The HIV-1 Tat protein recruits a ubiquitin ligase to reorganize the 7SK snRNP for transcriptional activation
- Tyler B Faust
- Amit Weiss
- Bhargavi Jayaraman
- Billy W Newton
- Nevan J Krogan
- Iván D'Orso
- Alan D Frankel
- University of California, San Francisco, United States
- University of Texas Southwestern Medical Center, United States
- J David Gladstone Institutes, United States
Figures
Figure 1 with 1 supplement
UBE2O is a critical regulator of Tat-dependent transcription and binds HEXIM1 of the 7SK snRNP.
(A) UBE2O and ZFP91 RNAi knockdown inhibit Tat-dependent transcription. Normalized, relative Tat activities ([p24host RNAi/FFLhost RNAi] / [p24N.S. RNAi/FFLN.S. RNAi]) for CCNT1, CDK9, UBE2O, and …
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Figure 1—source data 1
Source data for luciferase-normalized p24 values of siRNA knockdown coupled to Tat activity of Figure 1A.
- https://doi.org/10.7554/eLife.31879.004
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Figure 1—source data 2
Source data for proximal and distal Tat-dependent RNA species of Figure 1B.
- https://doi.org/10.7554/eLife.31879.005
Figure 1—figure supplement 1
RNAi-mediated protein knockdown and endogenous expression.
(A) Western blots demonstrate specific protein knockdown for the indicated siRNAs used in Figure 1A. (B) Protein expression comparison across multiple cell lines. 100,000 cells of HeLa, HEK 293T, …
Figure 2 with 1 supplement
Tat uses a novel interaction surface to interact with the 7SK snRNP and UBE2O.
(A) Tat transcriptional activity data collected from our systematic set of alanine point mutants (D'Orso et al., 2012; Fernandes et al., 2016) was mapped onto the crystal structure of the Tat-P-TEFb …
Figure 2—figure supplement 1
Protein input blots for Tat mutant IPs.
Input western blots for STREP IPs from Figure 2D using endogenous antibodies.
Figure 3 with 3 supplements
HIV-1 Tat hijacks UBE2O for the non-degradative monoubiquitination of HEXIM1.
(A) In vivo ubiquitination of transfected, STREP-tagged P-TEFb and 7SK snRNP proteins co-transfected with HA-ubiquitin ±co transfection of UBE2O-F or ZFP91-F (-F designates FLAG tagged protein). …
Figure 3—figure supplement 1
FLAG-tagged MEPCE and LARP7 AP-MS.
Peptide counts are shown for selected prey proteins from MEPCE (A) or LARP7 (B) duplicate (independent transfections on different days) FLAG purifications.
Figure 3—figure supplement 2
A catalytically inactive UBE2O does not induce HEXIM1 ubiquitination.
(A) In vivo ubiquitination of transfected HEXIM1 with the co-transfection of wild type or catalytically dead (CD) UBE2O in HEK 293T cells. HA western blot indicates the ubiquitinated species. (B) …
Figure 3—figure supplement 3
Tat induces the ubiquitination of endogenous HEXIM1 and MEPCE.
(A) HeLa cells were transfected with HIS-Ubiquitin and increasing amounts of Tat-STREP plasmid (0.33, 1, and 3 μg). A denaturing nickel pull-down followed by western blotting with specific …
Figure 4
UBE2O ubiquitinates the NLS of HEXIM1.
(A) Domain organization of HEXIM1. The positions of the 23 lysines in HEXIM1 are indicated by yellow bars in the C-terminal (CT) and N-terminal (NT) regions and the NLS. (Below) Alignment of the NLS …
Figure 5
UBE2O influences HEXIM1 subcellular localization.
(A) Subcellular localization of endogenous HEXIM1. HEK 293T were transfected ±3 μg UBE20-F plasmid and fractionated into cytoplasmic (Cyto.), soluble nuclear (Nucl. sol.), and nuclear pellet (Nucl. …
Figure 6
HEXIM1 ubiquitination is required for Tat-dependent release of P-TEFb from the inhibitory 7SK snRNP complex.
(A) Endogenous IP of HEXIM1 in non-silencing (n.s.) and UBE2O RNAi cells, ±Tat expression. Rabbit IgG was used as a negative IP control. Western blots were performed with the specific endogenous …
Figure 7
Tat utilizes UBE2O for the chromatin recruitment of P-TEFb released from a cytoplasmic pool of 7SK snRNP.
(A) Endogenous HEXIM1 IP using cytoplasmic and soluble nuclear fractions. One sample from each fraction was treated with RNAse A prior to the IP to evaluate RNA-dependence of interactions. (B) …
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Figure 7—source data 1
Source data for RIP assay of Figure 7C.
- https://doi.org/10.7554/eLife.31879.016
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Figure 7—source data 2
Source data for ChIP experiment in Figure 7E.
- https://doi.org/10.7554/eLife.31879.017
Figure 8
DRB releases P-TEFb from cytoplasmic 7SK snRNP.
(A) HeLa cells were treated with 100 μM DRB or an equal volume of DMSO for 1 hr prior to subcellular fractionation and Western blotting. (B) HEK 293 T cells were transfected with UBE2O-S. Cells were …
Figure 9
Model of basal and Tat-activated HIV-1 transcription.
