre-incubating synthetic medium with chelating beads, and adding back all metals, except iron or zinc. Cells shifted into LZM but not LIM exhibited downregulation of the RNAPI subunits Rpa190-TAP and Rpa135p (Fig.2D), indicating that the downregulation observed is specific to zinc deficiency. In agreement with a zinc-specific effect, we found that cells “pre-loaded” with excess zinc by preculture in a medium supplemented with 2mM zinc exhibited a slower RNAPI downregulation than cells precultured in non-supplemented medium (Fig.2E). In addition, a faster downregulation of RNAPI was observed in a strain lacking the Zap1p transcription factor (Fig.2F), which is genetically zinc-deficient (Lyons et al., 2000). This result shows that RNAPI downregulation does not require the Zap1p-mediated transcriptional response, but that genetically depriving the cells of zinc accelerates the kinetics of RNAPI downregulation.

Several RNAPI subunits have been shown to bind zinc (Treich et al., 1991). A general instability of nuclear zinc-binding proteins in zinc starvation conditions could account for the downregulation of RNAPI in these conditions. To test this hypothesis, we monitored the levels of two nuclear zinc-binding splicing factors unrelated to RNAPI, Sad1p and Luc7p. In contrast to what was observed for RNAPI, levels of Sad1p and Luc7p were unaffected by zinc starvation (Fig.2G). Similarly, the Rpb2p, Rpb3p and Rpb9p zinc-binding subunits of RNAPII were stable (Fig.2A/B). Taken together, these results demonstrate that zinc deficiency results in a specific downregulation of RNAPI.

Low zinc-dependent RNA Polymerase I downregulation is unrelated to stress pathways known to affect its activity and is not due to cellular death

To investigate whether the downregulation of RNAPI during zinc deficiency is mechanistically linked to stress conditions previously known to affect its activity (Grummt, 2003; Warner, 1999), we monitored RNAPI levels in a variety of conditions or mutants known to affect RNAPI activity. RNAPI levels were unaffected by amino acid starvation (Fig. S2A) although this condition is known to result in a reduction in rDNA transcription (Lempiainen and Shore, 2009). This result is consistent with recent data showing that Rpa190p levels are unaffected by nutrient starvation (Richardson et al., 2012). In addition, we also did not detect any RNAPI downregulation in cells grown in the absence of glucose (see below), which completely blocks cell division. RNAPI activity is also known to be affected by defects in secretion, in a pathway dependent on the Wsc family of plasma membrane sensors and the Pkc1p protein kinase (Li et al., 2000; Nierras and Warner, 1999). To investigate if the downregulation of RNAPI observed in zinc deficiency is mechanistically connected to this response, we studied the kinetics of downregulation of Rpa135p in a pkc1Δ strain. Although this strain exhibits lower levels of RNAPI in normal zinc conditions, zinc starvation resulted in normal RNAPI downregulation kinetics (Fig. S2B), showing that Pkc1p is not involved in the zinc-dependent downregulation of RNAPI. Similarly, RNAPI downregulation was not inhibited in wsc mutants (Fig. S2C), indicating that RNAPI downregulation during zinc deficiency is unrelated to the response that occurs as a result of defects in plasma membrane synthesis or secretory pathways (Li et al., 2000; Nierras and Warner, 1999).

Previous studies had shown that the downregulation of RNAPI transcriptional activity during nutrient deprivation is mediated by the TOR signal transduction pathway (Claypool et al., 2003; Powers and Walter, 1999). To investigate if the downregulation of RNAPI during zinc deficiency is mechanistically dependent on the TOR pathway, we used tor1Δ or fpr1Δ strains deficient in TOR signaling. We found that RNAPI downregulation during zinc deficiency does not require an intact TOR pathway, as it occurs normally in tor1Δ or fpr1Δ mutants (Fig. S2D). Taken together, these results show that RNAPI downregulation during zinc deficiency is unrelated to regulatory pathways previously described to affect ribosome biogenesis or integrity. Additionally, we found that RNAPI downregulation in zinc deficiency is not due to cell death following prolonged exposure to low zinc conditions, as cells shifted back to normal medium after growth in zinc-deficient medium quickly resumed growth and recovered RNAPI levels (Fig. S3).

