PRP19 transforms into a sensor of RPA-ssDNA after DNA damage and drives ATR activation via a ubiquitin-mediated circuitry

Abstract

PRP19 is a ubiquitin ligase involved in pre-mRNA splicing and the DNA damage response (DDR). Although the role for PRP19 in splicing is well characterized, its role in the DDR remains elusive. Through a proteomic screen for proteins that interact with RPA-coated single-stranded DNA (RPA-ssDNA), we identified PRP19 as a sensor of DNA damage. PRP19 directly binds RPA and localizes to DNA damage sites via RPA, promoting RPA ubiquitylation in a DNA-damage-induced manner. PRP19 facilitates the accumulation of ATRIP, the regulatory partner of the ataxia telangiectasia mutated and Rad3-related (ATR) kinase, at DNA damage sites. Depletion of PRP19 compromised the phosphorylation of ATR substrates, recovery of stalled replication forks, and progression of replication forks on damaged DNA. Importantly, PRP19 mutants that cannot bind RPA or function as an E3 ligase failed to support the ATR response, revealing that PRP19 drives ATR activation by acting as an RPA-ssDNA-sensing ubiquitin ligase during the DDR.

Fig. 1. A proteomic screen for proteins that associate with RPA-ssDNA

A. Schematic of the screen. Purified heterotrimeric RPA complex was used to coat ssDNA. B. RPA^WT- or RPA^t-11-coated, biotinylated ssDNA was incubated with HeLa nuclear extracts. The proteins captured by RPA-ssDNA were analyzed using the indicated antibodies. C. The proteins captured by RPA^WT-ssDNA, RPA^t-11-ssDNA, or beads without ssDNA attached were stained with Coomassie blue. D. Pie chart representation of the top biological functions amongst the RPA-ssDNA-binding proteins generated using the Ingenuity Pathway Analysis Software. Numbers in the pie slices indicate the number of proteins annotated in the specified category. E. A network of DDR and replication proteins was identified by the Ingenuity analysis. Red: proteins exclusively bound to RPA^WT-ssDNA; Yellow: proteins preferentially bound to RPA^WT-ssDNA; Green: proteins with no preference for RPA^WT-ssDNA; See legend of Fig. S1 for additional details on the labeling. Additional information of the proteins identified from the screen is shown in Fig. S1 and Tables S1–S3.

Fig. 2. PRP19 interacts with RPA at sites of DNA damage

A. The PRP19 complex specifically bound to RPA^WT-ssDNA but not RPA^t-11-ssDNA (left panel). Specific binding of PRP19 and CDC5L to RPA^WT-ssDNA was confirmed by Western blot analysis (right panel). B. HEK293T cells transiently expressing SFB-PRP19 were treated with 1 μM CPT for 3 hrs or mock treated. SFB-PRP19 was captured using streptavidin-coated magnetic beads (left panel), and RPA32 was immunoprecipitated (right panel). C. Nuclear extracts were prepared from HeLa cells treated with CPT or mock treated. RPA32 was immunoprecipitated from extracts, and the coprecipitated PRP19 was detected by Western blot. D. HEK293T cells expressing SFB-PRP19 were treated with CPT or mock treated. As indicated, 10 μM of VE-821 was added to cells 30 min before CPT treatment. SFB-PRP19 was captured and the coprecipitated RPA32 was analyzed by Western blot. E. HeLa cells were treated with the transcription inhibitor DRB to reduce the transcription-associated PRP19 signals, and microirradiated with UV laser. Cells were immunostained to visualize protein recruitment to laser tracts. See also Fig. S2.

Fig. 3. PRP19 recognizes DNA damage via its interaction with RPA

A. Schematic of the PRP19 fragments tested and their abilities to bind RPA and CDC5L. B. Purified untagged RPA complex was incubated with GST-WD40^WT or GST. The interaction between RPA and GST-WD40^WT was tested by GST pulldown. C. Cells expressing full-length SFB-PRP19^WT or SFB-PRP19^Y405A were treated with CPT, and SFB-tagged proteins were captured from cell extracts. The coprecipitated proteins were analyzed by Western blot. D. HeLa cells were nucleofected with PRP19 siRNA and the indicated plasmids, treated with DRB, and microirradiated with UV laser. See also Fig. S3.

