The deubiquitylase Ubp15 couples transcription to mRNA export
- Institut de recherches cliniques de Montréal, Canada
- St-Patrick Research Group in Basic Oncology, Laval University Cancer Research Center, Axe Oncologie du Centre de Recherche du CHU de Québec-Université Laval, Canada
- Département de Médecine, Faculté de Médecine, Université de Montréal, Canada
Figures
Figure 1 with 1 supplement
Ubp15 interacts with the phosphorylated RNAPII and with the NPC.
(A) A schematic representation of the proteomic experiment shown in panel B. (B) Volcano plot showing the average intensity versus relative abundance (log2) of proteins identified in RNAPII complexes purified from fcp1-1 versus WT cells using spectral counts as a proxy for relative protein abundance. RNAPII subunits are shown in black and other proteins of interest are indicated. Ubp15 is circled in red. In the interest of clarity, the maximum value of the y-axis was set at 100, resulting in Rpb1 and Rpb2 not appearing on the graph. See Supplementary file 1 for the complete list of values. (C) Spectral counts of the proteins identified in a TAP-tag purification of Ubp15. (D) Western blot confirming that Ubp15 is associated with a phosphorylated form of RNAPII (Rpb1-Ser5P).
Figure 1—figure supplement 1
Rpb1-TAP complexes purified from WT and fcp1-1 cells.
(A) A silver-stained SDS-PAGE gel showing RNAPII complexes purified from WT and fcp1-1 cells. Lanes 1 and 2 are control purifications from cells non-expressing Rpb1-TAP protein. The bait (Rpb1-TAP), the IgG (from the beads used to purify the complexes), and the TEV (used to cleave within the TAP-tag and eluate the complexes from the beads) are indicated. (B) Western blot showing Rpb1, Rpb1-Ser2P, Rpb3, and Spt16 in RNAPII complexes purified from WT and fcp1-1 cells. Lanes 1 and 2 are control purifications from cells non-expressing Rpb1-TAP protein.
Figure 2 with 1 supplement
The deletion of UBP15 suppresses the 6AU sensitivity of dst1Δ and spt4Δ cells.
(A) Serial-dilution growth assays assessing the 6AU sensitivity of WT, dst1Δ, and spt4Δ cells, alone and in combination with ubp15Δ. The indicated yeast strains were grown to saturation in YNB medium lacking uracil (−URA), washed, resuspended at the same density in water, serially diluted (fivefold series), and spotted on –URA in the absence or presence of 6AU as indicated. Plates were incubated at 30°C for 3 days. (B) Top: Serial-dilution growth assays assessing the requirement of the catalytic activity of Ubp15 for its effect on the 6AU sensitivity of WT, dst1Δ, and spt4Δ cells. Strains were deleted for UBP15, DST1, and SPT4, alone or in combinations, and transformed with empty vector, plasmids expressing Ubp15-3Flag (UBP15) or catalytic dead Ubp15-C214A-3Flag (ubp15-C214A) and spotted on −URA lacking histidine (−URA /−HIS) in the absence or presence of 6AU as indicated. Bottom: Equal expression levels of WT and catalytic dead versions of Ubp15-3Flag in WT, dst1Δ, and spt4Δ cells were confirmed by western blot. Note that the 6AU concentration varies and has been optimized for each mutant.
Figure 2—figure supplement 1
UBP15 deletion phenotypes and genetic interactions.
(A) Deletion of UBP15 suppresses the 6AU sensitivity of spt5-CTRΔ, spt6-1004, and hpr1Δ cells but not of bur2Δ, ctk1Δ, rtf1Δ, and cdc73Δ cells. Cells were grown, diluted, and spotted as described for Figure 2A. Note that the 6AU concentration varies and has been optimized for each mutant. (B) Serial-dilution growth assays assessing the sensitivity of WT and ubp15Δ cells to various conditions. Cells were grown to saturation in YPD medium, washed, resuspended at the same density in water, serially diluted (fivefold series), and spotted on the indicated media. For growth at 37°C and 15°C, spt6-11 was used as a positive control. For heat-shock, plates were placed for 30 min at 50°C before incubation at 30°C and mex67-5 was used as a positive control. For ultraviolet light (UV), the plates were subjected to the indicated doses of UV (UV Stratalinker 1800) right after spotting and rad6Δ was used as a positive control. For hydroxyurea (HU), bur2Δ was used as a positive control. For formamide and caffeine, rad6Δ was used as a positive control.