RNAPI is exported to the vacuole and degraded by vacuolar proteases in zinc deficiency

The downregulation of RNA polymerase I subunits could be due to transcriptional repression of the genes encoding these subunits or to post-transcriptional processes. We monitored the mRNA levels of genes encoding three RNAPI subunits (RPA135, RPA49 and RPA43) and found that their levels were unaffected during a shift to low zinc medium (Figure 3A). During the time course of this experiment, the ZRT1 mRNA was robustly induced (Fig.3A). Given the short half-life of this mRNA (Toesca et al., 2011), the steady accumulation of this mRNA, combined with the observation that RNAPI mRNAs are stably expressed show that that RNAPI downregulation is not an indirect consequence of a general decrease in RNAPII-mediated transcription in zinc deficiency, and is not due to transcriptional repression of RNAPI subunit genes or to a degradation of RNAPI subunit mRNAs. We next hypothesized that this downregulation was due to increased protein turnover and searched for proteases involved in zinc deficiency. Vacuolar proteases were previously shown to be upregulated during zinc deficiency (Lyons et al., 2000). To test their involvement in RNAPI downregulation, we monitored Rpa135p levels in the vacuolar protease mutant strains prb1Δ, pep4Δ or prc1Δ during a shift to low zinc medium. Fig.3B shows that Rpa135p downregulation in low zinc was rescued by inactivating Prb1p or Pep4p, but not Prc1p. The observation that inactivation of either Pep4p or Prb1p was sufficient to rescue the downregulation of RNAPI can be explained by the mutual requirement of these proteases for each other for proteolytic processing to their fully functional mature forms (Hirsch et al., 1992; Moehle et al., 1989). Similarly, the downregulation of GFP-tagged versions of Rpa135p or Rpa43p was rescued in a prb1Δ strain (Fig.3C). This effect was not due to a general involvement of vacuolar proteases in RNAPI subunits turnover under normal conditions, since RNAPI subunits half-life was identical in wild-type and vacuolar protease mutants grown in normal medium (Fig. S4). Based on these results, we hypothesized that RNAPI complexes are exported to the vacuole and degraded by vacuolar proteases during zinc deficiency. Using RNAPI subunits fused to GFP to monitor RNAPI localization during zinc starvation, we observed a major loss of GFP signal in wild-type cells (Fig.4A), consistent with the immunoblot analysis. By contrast, GFP-fused RNAPI accumulated inside the vacuolar membrane (stained with the red vital stain FM4–64; Vida and Emr, 1995) in the pep4Δ or prb1Δ strains (Fig.4A). This change of localization is highly specific to RNAPI and was not observed with the nucleolar protein Nop1p (Fig.4B), which is present in the same nucleolar fraction as RNAPI and physically associates with it (Fath et al., 2000). These experiments demonstrate that RNAPI is specifically exported to the vacuole during zinc deficiency, where it is degraded by vacuolar proteases. To investigate whether RNAPI disassembles prior to vacuolar export, we performed a TAP-tag purification (Rigaut et al., 1999) of RNAPI complexes from extracts prepared from a pep4Δ strain expressing Rpa190p-TAP grown in normal or low zinc medium. As shown in Fig.3D, the pattern of bands obtained after TAP purification was very similar before and after shift to zinc deficiency. In addition, we could detect by mass spectrometry the presence of all RNAPI subunits in these purified complexes in sub-stoichiometric proportions before and after the shift (Supplemental Table S1). Furthermore, several proteins that were shown to be present in the same nucleolar fraction and/or were co-immunoprecipitated with RNAPI such as Reb1p, Fpr3p, Rrp5p and Cbf5p (Fath et al., 2000) were degraded in the same Pep4p/Prb1p-dependent manner during zinc deficiency (Fig.3E/F). The downregulation of some RNAPI-associated rRNA processing factors such as Cbf5p and Rrp5p might contribute to the defects in rRNA synthesis described in Figure 1. By contrast, several other nuclear and nucleolar proteins were unaffected (Tfg1p, Smd1p, Smb1p, Fob1p), including Nop1p (Fig.3E). Interestingly, although Nop1p is co-immunoprecipitated by RNAPI (Fath et al., 2000), it remains localized in the nucleolus during zinc starvation (Fig.4B) and is unaffected by the downregulation (Fig.3E). This result shows that only a specific subset of RNAPI-associated proteins is degraded with RNAPI. We conclude that RNAPI and some of its associated proteins are likely to be exported during zinc deficiency as a complex to the vacuole, where they are degraded by vacuolar proteases.

