Volume 51, Issue 1, Pages 105-115 (July 2013) RNA Polymerase I Stability Couples Cellular Growth to Metal Availability  Yueh-Jung Lee, Chrissie Young Lee, Agnieszka Grzechnik, Fernando Gonzales-Zubiate, Ajay A. Vashisht, Albert Lee, James Wohlschlegel, Guillaume F. Chanfreau  Molecular Cell  Volume 51, Issue 1, Pages 105-115 (July 2013) DOI: 10.1016/j.molcel.2013.05.005 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 51, 105-115DOI: (10.1016/j.molcel.2013.05.005) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Downregulation of Ribosomal RNA Synthesis in S. cerevisiae Cells Grown in the Presence of the Metal Chelator BPS (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 an 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 hr were analyzed by transcriptional pulse-chase analysis using 3H-uracil to monitor the kinetics of rRNA production. An rnt1Δ strain grown in normal medium, which is deficient in rRNA synthesis, was included for comparison. This analysis showed that the rate of rRNA transcription was lower in wild-type cells shifted to BPS than in rnt1Δ cells. Molecular Cell 2013 51, 105-115DOI: (10.1016/j.molcel.2013.05.005) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Downregulation of RNA Polymerase I in Zinc Deficiency (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. A western blot analysis of Rpb1 using 8WG16 and a high-percentage acrylamide gel is shown (lower panel). (D) Downregulation of RNAPI subunits is due to zinc limitation. Western blots of Rpa135p or Rpa190-TAP levels in LZM or LIM are shown. (E) RNAPI downregulation is slower in cells preloaded with zinc. An Rpa135-GFP western analysis of wild-type cells pregrown in minimal medium with (2 mM) or without (0 mM) zinc supplement and shifted in a medium containing EDTA is shown. (F) Rpa135p downregulation occurs faster in a strain genetically zinc deficient. An Rpa135-GFP western analysis in wild-type and zap1Δ strains is shown. (G) Western blot analysis of the zinc-binding proteins Sad1p and Luc7p fused to GFP during zinc starvation. Molecular Cell 2013 51, 105-115DOI: (10.1016/j.molcel.2013.05.005) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Stabilization of RNAPI Subunits and Associated Proteins during Zinc Deficiency in Vacuolar Protease Mutants (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 an Rpa190p-TAP pep4Δ strain before or after a shift in low-zinc conditions. Extracts prepared from strains grown in normal medium or after 8 hr in an EDTA-containing medium were used for TAP purification. The protein eluates after TAP purification visualized by SYPRO Ruby staining are shown. (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 were 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, Nop1p) were stable. (F) Dependency on Pep4p and Prb1p for the downregulation observed for Reb1p, Fpr3p, Rrp5p, and Cbf5p. Legends as in (A). Molecular Cell 2013 51, 105-115DOI: (10.1016/j.molcel.2013.05.005) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Export of RNA Polymerase I to the Vacuole during Zinc Deficiency Is Dependent on Xpo1p (A) Localization of Rpa49-GFP in wild-type, pep4Δ, or prb1Δ strains in normal medium (0 hr in EDTA) or after 8 hr in EDTA. 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 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 hr at 37°C before a shift to EDTA, and the level of Rpa135-GFP was analyzed by western blot. (D) Analysis of Rpa135-GFP localization in wild-type and xpo1-1 strains grown at 25°C and shifted to 37°C for 2 hr or to 37°C with EDTA. Molecular Cell 2013 51, 105-115DOI: (10.1016/j.molcel.2013.05.005) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Loss of Rpa190p Ubiquitination Triggers RNAPI Downregulation during Zinc Deficiency (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 hr in EDTA. 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. Immunoblots of ubiquitinated proteins purified by chromatography on Ni-agarose beads from extracts prepared from strains expressing His-tagged ubiquitin and HA-tagged Rpa190p are shown. 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 nonpermissive temperatures. Molecular Cell 2013 51, 105-115DOI: (10.1016/j.molcel.2013.05.005) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Analysis of RNA Polymerase I Localization in ubp2Δ, ubp4Δ, and rsp5-1 Mutants (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 hr. Molecular Cell 2013 51, 105-115DOI: (10.1016/j.molcel.2013.05.005) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 Growth of Wild-Type and Ubiquitin Protease Mutant Strains in Normal Media and Various Stress Conditions 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. Molecular Cell 2013 51, 105-115DOI: (10.1016/j.molcel.2013.05.005) Copyright © 2013 Elsevier Inc. Terms and Conditions