(Top) Basal transcription. The inhibited P-TEFb-7SK snRNP complex resides in the cytoplasm and bound to RNAP II at the viral promoter. Inhibition of P-TEFb kinase activity pauses the RNAP II-7SK …
Author response image 1
Author response image 2
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Recombinant DNA reagent | UBE2O-FLAG (plasmid) | this paper | vector:pcDNA4/TO, insert: full-length Homo sapiens UBE2O | |
| Recombinant DNA reagent | HEXIM1-STREP (plasmid) | this paper | vector:pcDNA4/TO, insert: full-length Homo sapiens HEXIM1 | |
| Sequence-based reagent | ZFP91 siRNA #1 | Qiagen | SI05109223 | |
| Sequence-based reagent | ZFP91 siRNA #2 | Qiagen | SI05109230 | |
| Sequence-based reagent | ZFP91 siRNA #3 | Qiagen | SI05150985 | |
| Sequence-based reagent | UBE2O siRNA #1 | Qiagen | SI04153863 | |
| Sequence-based reagent | UBE2O siRNA #2 | Dharmacon | J-008979–05 | |
| Sequence-based reagent | UBE2O siRNA #3 | Dharmacon | J-008979–06 | |
| Sequence-based reagent | CDK9 siRNA | Qiagen | SI00605066 | |
| Sequence-based reagent | CCNT1 siRNA | Qiagen | SI02625707 | |
| Sequence-based reagent | Non-silencing siRNA | Qiagen | SI03650318 | |
| Cell line (Homo sapiens) | HeLa (provirusΔtat) | PMID: 28345603 | ||
| Peptide, recombinant protein | HA-ubiquitin | this paper | purified from E. coli BL21 | |
| Peptide, recombinant protein | HA-ΔK ubiquitin | this paper | purified from E. coli BL21 | |
| Peptide, recombinant protein | UBE2O | this paper | purified from HEK 293 T cells with high salt | |
| Peptide, recombinant protein | UBE2O, catalytically inactive | this paper | purified from HEK 293 T cells with high salt | |
| Peptide, recombinant protein | HEXIM1 | this paper | purified from HEK 293 T cells with high salt | |
| Commercial assay or kit | HiPerFect transfection reagent | Qiagen | used for transfecting siRNA | |
| Commercial assay or kit | PolyJet Transfection reagent | Signa Gen | used for transfecting DNA | |
| Commercial assay or kit | STREP-tactin superflow resin | IBA Lifesciences | ||
| Antibody | anti-HA | Covance | MMS-101P RRID:AB_2314672 | 1:2000 dilution |
| Antibody | anti-HEXIM1 | Abcam | ab25388 RRID:AB_2233058 | 1:2000 dilution, also used for IP |
| Antibody | anti-CDK9 | Santa Cruz Biotechnology | sc-13130 RRID:AB_627245 | used for ChIP |
| Antibody | anti-IgG | Millipore | 12–371 RRID:AB_145840 | used for ChIP |
| Antibody | anti-IgG | Santa Cruz Biotechnology | sc-2027 RRID:AB_737197 | For IP |
| Antibody | anti- RNA polymerase II | Santa Cruz Biotechnology | sc-899x RRID:AB_632359 | 1:2000 |
| Antibody | anti- CCNT1 | Santa Cruz Biotechnology | sc-8127 RRID:AB_2073892 | 1:2000 |
| Antibody | anti- CCNT1 | Santa Cruz Biotechnology | sc-10750 RRID:AB_2073888 | 1:2000 |
| Antibody | anti- MEPCE | Santa Cruz Biotechnology | sc-82542 RRID:AB_2266399 | 1:1000 |
| Antibody | anti- SP1 | Santa Cruz Biotechnology | sc-14027 RRID:AB_2171049 | 1:5000 |
| Antibody | anti-AFF1 | Bethyl | A302-344A RRID:AB_1850255 | 1:1000 |
| Antibody | anti- AFF4 | Bethyl | A302-538A RRID:AB_1998985 | 1:1000 |
| Antibody | anti- UBE2O | Bethyl | A301-873A RRID:AB_1309799 | 1:1000 |
| Antibody | anti-Ku70 | Bethyl | A302-624A-T RRID:AB_10554672 | 1:1000 |
| Antibody | anti- SFPQ | Bethyl | A301-322A-T RRID:AB_937995 | 1:1000 |
| Antibody | anti-LARP7 | Aviva | ARP40847_P050 RRID:AB_1294403 | 1:2000 |
| Antibody | anti-LARP7 | Santa Cruz Biotechnology | sc-515209 RRID:AB_2728652 | 1:2000 |
| Antibody | anti-HEXIM1 | Everest | EB06964 RRID:AB_2118260 | 1:5000 |
| Antibody | anti-CDK9 | Santa Cruz Biotechnology | sc-8338 RRID:AB_2260303 | 1:2000 |
| Antibody | anti-CDK9 | ProMab | 30212 RRID:AB_2728653 | 1:2000 |
| Antibody | anti-TUBULIN | Sigma-Aldrich | T6199 RRID:AB_477583 | 1:10,000 |
| Antibody | anti-FLAG | Sigma-Aldrich | F1804 RRID:AB_262044 | 1:5000 |
| Antibody | anti-RNA pol II phosphor-Ser2 | Abcam | ab5095 RRID:AB_304749 | 1:2000 |
| Antibody | anti-STREP-HRP | Millipore | 71591 RRID:AB_10806716 | 1:5000 |
| Antibody | anti-histone H4 | Active Motif | 39269 RRID:AB_2636967 | 1:5000 |
Additional files
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Supplementary file 1
Full HEXIM1, MEPCE, and LARP7 AP-MS lists.
Each tab is a biological replicate FLAG affinity purification for the indicated protein.
- https://doi.org/10.7554/eLife.31879.020
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Supplementary file 2
HEXIM1-Ub diGly data, related to Figure 4.
Tabs show raw data for all detected unmodified and modified HEXIM1 peptides (HEXIM1 tab) or the sum of MS/MS counts for peptides generated from trypsin or Arg-C treatment (HEXIM1 summ. tab).
- https://doi.org/10.7554/eLife.31879.021
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Transparent reporting form
- https://doi.org/10.7554/eLife.31879.022
