A Molecular Titration System Coordinates Ribosomal Protein Gene Transcription with Ribosomal RNA Synthesis
Benjamin Albert, Britta Knight, Jason Merwin, Victoria Martin, Diana Ottoz, Yvonne Gloor, Maria Jessica Bruzzone, Adam Rudner, and David Shore
Figure S1. Related to Figure 2
from stationary phase from Log phase
ifh1-AA
Figure S1: (A) Ten-fold serial dilutions of cells with the indicated IFH1 genotypes were spotted on rich media (YPD) and grown at 30°C. (B) Whole cell extracts from strains expressing Ifh1-TAP, ifh1-AA-TAP or Ifh1-Myc were subjected to IgG immunoprecipitation. Precipitates were fractionated by SDS-PAGE and silver stained. Arrows indicate positions of CURI complex subunits. (C) and (D) Ten-fold serial dilution of IFH1 or ifh1-AA strains with either normal expression or pGAL1 overexpression of the endogenous UTP22 (C) or RRP7 (D) genes, as indicated, were grown on rich medium (YP plates) with galactose as the sole carbon source. Plates were incubated at 30°C for 48 hr. (E) Ten-fold serial dilution of IFH1 or ifh1-AA cells transformed with a plasmid containing either the UTP22 or RRP7 gene under the control of a doxycycline-repressible promoter (pTet-UTP22 pTet-RRP7), or transformed with an empty pTet vector (-). Cells were spotted in the absence (-Dox) or presence (+Dox) of doxycycline and grown at 30°C for 48 hrs. (F) Rpa190 occupancy (qPCR-ChIP) on the indicated rDNA regions (fold enrichment relative to Y’-ARS control), in a strain carrying a plasmid with the UTP22 gene under the control of the Tet promoter (Tet-OFF system; pTet-UTP22) and grown at the indicated doxycycline concentrations. (G) Rap1 (left panel) and Fhl1 (right panel) occupancy (qPCR-ChIP) on RPL30 and RPL37A promoters (fold enrichment relative to ACT1 control), in a strain carrying pTet-UTP22 and grown at the indicated doxycycline concentrations.
Figure S2. Related to Figure 3
Ifh1tnemhcirne dlof PIhC 0=t ot evitaler 0 0.2 0.4 0.6 0.8 1
0 5 20 0 5 20
Rrp7 depletion ckb2Δ
RPL37A RPL30
time after rapamycin treatment [min]
Figure S2: Ifh1 occupancy (qPCR-ChIP; fold-enrichment relative to ACT1 plotted relative to t=0 values, normalized to 1) on RPL30 and RPL37A promoters at the indicated times following rapamycin treatment in a pGAL1-RRP7 strain grown for 3 hr on glucose, or in a ckb2∆ strain.
Figure S3. Related to Figure 4
GBD-Rrp7 empty GBD-Rrp7 empty GBD-Rrp7 empty
GAD
Figure S3: (A) Ten-fold serial dilutions of cells with the indicated IFH1 alleles were spotted on YPD plates and grown at 30°C for 2 days. (B) Yeast two-hybrid interaction of Ifh1 mutants with Rrp7. Each row contains PJ69-4A cells expressing GBD-Rrp7 (or the empty GBD vector) and the indicated GAD-Ifh1 mutant. Ten-fold serial dilutions were spotted onto the indicated selective synthetic media, as described in Figure 4B. (C) Ten-fold serial dilutions of the indicated IFH1 wild type or mutant cells transformed with the pTet-UTP22 plasmid,or transformed with an empty vector (-), and grown at 30°C for 2 days.
Cells are spotted in absence or presence of doxycycline (Dox), as indicated. (D) Growth curves on YPD liquid medium of either wild type IFH1 (blue), ifh1-s (red), or ifh1-6 (green) strains. Doubling times (min) during exponential phase growth are noted. (E) Ten-fold serial dilution of ifh1-s mutant cells transformed with the pTet-UTP22 plasmid or transformed with an empty vector (-). Cells were spotted in the absence or presence of doxycycline (Dox), as indicated, and grown at 30°C for 2 days.
Figure S4. Related to Figure 5
ckb2Δ
pTet-UTP22 – gcn5Δ –
+Dox 48h -Dox 48h
pTet-UTP22 ckb2Δ –
ifh1-AA
+Dox 80h -Dox 80h
sch9Δ
pTet-UTP22
– -Dox 120h +Dox 120h
sfp1Δ
+Dox 120h -Dox 120h
pTet-UTP22 – pTet-UTP22
Figure S4: Ten-fold serial dilutions of the indicated IFH1 wild type or mutant cells transformed with pTet-UTP22 an empty vector (-) were spotted onto selective medium in the absence (-) or presence of doxycycline (Dox), as indicated, and grown at 30°C for the indicated times.