Figure 3 with 1 supplement
UBP15 deletion does not rescue RNAPII processivity in dst1Δ cells.
(A) RNAPII processivity, defined as the ratio of the % of Input detected in the 3’ amplicon divided by the % of Input detected in the 5’ amplicon, after 30 min treatment with 6AU (dark gray) and absence of 6AU (light gray), as determined by ChIP-qPCR. Experiments were performed in two biological replicates. Bars show the average and circles show individual replicates. The position of PCR amplicons over the YLR454W gene used for the qPCR is indicated on the sketch above the graphs. (B) Aggregate profiles of RNAPII (Rpb3) occupancy over highly expressed yeast genes longer than 1 kb (n = 234) as determined by ChIP-chip after 1 hr treatment with 6AU. TSS, transcription start site; pA, polyadenylation site.
Figure 3—figure supplement 1
Violin plot showing the RNAPII processivity, as determined by the log2 ratio of RNAPII (Rpb3) occupancy in the last 300 bp versus the first 300 bp of each gene, as determined by ChIP-chip.
All genes with measurable values are shown (n = 4889).
Figure 4 with 1 supplement
Deletion of UBP15 rescues phenotypes of rsp5-1 mutants.
(A) Serial-dilution growth assays assessing the sensitivity of rsp5-1 cells, alone or in combination with dst1Δ and or ubp15Δ, to 6AU. The indicated yeast strains were grown to saturation in YNB medium lacking uracil (−URA), washed, resuspended at the same density in water, serially diluted (fivefold series), and spotted on –URA in the absence or presence of 6AU as indicated. Plates were incubated at 30°C for 3 days. (B) Serial-dilution growth assays assessing the effect of UBP15 deletion on the viability of rsp5-1 cells at 33°C. The indicated yeast strains were grown to saturation in YPD, washed, resuspended at the same density in water, serially diluted (fivefold series), and spotted on YPD. Plates were incubated for 3 days at 30°C or 33°C as indicated. (C) Serial-dilution growth assays assessing the contribution of the catalytic activity of Ubp15 to the genetic interaction between RSP5 and UBP15 shown in panel B. Strains deleted for UBP15, alone or in combination with the rsp5-1 mutation, were transformed with empty vector, plasmids expressing Ubp15-3Flag (UBP15) or catalytic dead Ubp15-C214A-3Flag (ubp15-C214A), spotted on YNB medium lacking histidine (−HIS), and incubated at 30°C or 33°C as indicated. (D) RNA FISH experiments looking at bulk polyA RNAs in WT, ubp15Δ, rsp5-1, and ubp15Δ/rsp5-1 cells (FY genetic background). The indicated strains were grown at 30°C in YPD then shifted to 37°C for 3 hr before being analyzed by FISH using Cy5-oligo-dT45. DNA was stained with DAPI. Scale bar, 10 µm. The percentage of cells (from at least 200 cells in each strain) with retention of polyA RNA in the nucleus is indicated on the graphic shown at the bottom (WT: 0/200, ubp15Δ: 0/200, rsp5-1: 172/222, ubp15Δ/rsp5-1: 11/227).
Figure 4—figure supplement 1
A screen for E3 ligases genetically connected to UBP15.
