Defective RNA polymerase III is negatively regulated by the SUMO-Ubiquitin-Cdc48 pathway
- Salk Institute for Biological Studies, United States
- The Scripps Research Institute, United States
Figures
Figure 1 with 1 supplement
A reverse suppressor screen using the dominant SMT3-Q56K mutant.
(A) A mot1-301 strain was transformed with CEN LEU2 vectors carrying indicated genes, then selected for transformants on SC-Leu plates. Wild-type MOT1 or SMT3-Q56K made mot1-301 cells grow faster. (B…
Figure 1—figure supplement 1
mot1-301 suppressor mutations.
* Dominant mutation.
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Figure 1—figure supplement 1—source data 1
Source data for Figure 1—figure supplement 1
- https://doi.org/10.7554/eLife.35447.004
Figure 2 with 3 supplements
Disrupting sumoylation rescues Pol III mutations.
(A) Tetrad analysis of a cross between rpc160-M809I and siz1Δ. Tetrads were dissected on YPD, then incubated at 30°C for 4 days. The offspring of one representative tetrad was shown with genotypes …
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Figure 2—source data 1
Raw Ct values for Figure 2E.
- https://doi.org/10.7554/eLife.35447.012
Figure 2—figure supplement 1
Position of the mutated residues in Pol III structure.
https://doi.org/10.7554/eLife.35447.007-
Figure 2—figure supplement 1—source data 1
Source data for Figure 2—figure supplement 1
- https://doi.org/10.7554/eLife.35447.008
Figure 2—figure supplement 2
siz1Δ rescued a wide spectrum of Pol III mutations.
(A) Plasmid shuffle experiments to test the growth phenotype of known rpc160 and rpc31 mutants in wild-type SIZ1 or siz1Δ background. The RPC160 or RPC31 alleles were on LEU2 vectors. The parental rp…
Figure 2—figure supplement 3
Growth phenotype of Pol III disease mutations in yeast.
CEN LEU2 plasmids containing the indicated mutant alleles (e.g. rpc160 mutants) were transformed into a corresponding null strain in wild-type SIZ1 or siz1Δ background (e.g. rpc160Δand rpc160Δ siz1Δ …
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Figure 2—figure supplement 3—source data 1
Source data for Figure 2—figure supplement 3
- https://doi.org/10.7554/eLife.35447.011
Figure 3
Specificity of the rescue effect.
(A) The rpa190Δ, rpb1Δ, or rpc160Δ strain carries a URA3 plasmid carrying wild-type RPA190, RPB1, and RPC160 gene, respectively, in order to maintain viability. These strains were then transformed …
Figure 4 with 1 supplement
SUMO represses Pol III by modifying Rpc53.
(A) Total protein extracted from the indicated strains was subjected to anti-Flag IP to purify Flag-tagged Rpc160 Pol III complexes and associated proteins. Precipitated proteins were eluted with …
Figure 4—figure supplement 1
Summary of sumoylated polymerase subunits.
https://doi.org/10.7554/eLife.35447.015-
Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1
- https://doi.org/10.7554/eLife.35447.016
Figure 5 with 2 supplements
Pol III is repressed by ubiquitylation and p97/Cdc48.
(A–C) The indicated rpc160 mutant strains were crossed with slx5Δ, slx8Δ, or ubc4Δ strain, respectively, followed by tetrad analysis. The cross between slx8Δ and rpc160-G1297D was shown, because slx8…
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Figure 5—source data 1
Raw Ct values for Figure 5G.
- https://doi.org/10.7554/eLife.35447.020
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Figure 5—source data 2
Raw Ct values for Figure 5H.
- https://doi.org/10.7554/eLife.35447.021
Figure 5—figure supplement 1
ulp2-101 rescued rpc160-M809I without abolishing Rpc53 sumoylation.
(A) An rpc160-M809I strain was crossed with a wild type (left) or an ulp2-101 mutant strain (right), followed by tetrad analysis. The growth of five representative tetrads were shown. The cross …
Figure 5—figure supplement 2
Genetic relationship between rpc160, cdc48, and ufd1.
(A–C) Tetrad analysis between rpc160-M809I and cdc48-sim, ufd1-sim, and ufd1-1, respectively, showing none of these mutations rescued rpc160-M809I.
Figure 6
Pol III repression by ubiquitylation is partially mediated through Rpc160.
(A) CEN URA3 plasmids expressing HA-tagged wild type or mutant Rpc160, as indicated, were transformed into a wild-type strain, and their stabilities were assayed during a cycloheximide (CHX) chase …
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Figure 6—source data 1
Raw Ct values for Figure 6C.
- https://doi.org/10.7554/eLife.35447.023
Figure 7
DNA is involved in Pol III repression.