0
Ctrl1 Prom ITS2 25S-1 25S-2 Ctrl2 60 Veh
time after rapamycin treatment [min]
Sfp1-TAP ChIP fold enrichment
ChIP Utp22-TAP ChIP Rrp7-TAP ChIP Ifh1 time after rapamycin treatment [min]
Figure S5: (A) Rrn3 (left panel) or Rpa190 (right panel) occupancy at the indicated rDNA regions (qPCR-ChIP; fold enrichment relative to Y’ARS control) at different times following rapamycin treatment. (B) Rpa190-FLAG occupancy at the rDNA locus in sch9-as cells (qPCR-ChIP, as in (A)) at the indicated times following 1NM-PP1 treatment. (C) Utp22, Rrp7 and Ifh1 occupancy (qPCR-ChIP of the indicated proteins; fold-enrichment relative to ACT1) at the RPL30 and RPL37A promoters, at the indicated times following rapamycin treatment. (D) Sfp1 promoter occupancy on RPL30 and RPL37A (qPCR-ChIP) at the indicated times following rapamycin treatment, in wild type (WT) and CARA cells. (E) Ten-fold serial dilutions of strains with the indicated genotypes were spotted onto YPD medium with or without rapamycin (Rapa) and grown at 30°C for 48 hr. (F) Utp22 occupancy (qPCR-ChIP; fold enrichment relative to Y’ARS) on the indicated rDNA regions in an RPA135-FRB strain at 60 min following addition of rapamycin (Rap) or vehicule (Veh). (G) Ten-fold serial dilutions of strains with the indicated genotypes were spotted onto YPD medium alone (-), or YPD plus the indicated drug (auxin or rapamycin), and grown at 30°C for 2 days. (H) Western blot of SDS-PAGE fractionated whole cell extracts from strains of the indicated genotypes, prepared 0, 30 and 60 min following auxin treatment and probed with an anti-AID antibody (upper panel) or anti-actin (lower panel), as a loading control.
Utp22-FLAG ChIP fold enrichment
Rpa190-MYC ChIP fold enrichment fold enrichment
Rpa190-FLAG ChIP fold enrichment
Rrn3-FLAG ChIP fold enrichment
Figure S5. Related to Figure 6
Figure S6. Related to Figure 7
anti-MYC anti-FLAG
Rapamycin – – + – – +
IP:MYC input
Ifh1 Utp22-FLAGIfh1-MYC Utp22-FLAGIfh1 Utp22-FLAGIfh1-MYC Utp22-FLAG
anti-MYC
IP:MYC input
anti-FLAG
Ifh1 Utp22-FLAG
Ifh1-MYC Utp22-AID+ Ifh1 Utp22-FLAGIfh1-MYC Utp22-AIDIfh1 Utp22-FLAGIfh1-MYC Utp22-AID+ Ifh1 Utp22-FLAGIfh1-MYC Utp22-AID
Rap
Figure S6: (A) Western blot using a FLAG monoclonal antibody (anti-FLAG) following immunoprecipitation with a Myc mono-clonal antibody (anti-Myc) from cells expressing Ifh1-Myc and Utp22-FLAG, or from control cells expressing only Utp22-FLAG, as indicated. Extracts were prepared before (-) or after (+) 20 min of rapamycin treatment. (B) Western blot using a FLAG monoclo-nal antibody (anti-FLAG) following immunoprecipitation with anti-Myc antibody from cells lysate expressing Ifh1-Myc and Utp22-AID, Ifh1 and Utp22-FLAG, or from the two cell lysates (Ifh1-Myc - Utp22-AID and Ifh1 - Utp22-FLAG) mixed together.
Cells containing Utp22-AID were treated with auxin for 1 hr before extract preparation. (C) Cells carrying a genomic GFP-tagged allele (IFH1, FHL1, UTP22, or RRP7) and an RFP-tagged NOP56 gene (encoding a nucleolar protein) were grown to exponential phase, and treated with vehicle (Veh) or rapamycin (Rap) for 30 min. Merged images are shown to the right of the RFP and GFP panels, as marked. (D) Schematic representation of regions conserved between Ifh1 and Crf1 (dashed box). (E) Ten-fold serial dilution of sch9-as cells in the TB50 background transformed with the pTet-UTP22 plasmid or with an empty pTet plasmid (-).
Cells were spotted in the presence (+Dox) or absence( -Dox) of doxycycline, without (left) or with (right) 200 nM 1NM-PP1, and incubated at 30°C for 40 hrs.