Figure 3. Stabilization of RNAPI subunits and associated proteins during zinc deficiency in vacuolar protease mutants.

Figure 3

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A. Northern analysis of RPA135, RPA49, RPA43 and ZRT1 mRNAs during zinc deficiency. SCR1 was used as a loading control. B. Immunoblot analysis of Rpa135p in wild-type and vacuolar protease mutants. C, same as B but Rpa135-GFP and Rpa43-GFP were used. D. TAP purification of RNAPI complexes in a Rpa190p-TAP pep4Δ strain before or after a shift in low zinc conditions. Extracts prepared from strains grown in normal medium or after 8 hours in an EDTA-containing medium were used for TAP purification. Shown are the protein eluates after TAP purification visualized by Sypro-Ruby staining.

E. Analysis of RNAPI-associated proteins and other nuclear proteins during a low zinc shift. Cells expressing TAP-tagged subunits of proteins known to physically associate with RNAPI (Reb1p, Fpr3p, Rrp5p, Cbf5p; (Fath et al., 2000), and other nucleolar (Fob1p) or nuclear (Tfg1p, Smd1p, Smb1p) proteins were shifted in a medium containing EDTA for the indicated times, and protein levels analyzed. Extracts prepared from wild-type cells were also analyzed by immunoblotting using anti-Nop1p monoclonal antibodies. RNAPI associated proteins (Reb1p, Fpr3p, Rrp5p, Cbf5p) showed downregulation, while other control proteins (Tfg1p, Smd1p, Smb1p, Fob1p, Nop1) were stable.

F. Dependency on Pep4p and Prb1p for the downregulation observed for Reb1p, Fpr3p, Rrp5p, and Cbf5p. Legends as in A.

Figure 4. Export of RNA polymerase I to the vacuole during zinc deficiency is dependent on Xpo1p.

Figure 4

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A. Localization of Rpa49-GFP in wild-type, pep4Δ or prb1Δ strains in normal medium (0 hrs in EDTA) or after 8 hours in low zinc medium. Green, Rpa49-GFP localization. Red, vacuolar membrane stained with FM4–64.

B. Localization of Nop1-GFP in normal and low zinc conditions. Legends as in A, except that the strains were transformed with a plasmid expressing Nop1-GFP.

C. Genetic Inactivation of Xpo1p rescues RNAPI downregulation. Wild-type or xpo1-1 cells were shifted for 2 hours at 37°C before a shi ft to low zinc medium, and the level of Rpa135-GFP analyzed by western blot.

D. Analysis of Rpa135-GFP localization in wild-type and xpo1-1 strains grown at 25°C, shifted to 37°C for 2 hours, or to 37°C with EDTA.

Export of RNAPI to the vacuole requires the exportin Xpo1p but is independent of known autophagy pathways

We next investigated whether RNAPI complexes are exported from the nucleus to the vacuole via nucleus-vacuole junctions, which are formed during piecemeal microautophagy of the nucleus (PMN) in starvation conditions (Kvam and Goldfarb, 2007). The nvj1Δ and vac8Δ mutant strains defective in the PMN pathway did not rescue RNAPI downregulation during zinc starvation (Fig. S2E), showing that PMN is not required for translocation of RNAPI to the vacuole. We next investigated a potential function of the nuclear export factor Xpo1p (Stade et al., 1997) in this process and found that genetic inactivation of Xpo1p prior to a shift to low zinc medium inhibited the downregulation of RNAPI (Fig.4C) by preventing RNAPI export out of the nucleus (Fig.4D). Although we cannot completely rule out that RNAPI vacuolar degradation is due to a failure of newly synthesized protein to fold or assemble during zinc starvation, these results support the hypothesis that Xpo1p mediates the export of the RNAPI complex out of the nucleus for subsequent import into the vacuole during zinc deficiency. To understand the mechanism of vacuolar import of RNAPI during zinc deficiency, we screened mutant strains defective in various vacuolar import pathways. Neither of the atg mutants analyzed (atg1Δ, atg7Δ), which are defective in the autophagy or cytoplasm to vacuole targeting (CVT) pathways showed a rescue or delay in the downregulation of RNAPI subunits during zinc deficiency (Fig. S2F). Thus RNAPI vacuolar degradation in zinc deficiency does not require any of the known autophagy or CVT pathways.