(A) Chromatin IP of Siz1-Flag. An untagged strain was used as negative control. Chromatin association was determined by real-time PCR of the indicated genomic loci, using the percent of input …
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Figure 7—source data 1
Raw Ct values for Figure 7A–D.
- https://doi.org/10.7554/eLife.35447.025
Figure 8
Model of Pol III regulation by SUMO, ubiquitin, and Cdc48.
A stable interaction between chromatin and the SAP domain of Siz1 stimulates its activity to modify Rpc53 with SUMO (S). Rpc53 sumoylation triggers ubiquitin (U) modification of Rpc160 and …
Tables
Table 1
Summary of mutations rescued by SMT3-Q56K.
https://doi.org/10.7554/eLife.35447.005| Gene | Protein | # of alleles | Mutations |
|---|---|---|---|
| MOT1 | Negative regulator of TBP | 3 | mot1-399 (Q1587 Stop) |
| mot1-517 (G1410R) | |||
| mot1-753 (G1300S) | |||
| SMT3 | SUMO | 4 | Not sequenced |
| AOS1 | SUMO E1 | 1 | aos1-492 (G56S) |
| ULP2 | SUMO protease | 4 | ulp2-4 (S108 Stop) |
| ulp2-253 (G265D) | |||
| ulp2-527 (W532 Stop) | |||
| ulp2-63 (W532 Stop) | |||
| RPC160 | RNA Pol III subunit | 8 | rpc160-58 (M809I) |
| rpc160-85 (G1297D) | |||
| rpc160-33 (T379I) | |||
| rpc160-419 (A880T) | |||
| rpc160-426 (E282K) | |||
| rpc160-480 (G1098D) | |||
| rpc160-628 (R365K) | |||
| rpc160-211 (G606S) | |||
| RPC128 | RNA Pol III subunit | 2 | rpc128-202 (A704T) |
| rpc128-578 (D501N) | |||
| BRF1 | TFIIIB subunit | 1 | brf1-137 (S271L) |
| TFC1 | TFIIIC subunit | 1 | tfc1-321 (N255K, Fs) |
| (AAC-AAAC, Ins, Fs) | |||
| TFC6 | TFIIIC subunit | 1 | tfc6-192 (G391E) |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | anti-HA | Santa Cruz | SC-7392 | |
| Antibody | anti-HA beads | Sigma | A2095 | |
| Antibody | anti-Flag | Sigma | F3165 | |
| Antibody | anti-Flag beads | Sigma | A2220 | |
| Antibody | anti-Myc | This study | ||
| Antibody | anti-G6PDH | Sigma | A9521 | |
| Antibody | anti-Smt3 (SUMO) | Santa Cruz | SC-28649 | |
| Antibody | GFP-Trap beads | chromotek | gta-20 | |
| Antibody | Alexa Fluor 680 Goat anti-Mouse IgG | Fisher Scientific | A21058 | |
| Antibody | Alexa Fluor 680 Goat anti-Rabbit IgG | Fisher Scientific | A21109 | |
| Antibody | DyLight 800 Goat anti-Rabbit IgG | Fisher Scientific | SA535571 | |
| Antibody | DyLight 800 Goat anti-Mouse IgG | Fisher Scientific | SA535521 | |
| Peptide, recombinant protein | 2x HA peptide | This study | ||
| Peptide, recombinant protein | 2x Flag peptide | This study | ||
| Commercial assay or kit | cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail | Sigma | 11836170001 | |
| Commercial assay or kit | Power SYBR Green PCR Master Mix | Fisher Scientific | 4367659 | |
| Commercial assay or kit | SuperScript III First-Strand Synthesis System | Invitrogen | 18080–051 | |
| Chemical compound, drug | N-Ethylmaleimide (NEM) | Sigma | E3876 | |
| Chemical compound, drug | 5-Fluoroorotic Acid (5-FOA) | Toronto Research Chemicals | F595000 | |
| Chemical compound, drug | MG132 | Fisher Scientific | 50-833-9 | |
| Chemical compound, drug | Cycloheximide | Sigma | C7698 | |
| Software, algorithm | Excel | Microsoft |
Additional files
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Supplementary file 1
Yeast strains used in this study.
- https://doi.org/10.7554/eLife.35447.027
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Supplementary file 2
Plasmids used in this study.
- https://doi.org/10.7554/eLife.35447.028
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Supplementary file 3
Primers used in this study.
The same primers were used in RNA level measurement and in chromatin IP experiments. * Used as gene-specific primer in reverse transcription.
- https://doi.org/10.7554/eLife.35447.029
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Transparent reporting form
- https://doi.org/10.7554/eLife.35447.030