(A) Serial-dilution growth assays to screen various E3 ligases for genetic interaction with DST1. The indicated yeast strains were grown to saturation in YNB lacking uracil (−URA), washed, resuspended at the same density in water, serially diluted (fivefold series), and spotted on −URA in the absence or presence of 6AU as indicated. Plates were incubated for 3 days at 30°C. (B) Serial-dilution growth assays assessing the effect of UBP15 deletion on the viability of rsp5-1, rsp5-sm1, and rsp5-3 cells at 33°C or 37°C. The indicated yeast strains were grown to saturation in YPD, washed, resuspended at the same density in water, serially diluted (fivefold series), and spotted on YPD. Plates were incubated for 3 days at 30°C or 33°C as indicated. (C) Serial-dilution growth assays assessing the effect of Rsp5-1 overexpression (when expressed under the ADH1 promoter) on the viability of rsp5-1 cells at 33°C. The indicated yeast strains were grown to saturation in YPD, washed, resuspended at the same density in water, serially diluted (fivefold series), and spotted on YPD. Plates were incubated for 3 days at 30°C or 33°C as indicated. (D) Western blot showing HA-Rsp5 levels in WT, rsp5-1, ubp15Δ, and ubp15Δ/rsp5-1 cells. The expression of Rsp5-1 under the control of the ADH1 promoter (used in ‘C’) is also shown.
Figure 5 with 2 supplements
The ubiquitylation of Mex67 is regulated by Ubp15 and Rsp5.
(A) A schematic representation of the in vivo ubiquitylation assay used in panels B and C. A plasmid expressing polyhistidine-tagged ubiquitin (6His-Ub) under the control of a copper-inducible promoter was transformed in WT and mutant cells. 6His-Ub expression was induced with copper sulfate (CuSO4) and His-tagged ubiquitin-conjugated proteins were purified using Ni-NTA beads and analyzed by western blot. (B) Western blots for Mex67-6HA levels from His-tagged ubiquitin-conjugated protein pulldowns (Ni-NTA) and their inputs expressing (+) or not (−) 6His-Ub in WT, ubp15Δ, and rsp5-1 cells. (C) Same as panel B but for WT, ubp15Δ, rsp5-1, and ubp15Δ/rsp5-1 cells. (D) Ubp15 regulates the interaction of THO and CBC with Mex67. A volcano plot showing the significance versus the log2 fold change for the proteins identified in Mex67-3Flag purifications by MS in ubp15Δ versus WT cells. Gray dots show the bulk of the data and the regions for significant (p value<0.1) twofold changes are boxed. THO, CBC, and NPC subunits are labeled in red, gold, and blue, respectively. See Supplementary file 2 for the complete list of values. (E) A graphical model illustrating how the ubiquitylation/deubiquitylation of Hpr1 and Mex67 by Rps5, Ubp15, and likely other deubiquitylases, may enable dynamic interactions between THO and Mex67. Both promoting and disrupting these interactions are surmise to be required for the mRNPs to mature into export-competent particles.
Figure 5—figure supplement 1
In vivo ubiquitylation assays testing for various Ubp15 possible substrates.
(A) Western blots for NPC components (Nup82-6HA, Nup133-6HA, Nup57-6HA, Nup120-6HA, Nup145-6HA, and Nup159-6HA) levels from His-tagged ubiquitin-conjugated protein pulldowns (Ni-NTA) and their inputs expressing (+) or not (−) 6His-Ub in WT and ubp15Δ cells. (B) Western blots for nuclear export factors (Hpr1-6HA, Nab2-6HA, Npl3-6HA, and Mtr2-6HA) levels from His-tagged ubiquitin-conjugated protein pulldowns (Ni-NTA) and their inputs expressing (+) or not (−) 6His-Ub in WT and ubp15Δ cells.
Figure 5—figure supplement 2
A silver-stained SDS-PAGE gel showing Mex67 complexes purified from WT and ubp15Δ cells.
Lanes 1 and 2 are control purifications from cells non-expressing any Mex67-3Flag protein. The bait (Mex67-3Flag) and IgG (from the beads used to purify the complexes) are indicated.
Author response image 1
Mex67 occupancy on active genes is not affected by the deletion of UBP15.