Fig. 4. The PRP19 complex promotes ATR activation and the replication stress response

A. Cells transfected with siRNAs were treated with CPT for 1 hr or mock treated. The levels of various proteins and phosphorylated proteins were analyzed. B–C. Cells transfected with the indicated siRNAs were treated with 2 mM HU for 16 hrs and then released into fresh media. Cells were immunostained for γH2AX foci at 0 and 12 hrs post release (B), and γH2AX-positive cells were quantified (C). For each condition, ≥ 400 cells were counted. The error bars represent standard deviations (SD) from two independent experiments (n=2). D. Cells were labeled with CldU for 30 min in the absence of CPT, and then labeled with IdU for 60 min in the presence or absence of 2.5 μM CPT. DNA fibers were spread onto glass slides, stained, and imaged. The lengths of individual DNA replication tracts were measured. The results of a representative experiment are shown. Numbers in red or green are the median tract lengths for CldU and IdU, respectively. E. The average IdU/CldU length ratios in the indicated cell populations were determined from multiple independent fiber assays. The error bars represent standard deviations (SD) from multiple independent experiments (Control and PRP19: n=3; CDC5L: n=2). See also Fig. S4.

Fig. 5. PRP19 promotes ATR activation and the replication stress response as an RNA-ssDNA-sensing ubiquitin ligase

A–B. Cells were nucleofected with control or PRP19 siRNA and plasmids expressing siRNA-resistant SFB-PRP19^WT, SFB-PRP19^Y405A (A), or SFB-PRP19^ΔUBOX (B). Cells were subsequently treated with 1 μM CPT for 2 hrs, and analyzed by Western blot using the indicated antibodies. C–D. Cells were nucleofected as in (A) and then treated with 2 mM HU for 16 hrs. Cells were then released into fresh media and immunostained for γH2AX and RAD51 foci at 0 and 12 hrs. For each condition, ≥ 300 cells were counted. The error bars represent standard deviations (SD) from two independent experiments (n=2). See also Fig. S5.

Fig. 6. PRP19 is required for DNA damage-induced RPA ubiquitylation

A. Cells were transfected with the indicated plasmids, treated with 1 μM CPT for 3 hrs or mock treated, and then processed for anti-HA immunoprecipitation under denaturing conditions. B. Cells were first transfected with the indicated plasmids and subsequently retransfected with control or PRP19 siRNA, treated with CPT and processed for anti-HA immunoprecipitation under denaturing conditions. C–D. Cells were transfected with the indicated plasmids and subsequently retransfected with control or PRP19 siRNA. Cells were then treated with CPT and processed for Ni-NTA pulldown under denaturing conditions. E. The PRP19 complex was affinity purified from HEK293T cells expressing SFB-PRP19. The ubiquitylation reactions were performed by incubating the PRP19 complex with in vitro translated HA-RPA32, ubiquitin, ubiquitin aldehyde, ATP and energy regeneration system at 30°C for 30 min. UbcH5c or the Ubc13-Mms2 complex were used as E2. See also Fig. S6.

Fig. 7. The PRP19 complex promotes the recruitment of ATR-ATRIP to sites of DNA damage

A. HCT116 cells transfected with control, PRP19, or CDC5L siRNA were treated with CPT for 2 hrs or mock treated. Endogenous ATR was immunoprecipitated from cell extracts and analyzed with ATR and ATR pT1989 antibodies. B. HeLa cells were transfected with the indicated siRNAs and a plasmid expressing GFP-ATRIP. Cells were treated with 1 μM CPT for 1 hr and analyzed for GFP signals. C. GFP-positive cells with GFP-ATRIP foci were quantified and shown. For each condition, ≥ 400 cells were counted. The error bars represent standard deviations (SD) from two or three independent experiments (Control and PRP19: n=3; CDC5L: n=2). D. HeLa cells were transfected with the indicated siRNAs, and 24 hrs later were retransfected with a GFP-ATRIP-expressing plasmid together with the indicated plasmids. The levels of the relevant proteins are shown in Fig. S7A. Transfected cells were treated with CPT and analyzed for GFP signals. For each condition, ≥ 250 cells were counted. The error bars represent standard deviations (SD) from three independent experiments. An unpaired two-tailed t-test was used to evaluate significance *: P ≤ 0.05, **: P ≤ 0.01. E. Biotinylated tetra-ubiquitin chains were incubated with nuclear extracts from HeLa cells. Proteins bound to ubiquitin chains were pulled down using streptavidin-conjugated magnetic beads and analyzed by Western Blot. F. A model for the ATR- and PRP19-mediated feed-forward loop that promotes ATR-ATRIP recruitment and activation. See also Fig. S7.