Downregulation and vacuolar import of RNAPI in zinc deficiency requires deubiquitination of the large subunit Rpa190p

Recent results showed that stability of the RNAPI large subunit Rpa190p is affected by ubiquitination (Richardson et al., 2012). To investigate if this modification is involved in RNAPI vacuolar degradation during zinc deficiency, we probed blots of TAP-purified RNAPI complexes (prepared from a pep43Δ strain) with an anti-ubiquitin antibody. We detected high molecular mass (>190 kDa) bands (Fig.5A), which, given their mobility were likely to correspond to ubiquitinated Rpa190p. These bands disappeared after 8 hours in low zinc medium, suggesting that Rpa190p becomes deubiquitinated in low zinc conditions. To further demonstrate this, we used a strain expressing His-tagged ubiquitin and an HA-tagged version of Rpa190p (Richardson et al., 2012). After introducing a pep4Δ deletion in this strain, we purified ubiquitinated proteins under denaturing conditions using Ni-agarose beads. We confirmed the presence of ubiquitinated HA-tagged Rpa190p in normal growth conditions (Fig.5B). However we observed that the levels of ubiquitinated Rpa190p decreased during zinc starvation (Fig.5B). This decrease could also be detected in crude extracts based on the disappearance of the high molecular mass smear in low zinc conditions (Fig.5B). These observations suggest that deubiquitination of Rpa190p might be required for nuclear export and subsequent vacuolar degradation of the RNAPI complexes. To test this hypothesis, we analyzed a number of ubiquitin protease mutants for their ability to mediate RNAPI degradation in low zinc. We found that inactivation of the Ubp2p and Ubp4p deubiquitinases, which are required for sorting proteins to the vacuole (Amerik et al., 2000; Dupre and Haguenauer-Tsapis, 2001) resulted in greater stability of RNAPI in low zinc conditions (Fig.5C). By contrast, inactivation of Ubp3p had no effect (Fig.5C), showing that RNAPI vacuolar degradation does not require Ubp3p and thus is mechanistically unrelated to ribophagy, a process in which mature ribosomes are targeted to the vacuole during starvation (Kraft et al., 2008). Similarly, Rpa190p levels were not rescued upon inactivation of Ubp8p (Fig.5C) and Ubp14p (Fig. S5A), showing that the stabilization observed in the ubp2Δ and ubp4Δ strains was specific to these two ubiquitin proteases. Recently, Ubp10p was shown to stabilize Rpa190p (Richardson et al., 2012), suggesting that this ubiquitin protease might also be involved in controlling RNAPI stability during zinc starvation. However, we found that Ubp10p inactivation had no effect on Rpa190p downregulation during zinc starvation (Fig.5C). This result demonstrates that the deubiquitination events involved in stabilizing RNAPI during normal growth conditions and promoting degradation in zinc starvation are mechanistically distinct. The stabilization of RNAPI in the ubp2Δ and ubp4Δ strains was specific to zinc starvation, as inactivation of these ubiquitin proteases had no effect on Rpa190p levels during glucose or amino acid starvation (Fig. S5B). Finally, we found that growth of ubp2Δ and ubp4Δ strains in low zinc resulted in inhibition of RNAPI vacuolar import, as the majority of GFP-tagged Rpa190p localized outside the vacuole in these strains (Fig.6A). These experiments demonstrate that the activity of the Ubp2p and Ubp4p ubiquitin proteases is required for vacuolar import and degradation of RNAPI during zinc deficiency and is consistent with their demonstrated role in sorting vacuolar proteins and multi-vesicular bodies (Amerik et al., 2000; Dupre and Haguenauer-Tsapis, 2001).

Figure 5. Loss of Rpa190p ubiquitination triggers RNAPI downregulation during zinc deficiency.

Figure 5

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A. Analysis of ubiquitin in TAP-purified RNAPI complexes. RNAPI was TAP-purified from pep4Δ cells expressing a TAP-tagged version of Rpa190p grown in normal medium or after 4 or 8 hours in a low zinc medium. TAP-purified RNAPI complexes were visualized by Sypro-Ruby staining (right), or analyzed by immunoblot using anti-ubiquitin antibody, or anti-Rpa135 monoclonal antibody. Whole cell extracts were analyzed by immunoblot using the anti-ubiquitin antibody.