A) RNAPII (Rpb3) occupancy, as determined by ChIP-chip, over genes longer than 1 kb and with an average RNAPII occupancy (log2 ratio) over 1 (n=234), in WT and ubp15Δ cells. B) Mex67-3xFLAG occupancy, as determined by ChIP-chip, over genes longer than 1 kb and with an average RNAPII occupancy (log2 ratio) over 1 (n=234), in WT and ubp15Δ cells. The left panel displays the data using the same y-axis as panel A, while the right panel shows a zoom into the -0.3-0.2 log2 ratio range.
Author response image 2
Deletion of UBP15 does not lead to increased levels of aberrant mRNAs in the cytoplasm.
A) % of intron-containing mRNAs leaking into the cytoplasm, as measured using the “Leakage assay” described before (Legrain and Rosbash, 1989, Rain and Legrain, 1997) in WT, ubp15Δ and mlp1Δ cells. mpl1Δ is used as a positive control. B) The indicated mutants were grown overnight in YPD, serial-dilated (10-fold), and plated on YMB containing a complete set of amino acids (complete) and YNB lacking lysine (-LYS) Growth on – LYS indicates that the defective (but still functional) lys2-370 transcript is exported in the cytoplasm. rrp6Δ is used as a positive control.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (S. cerevisiae) | Various | This paper | NCBITaxon:4932 | See Supplementary file 3 |
| Recombinant DNA reagent | Various | This paper | Plasmids | See Supplementary file 4 |
| Antibody | anti-Flag M2 mouse monoclonal antibody | Sigma | Cat# F3165, RRID:AB_259529 | 34 µg per IP |
| Antibody | anti-Rpb3 mouse monoclonal antibody (W0012) | Neoclone/Biolegend | Cat# 665003, RRID:AB_2564529 | 3 μL per ChIP |
| Antibody | anti-HA F7 mouse monoclonal antibody | Santa Cruz Biotechnology | Cat# sc-7392, RRID:AB_627809 | (1:1000) for WB |
| Antibody | rabbit IgG | Sigma | Cat# I5006, RRID:AB_1163659 | 50 mg coupled to 2 × 1010 Dynabeads M-270 Epoxy |
| Chemical compound, drug | Pan Mouse Dynabeads | Thermo Fisher Scientific | Cat# 11042 | 50 μL per ChIP 200 µL per IP |
| Chemical compound, drug | Dynabeads M-270 Epoxy | Thermo Fisher Scientific | Cat# 14302D | 200 µL per IP |
| Chemical compound, drug | Ni-NTA agarose beads | Qiagen | Cat# 30210 | 100 µL per purification |
| Chemical compound, drug | mono-reactive NHS ester fluorescent Cy5 and Cy3 dyes | GE Healthcare | Cat# PA23001 Cat# PA25001 | |
| Software, algorithm | Versatile Aggregate Profiler (version 1.1.0) | Brunelle et al., 2015; Coulombe et al., 2014 | http://lab-jacques.recherche.usherbrooke.ca/vap |
Additional files
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Supplementary file 1
List of the proteins associated with RNAPII and their differential association in fcp1-1 cells.
- https://cdn.elifesciences.org/articles/61264/elife-61264-supp1-v1.xlsx
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Supplementary file 2
List of the proteins associated with Mex67 and their differential association in ubp15Δ cells.
- https://cdn.elifesciences.org/articles/61264/elife-61264-supp2-v1.xlsx
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Supplementary file 3
List of yeast strains used in this study.
- https://cdn.elifesciences.org/articles/61264/elife-61264-supp3-v1.xlsx
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Supplementary file 4
List of plasmids used in this study.
- https://cdn.elifesciences.org/articles/61264/elife-61264-supp4-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/61264/elife-61264-transrepform-v1.docx
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The deubiquitylase Ubp15 couples transcription to mRNA export
eLife 9:e61264.
https://doi.org/10.7554/eLife.61264
