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 tRNAArg (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 tRNAArg transcribed from a cloned gene for the indicated times (in min), as previously described50. 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 tRNAArg 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.