B. Rpa190p is deubiquitinated in low zinc conditions. Shown are immunoblots of ubiquitinated proteins selected by Ni-agarose beads chromatography of extracts prepared from strains expressing His-tagged ubiquitin and HA-tagged Rpa190p. Two exposures of the same blot are shown.

C. Immunoblot analysis of RNAPI downregulation in low zinc in various ubiquitin proteases mutant strains.

D. Immunoblot analysis of RNAPI levels in the rsp5-1 and rsp5-1pep4Δ mutants at permissive and non-permissive temperatures.

Figure 6. Analysis of RNA Polymerase I localization in ubp2Δ, ubp4Δ and rsp5-1 mutants.

Figure 6

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A. Analysis of Rpa190-GFP localization in wild-type, ubp2Δ and ubp4Δ mutants. Rpa190-GFP (green) localization was compared to the vacuole (red) stained with the vital dye FM4–64 before (T=0) or after (T=8) a shift to low zinc conditions.

B. Analysis of Rpa135-GFP localization in wild-type, rsp5-1, and rsp5-1pep4Δ grown at 25°C or shifted to 37°C for 5 hours.

Inactivation of the ubiquitin ligase Rsp5p uncouples degradation of RNAPI from zinc deficiency

The Rsp5p ubiquitin ligase was previously implicated in ribosomal RNA biogenesis and ribosome stability (Neumann et al., 2003; Shcherbik and Pestov, 2011). In addition, Rsp5p exhibits a nuclear localization and was found to interact with RNA Polymerase subunits (Neumann et al., 2003). Thus, Rsp5p was a prime candidate to mediate the ubiquitination of RNAPI. If deubiquitination of RNAPI is a major determinant of RNAPI degradation in the vacuole, we predicted that inactivation of Rsp5p might result in Pep4p/Prb1p-dependent degradation of RNAPI complexes independently of zinc levels. Indeed, the abundance of Rpa190p quickly decreased in a strain carrying an rsp5-1 ts allele grown in normal medium but shifted to non-permissive temperature (Fig.5D). Strikingly, this downregulation was abolished when a PEP4 deletion was introduced in this mutant (Fig.5D). We monitored Rpa190p localization in these strains grown in normal medium, before or after a shift to 37°C. Unlike the wild-type control, we observed a loss of GFP signal in the rsp5-1 mutant shifted to 37°C (Fig.6B). However, in the rsp5-1 pep4Δ double mutant, the GFP signal was recovered and accumulated in both the vacuole and the nucleolus (Fig.6B). The partial nucleolar signal is likely due to incomplete inactivation of Rsp5p at restrictive temperature. Taken together, these results show that genetic inactivation of Rsp5p results in vacuolar degradation of RNAPI that is mechanistically indistinguishable to what is observed during zinc deficiency, and demonstrate that deubiquitination of Rpa190p is a main determinant in triggering vacuolar export and degradation of the RNAPI complex.

The Ubp2p and Ubp4p ubiquitin proteases are required for optimal fitness or survival in zinc starvation conditions

RNA Polymerase I contains multiple zinc-binding subunits (Treich et al., 1991). Because the vacuole is the site of zinc storage and redistribution (Simm et al., 2007), we hypothesized that export of RNAPI to the vacuole for degradation might provide a last resort zinc reservoir during zinc starvation. If this hypothesis were true, inhibiting export of RNAPI was predicted to severely hamper cellular growth in zinc starvation conditions by limiting the amount of zinc that could be recovered from RNAPI zinc-binding subunits. Indeed, the ubp2Δ strain exhibited a growth defect in a medium containing EDTA, which could be remedied by zinc supplementation (Fig.7). More strikingly, the ubp4Δ strain, which showed a severe growth defect in normal media (SDC or YPD), was inviable in the presence of EDTA, but its growth could be restored by adding back zinc. These growth phenotypes were specific to zinc starvation as the ubp2Δ and ubp4Δ strains did not exhibit any specific growth defect in other stress conditions, such as high iron or copper (Fig.7), and grew similarly to a wild-type strain in amino acid or glucose starvation conditions (Fig. S5B). In contrast, the pep4Δ strain did not show any growth defect in the presence of EDTA (Fig.7), suggesting that degradation of RNAPI might not be required to release zinc from RNAPI subunits once they have been imported to the vacuole. Taken together, these data demonstrate that the export of RNAPI to the vacuole mediated by Ubp2p and Ubp4p is required for efficient growth in the absence of zinc.

