A role of human RNase P subunits, Rpp29 and Rpp21, in homology directed-repair of double-strand breaks

Abstract

DNA damage response (DDR) is needed to repair damaged DNA for genomic integrity preservation. Defective DDR causes accumulation of deleterious mutations and DNA lesions that can lead to genomic instabilities and carcinogenesis. Identifying new players in the DDR, therefore, is essential to advance the understanding of the molecular mechanisms by which cells keep their genetic material intact. Here, we show that the core protein subunits Rpp29 and Rpp21 of human RNase P complex are implicated in DDR. We demonstrate that Rpp29 and Rpp21 depletion impairs double-strand break (DSB) repair by homology-directed repair (HDR), but has no deleterious effect on the integrity of non-homologous end joining. We also demonstrate that Rpp29 and Rpp21, but not Rpp14, Rpp25 and Rpp38, are rapidly and transiently recruited to laser-microirradiated sites. Rpp29 and Rpp21 bind poly ADP-ribose moieties and are recruited to DNA damage sites in a PARP1-dependent manner. Remarkably, depletion of the catalytic H1 RNA subunit diminishes their recruitment to laser-microirradiated regions. Moreover, RNase P activity is augmented after DNA damage in a PARP1-dependent manner. Altogether, our results describe a previously unrecognized function of the RNase P subunits, Rpp29 and Rpp21, in fine-tuning HDR of DSBs.

Figure 1

Rpp29 promotes repair of IR-induced DNA damage. (A) Real-time PCR analysis shows ~90% reduction in the steady state levels of Rpp29 mRNA in cells transfected with two different Rpp29 siRNAs, #23 and #25, and compared with those in cells transfected with control (CtRL) siRNA. P-values were calculated by two-sided Student’s t-test relative to ctrl siRNA; ***p < 0.001. (B) Control and Rpp29 depleted cells were exposed to 10 Gys of ionizing radiation (IR), harvested at the indicated time points after IR, and subjected to neutral comet assay. Quantitation of the DNA percentage in comet tail reveals DSB-repair deficiency in Rpp29 depleted cells. Error bars represent standard deviation (SD) from three independent experiments (n = 80 cells). Two-way ANOVA was used to test for differences at each dose; ** and **** indicate significance at p < 0.01 and p < 0.0001, respectively.

Figure 2

Rpp29 promotes homology-directed repair of double-strand breaks. (A) Quantitative real-time PCR shows the efficiency of Rpp29 knockdown in U2OS-TLR cells. Cells were transfected with control siRNA (Ctrl) or three different Rpp29 siRNAs. The y-axis represents the relative Rpp29 mRNA level normalized to that of GAPDH. P-values were calculated by two-sided Student’s t-test relative to Ctrl siRNA; ***p < 0.001. (B) Rpp29 knockdown increases IR-induced levels of pRPA2 S4/S8 phosphorylation, γH2AX, and NBS1 phosphorylation levels. Control and Rpp29-depleted U2OS cells were exposed to IR (10 Gys) and protein extracts were prepared at the indicated time points and subjected to western blot analysis using antibodies directed against the indicated proteins. Total RPA and β-actin were used as internal controls. The bands intensities of γH2AX, pNBS1 or pRPA were normalized relative to the intensities of their respective β-actin or total RPA bands, respectively. The ratios are shown at the bottom of the blot. Results are representative of two independent experiments. (C) Rpp29 knockdown impairs HDR of DSBs generated by I-SceI endonuclease. A reduction of ~50% in GFP-positive cells was observed after Rpp29 depletion. Caffeine was used as a positive control. Results shown are typical of three independent experiments. Error bars represent SD. P-values were calculated by two-sided Student’s t-test relative to Ctrl siRNA; ** and **** indicate significance at P < 0.01 and 0.0001. Given that HDR can occur only in S/G2 cell phase, data were corrected to flow-cytomertry S/G2 values. (D) TLR results for DSBs repair by NHEJ. Rpp29 knockdown had no significant effect on the integrity of NHEJ. Data represents SD for three independent experiments. (E) Percentage of control and Rpp29-depleted cells with more than five 53BP1 foci. Data shown represent at least 300 cells. Two-way ANOVA was used to test for differences at each dose; *p < 0.05.

Figure 3

Rpp21 and Rpp29 show rapid and transient recruitment to DNA damage sites. (A) Time-lapse images show the localization of EGFP-Rpp29 at the indicated time points after laser micro-irradiation of a single region, marked by a white arrow. Graph (Right) shows fold increase in the relative fluorescence intensity of EGFP-Rpp29 at laser-microirradiated sites. Each measurement is representative of at least 10 cells. Error bars indicate SD. (B) Representative images showing IRIF enriched with endogenous Rpp29 protein. U2OS cells were exposed to IR (5 Gy), fixed after 3 min recovery and stained for DNA (blue), Rpp29 (green) and γH2AX (red). A merged image is seen on the right. Results shown are typical of three independent experiments and represent at least 15 different cells. (C) As in (A), time-lapse images showing the recruitment of EGFP-Rpp21 to laser microirradiated sites. (D,E) Graphs display the percentage of Rpp21 (D) and Rpp29 (E) deficient cells showing recruitment of EGFP-Rpp29 and EGFP-Rpp21 to damaged sites, respectively, as compared with cells transfected with control siRNA. Error bars represent the SEM from two independent experiments.

Figure 4

Rpp29 and Rpp21 bind poly(ADP-ribose) (PAR) in vitro. (A) Protein alignment reveals that Rpp29 and Rpp21 contain a PAR-binding consensus (bold letters). (B) PAR-binding assay with full-length, 6 × His-tagged recombinant Rpp29 and Rpp21 proteins. 6 × His-Rpn8 and BSA are shown as negative controls, whereas histone H3 is used as a positive control. IB: Immunoblot. ³²P: phosphate radiolabeled PAR.