Figure 7. Growth of wild-type and ubiquitin protease mutant strains in normal media and various stress conditions.

Figure 7

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Strains shown were derived from a strain expressing His-tagged ubiquitin and an HA-tagged Rpa190p, in which the PEP4, UBP2 or UBP4 genes were knocked out by targeted disruption. The plates were incubated on the different media for the indicated days at 30°C.

Discussion

The results presented in this study show that zinc deficiency results in vacuolar degradation of RNAPI and some of its associated proteins in the yeast S. cerevisiae. This regulation dramatically decreases ribosomal RNA transcription, which accounts quantitatively for most of the transcriptional activity in rapidly growing cells. The levels of most RNA polymerase II subunits were unaffected (Fig.2), and the transcription of the RNAPI subunits and ZRT1 mRNAs were robust (Fig.3A), showing that RNAPII transcription is fully functional in low zinc conditions to allow for the expression of genes induced during zinc deficiency (Lyons et al., 2000). Thus, despite the apparent down-regulation of the Rpb6p subunit shared by all three RNA Polymerases (Fig.2A), the level of this subunit must be sufficient to fulfill the RNAPII activity necessary to robustly express low-zinc response genes. These results are consistent with previous observations in Euglena showing that zinc deficiency results in the formation of only one peak of RNA polymerase activity, which is likely to be an alpha-amanitin resistant form of RNAPII (Falchuk et al., 1985). Our results might also shed light on older studies showing that feeding rats a zinc-deficient diet results in overall reduced RNA polymerase activity (Terhune and Sandstead, 1972), although these results could be due to zinc depletion from the polymerases rather than to the active degradation mechanism demonstrated here. Similar responses to lower RNAPI activity in zinc deficiency across different eukaryotic lineages suggest the existence of a conserved strategy of zinc economy that slows cellular growth and spares an essential metal for proteins whose functions are needed to survive these adverse conditions. The proteins that we identified in this study that are involved in RNAPI down-regulation were not found in a large scale study that characterized mutant strains in zinc deficiency (North et al., 2012). This discrepancy might be explained by the fact that in their study, North et al. investigated the effect of limiting zinc concentrations, while in this study, zinc is completely lacking. Thus the factors that influence survival in each of these growth conditions might be different.

A mechanism for RNAPI degradation in zinc deficiency

Our results support a model in which Rpa190p deubiquitination triggers vacuolar import of the RNAPI complex. This regulated vacuolar degradation can be uncoupled from zinc deficiency by inactivating Rsp5p (Fig.5D). The destabilization of the RNAPI complex in the rsp5-1 mutant might explain some of the phenotypes described in this mutant, such as a decrease in rRNA biogenesis and a reduction in overall ribosome levels (Shcherbik and Pestov, 2011). This result contrasts with the general role of ubiquitination in triggering protein degradation, and also with the recent findings that deubiquitination of Rpa190p by Ubp10p strongly stabilizes Rpa190p (Richardson et al., 2012). The likely explanation is that the mono- or poly-ubiquitination sites that destabilize RNAPI under normal growth conditions (Richardson et al., 2012) are distinct from the sites that stabilize RNAPI and that are deubiquitinated under low zinc conditions (this study). This hypothesis is supported by the observation that Upb10p has no influence on RNAPI downregulation during zinc starvation (Fig.5C). One specific mechanism that remains to be determined is what controls the deubiquitination of RNAPI under low zinc conditions. A decrease in intracellular zinc concentration might trigger a sensing mechanism that exposes the RNAPI complex to the action of ubiquitin proteases for vacuolar import. Such a mechanism might involve conformational changes in the RNAPI complex upon a loss of zinc by some zinc-binding RNAPI subunits, which might trigger the binding of Ubp2p or Ubp4p. Alternatively, it is possible that the activity of one of the proteins that promote Rsp5p-mediated ubiquitination or Ubp2p/Ubp4p-mediated deubiquitination of RNAPI is directly controlled by zinc level, which would directly affect deubiquitination of Rpa190p.

Why degrade RNAPI in zinc deficiency?

A frequent strategy adopted by cells during metal deficiency is to reduce the expression or stability of specific metalloproteins in order to spare particular metals and/or redistribute them to other proteins (Bird et al., 2006; Merchant and Bogorad, 1986; Puig et al., 2005). Biochemical and structural studies have shown that many subunits of all three eukaryotic RNA polymerases, including RNAPI contain multiple zinc atoms (Cramer et al., 2001; Donaldson and Friesen, 2000; Naryshkina et al., 2003; Treich et al., 1991). Given the large amounts of RNAPI subunits in the cell (~104/cell; Ghaemmaghami et al., 2003), degradation of these subunits in the vacuole, the site of zinc storage and redistribution (Simm et al., 2007) might provide a last-resort zinc reservoir during prolonged zinc starvation. This model is supported by the observation that the ubp2Δ and ubp4Δ strains exhibit specific growth defects during zinc starvation (Fig.7). The degradation of RNA polymerase subunits during zinc deficiency might represent an extreme example of metal sparing, indicating that the functions of other cellular zinc-binding proteins are prioritized over those involved in ribosomal RNAs production. In addition, downregulation of RNAPI and of rRNA transcription would ultimately result in ribosome depletion and turnover, which could also provide a zinc supply given the abundance of zinc-binding proteins in ribosomes. When zinc is rate-limiting for growth, degradation of zinc-rich macromolecules such as RNAPI and ribosomes may provide an efficient mechanism to coordinately slow cellular growth, while at the same time release an intracellular zinc reservoir to proteins needed for cell survival in low zinc. We speculate that some of this zinc might be redistributed to RNA Polymerase II to allow for expression of genes required for survival in these conditions. Because RNAPI downregulation is quickly reversible (Fig. S3), it provides cells with the ability to fully restore ribosome biogenesis when zinc is re-introduced to cells. The finding that yeast cells degrade the most active transcriptional machinery during zinc deficiency underscores the importance of zinc economy in cellular metabolism and demonstrates an unprecedented mechanism of cellular growth control.

Experimental Procedures

For zinc starvation, S. cerevisiae cells were grown to log phase in synthetic defined (SD) media, washed twice with sterile water and shifted to SD media containing either 50uM BPS or 1mM EDTA for the indicated times. For temperature sensitive mutants, cells were first grown in SD at 25°C and shifted to 37°C for the indicated times. For the temperature sensitive mutants in low zinc condition, cells were grown to log phase at 25°C and shifted to 37°C for two hours. Cells were then washed twice with pre-warmed (37°C) sterile water and shifted to pre-warmed low zinc media for the indicated times.

Protein and RNA analysis

For protein analysis, cells were harvested and resuspended in lysis buffer (200mM Tris-HCl pH 8.0; 320mM Ammonium sulfate; 5mM MgCl2; 10mM EGTA pH 8.0; 20mM EDTA pH 8.0; 1mMDTT; 20% glycerol; 1mM PMSF; 2mM benzamidine HCl and protease inhibitor cocktail) and vortexed with glass beads for 8 min at 4°C. Supernatants were collected by centrifugation and total protein concentration was measured using the Bradford method. Protein samples from these crude extracts were analyzed by 8% SDS-PAGE and transferred to PVDF membranes for Western blot analysis. Antibodies used for western blot analysis were: anti-Rpa135 (1:5000; gift from M. Oakes and M. Nomura); 8WG16 (1:5000; Covance); N200 antibody (1:2500; gift from Steve Hahn); JL-8 anti-GFP (1:10000; Clontech); peroxidase anti-peroxidase (PAP; 1:1000; Rockland), anti-HA (1:10000; Santa Cruz Biotechnology), anti-Nop1 (1:10000; EnCor Biotechnology), and anti-Ubiquitinylated proteins (1:5000; clone FK2, Millipore). All antibodies were diluted in blocking solution (1XPBST, 5% milk).