Figure 5

Rpp29 and Rpp21 are recruited to DNA damage sites in a PARP1-dependent manner. (A) Western blot analysis shows the efficiency of siRNA knockdown of PARP1. Protein extracts were prepared from mock and PARP1 siRNA-transfected U2OS cells and immunoblotted with PARP1 antibody. β-actin is used as a loading internal control. (B,C) Representative time-lapse images show subcellular distributions of EGFP-Rpp29 and EGFP-Rpp21 after laser microirradiation of mock and PARP1-depleted cells (Left). Line graphs (middle) depict fold increase in the relative fluorescence intensity of Rpp29 and Rpp21 at laser-microirradiated sites in mock and PARP1-siRNA transfected cells. Each measurement is representative of at least 10 cells. Error bars indicate SD. Column graphs (right) display the percentage of PARP1-depleted cells exhibiting accumulation of EGFP-Rpp29 and EGFP-Rpp21 at damage sites, as compared with mock-transfected cells. Error bars represent the SEM from two independent experiments. P-values were calculated by two-sided Student’s t-test relative to mock; ***p < 0.001. (D,E) Representative time-lapse images (left) show localization of EGFP-Rpp29 (D) and EGFP-Rpp21 (E) fusions to laser-microirradiated sites (marked by red arrow) in U2OS cells treated for 1 h with either DMSO or 5 μM of the PARP1/2 inhibitor (Ku- 0059436)(PARPi). Results shown are typical of two independent experiments and represent at least 30 different cells. Line graphs (middle) show fold increase in the relative fluorescence intensity of EGFP-Rpp29 and EGFP-Rpp21 at laser-microirradiated sites in untreated and PARPi-treated cells. Column graphs (right) display the percentage of PARPi-treated cells with accumulation of EGFP-Rpp29 and EGFP-Rpp21 at damage sites, as compared with untreated cells. Error bars express the SEM from two independent experiments. P-values were calculated by two-sided Student’s t-test relative to DMSO; ***p < 0.001.

Figure 6

RNase A treatment of cells disrupts the recruitment of EGFP-Rpp29 and EGFP-Rpp21 to laser microirradiated sites. (A,B) Representative time-lapse images (left) of mock- and RNase A-treated U2OS cells showing the localization of EGFP-Rpp29 (A) and EGFP-Rpp21 (B) at the indicated times after laser-microirradiation of a single region marked by white arrow. Results shown are typical of three independent experiments and represent at least 30 different cells. Graphs (middle) show the increase in the relative fluorescence intensity of EGFP-Rpp29 and EGFP-Rpp21 at laser-microirradiated sites in mock and RNase A-treated cells. Each measurement is representative of at least 10 cells. Error bars indicate SD. Graphs (right) display the percentage of RNase A-treated cells showing subcellular distributions of EGFP-Rpp29 and EGFP-Rpp21, as compared with those seen in untreated cells. Error bars depicts the SEM from two independent experiments. P-values were calculated by two-sided Student’s t-test relative to DMSO; **p < 0.01. (C) As in A, except for that the laser microirradiation was applied on U2OS cells expressing MonomerRed-PARP1 (MR-PARP1).

Figure 7

Knockdown of H1 RNA inhibits the recruitment of EGFP-Rpp29 and EGFP-Rpp21 to laser-microirradiated sites. (A) TaqMan-based Real-Time PCR shows ~65% knockdown of H1 RNA. RNA was extracted from U2OS cells transfected with either a scramble shRNA or H1 RNA shRNA#165. Real-time PCR was performed to measure H1 RNA level. The y-axis represents the relative RNA level of H1 RNA, which was normalized to that of GAPDH. Error bars represent the SEM from two independent experiments. (B,C) Recruitment of EGFP-Rpp29 (B) and EGFP-Rpp21 (C) to DNA damage sites cells with shRNA knockdown of H1 RNA or control cells expressing a scramble shRNA. Graphs display the percentage of cells exhibiting accumulation of the two fusion proteins at DNA damage sites. Error bars show the SEM from two independent experiments and represent at least 30 different cells. P-values were calculated by two-sided Student’s t-test relative to scramble shRNA; ** and *** indicates significance of p < 0.01 and 0.001.

Figure 8

RNase P activity is induced after DNA damage in a PARP1-dependent manner. Whole cell extracts were prepared from untreated (A) and PARPi treated cells (B), and mature tRNA and ptRNA bands, seen in Supplementary Fig. S10, were quantified and ratios of product/substrate were plotted. (C) Processing of a nascent precursor tRNA^Arg (UCU) is inhibited by PARP1 inhibitor. U2OS cells were treated as in Supplementary Fig. S10, and dialyzed S20 extracts were assayed for processing of nascent precursor tRNA^Arg transcribed from a cloned gene for the indicated times (in min), as previously described. Labeled RNAs were resolved in an 8% polyacrylamide sequencing gel. The positions of the primary transcript, 93 nt in length, and transcript processed at 5′ end by RNase P, 88 nt in size, are shown. Extracts of U2OS cells are not as efficient as HeLa cells in tRNA gene transcription and splicing to mature tRNA (not shown), thus producing weak labeled RNA signals. (D) The 93- and 88-nt transcript bands seen in C were quantitated and the ratios of processed to unprocessed tRNA^Arg were plotted. P-values were calculated by two-sided Student’s t-test relative to scr shRNA; *, **, and *** indicates significance of p < 0.05, 0.01 and 0.001, respectively. (E) A hypothetical model shows that local ADP-ribosylation at DSB site and H1 RNA molecule underpin the recruitment of Rpp29 and Rpp21 to promote HDR of DSBs.