RNA extraction and northern analysis was performed as described previously (Lee et al., 2005). 10 μg of total RNA were denatured either with formaldehyde buffer (formamide and formaldehyde) or glyoxal treatment, in which total RNA were incubated with glyoxal mixture (DMSO, glyoxal, PIPES, Bis-Tris, EDTA, and glycerol) at 55°C for 1 hour. The denatured total RNAs were then resolved on 1.2% agarose gels. Gels were transferred to nylon membrane (N+, GE Healthcare). Probes were generated by PCR products or by 5′-end labeling of oligonucleotides for rRNA and hybridization was performed at 65°C overnight.

Tandem Affinity Purification

TAP purification was performed as described previously (Rigaut et al., 1999) with the following modifications. First, cells were harvested and resuspended in IPP50 or IPP150 buffer with 25% glycerol. Cell suspension was immediately frozen in liquid N2 and stored at −80°C. Cells were lysed by manual grinding in a mortar with pestle in liquid N2. Grinding-freezing was repeated until the cells became powder-like. Cell lysates were thawed on ice before centrifugation. Second, Calmodulin beads were incubated with elution buffer at 4°C for 30min before samples were eluted.

Vacuolar Membrane Staining and Microscopy

Cells expressing RNAPI-GFP or Nop1p-GFP fusion proteins were grown to log phase, harvested, resuspended in fresh medium and labeled with the vacuolar membrane dye FM4–64 (Vida and Emr, 1995; 20μM, Invitrogen). Cells were incubated with shaking at 30°C for 20 min and washed with fresh media twice to remove excess dye. Cells were then resuspended into fresh medium at 30°C for 2 ho urs and analyzed by fluorescence microscopy. For low zinc or non-permissive temperature shift, cells were first shifted to low zinc media or non-permissive temperature. Two hours prior to the indicated times, cells were harvested and labeled with FM4–64 dye as described above for two hours and visualized immediately by fluorescent microscope.

Ubiquitin pull-down in denaturing conditions

Cells expressing Rpa190–3HA and 8HIS-Ubiquitin (Richardson et al., 2012) were grown to OD600=0.8. 250 mL of cells were harvested and resuspended in 40mL of cold lysis buffer (1.85N NaOH, 7.5% 2-mercaptoethanol) and incubated on ice for 10 minutes. Proteins were precipitated by 100%TCA (final 20%TCA) on ice for 10 minutes. A protein pellet was obtained by centrifugation and was washed with ice-cold acetone. The pellet was resuspended in Buffer A (6M Guanidine-HCl; 100mM Tris-HCl pH 8.0; 20mM Imidazole). Lysates were clarified by centrifugation. Cleared lysates were incubated with Ni-NTA agarose beads (Qiagen) overnight at room temperature. Proteins bound to Ni-beads were washed with Buffer A and Buffer B (8M Urea, 100mM Tris-HCl pH8.0; 20 mM Imidazole) three times each. Bound proteins were eluted from Ni-beads by heating at 100°C for 20 min in SDS loading buffer and analyzed by SDS-PAGE and Western blot analysis.

Supplementary Material

01

Highlights.

  • Zinc deficiency triggers a down regulation of ribosomal RNA synthesis

  • RNA Polymerase I is exported and degraded by vacuolar proteases during zinc deficiency

  • Deubiquitination of the RNA Polymerase I large subunit by Ubp2/Ubp4 controls its export to the vacuole and degradation during zinc deficiency

  • The Ubp2 and Ubp4 ubiquitin proteases are required for optimal growth during zinc starvation

Acknowledgments

We thank M. Oakes and M. Nomura for the gift of anti-Rpa135p monoclonal antibody; R. Gardner, A. Goldfarb, S. Hahn, C. Guthrie, E. Jones, M. Lund, K. Severinov, J. Warner and C. Woolford, for gifts of plasmids, strains or antibodies, M. Carey, E. de Robertis, S. Merchant, G. Payne, E. Gralla and J. Valentine for helpful discussions; D. Eide for sharing results prior to publication and M. Carey, A. Courey, J. Coller and K. Roy for comments on the manuscript. C.Y.L was supported by a Ruth L. Kirschstein National Research Service Award GM07185 and by a UCLA Dissertation Year Fellowship. A.L. was supported by the UCLA Chemistry-Biology Interface training program and by a Fellowship from the American Heart Association Western States Affiliate. Supported by NIH grant GM61518 to G.C. and GM089778 to J.W.

Footnotes

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