Journal: Nature

Article Title: DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis

doi: 10.1038/s41586-020-2041-2

Figure Lengend Snippet: a, Representative colony formation assay for DNA-PKcs5A/5A ES cells. Note the frequent accumulation of small colonies in DNA-PKcs5A/5A cells, which is rescued by deletion of KU80. b, Quantification of colony size (A.U., arbitrary units) from DNA-PKcs5A/5A and control ES cells. Two independently derived ES cells were assayed. c, Cell cycle analyses of two independently derived DNA-PKcs5A/5A and control ES cell lines. The percentage of BrdU+ cells is shown. d, Frequency of metaphase with telomere abnormalities (see below for definition). e, Representative telomere fluorescence in situ hybridization (FISH) images of a normal mouse chromosome (top, with four telomere dots), a chromosome with a chromatid break (middle, showing loss of one telomere signal among the two sister chromatids), or a chromatid fusion without telomere signal (bottom). f, Quantitative analyses of telomere instability and chromosomal breaks in metaphase. Telomere FISH analyses of MEFs were performed with the telomere-specific PNA probe as previously described2. Normal mouse chromosomes have four discrete telomere signals (e, top). Telomere instability or breaks considered include: i) telomere instability (indicated by more than one telomere signal per chromatid), ii) telomere/chromosome fusion (e, bottom; with telomere at the fusion junction (telomere fusion) or without telomere signal at the fusion junction (non-telomere fusion)), iii) chromosome breaks (S.B.; loss of both telomere signals on the paired sister chromatids) and iv) chromatid breaks (T.B.; loss of one of the two chromatids (e, middle)). The number of metaphases with at least one telomere instability, break or fusion is shown in d as a percentage of metaphases with abnormalities. Data derived from four independent MEF lines of each genotype. g, Representative flow cytometry analyses of erythroblasts from age-matched (2 weeks) DNA-PKcs+/+ and DNA-PKcs−/− mice. h, Representative protein translation analyses of S1 (CD71+Ter119−), S2 (CD71+Ter119mid) and S3 erythroblasts (CD71+Ter119high) from 2-week-old DNA-PKcs+/+, DNA-PKcs−/− and Ku70−/− mice. Quantification is shown in Fig. 2i. b, d, Mean ± s.e.m.; two-sided unpaired Student’s t t-test, **P < 0.01, *P < 0.05, n.s. P > 0.05. Exact P values and defined sample sizes (n) are provided in Supplementary Data 1.

Article Snippet: Fixed cells were then incubated with primary antibodies in 3% BSA for 1 h at 25 °C, including mouse anti-human KU86 (ThermoFisher, MA5–12933, 1:100), rabbit anti-human DDX21 (Novus, NB100–1718, 1:500) or anti-DNA-PKcs (ThermoFisher, Ab-4(cocktail)), followed by fluorophore-conjugated secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit, Alexa Fluor 594-conjugated anti-rabbit, and cyanine3-conjugated anti-mouse, Invitrogen, 1:500) for 1 h at room temperature.

Techniques: Colony Assay, Control, Derivative Assay, Fluorescence, In Situ Hybridization, Flow Cytometry

Journal: Nature

Article Title: DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis

doi: 10.1038/s41586-020-2041-2

Figure Lengend Snippet: a, b, Immunofluorescence staining of endogenous KU86 (a) and DNA-PKcs (b) in U2OS cells. DDX21 RNA helicase is used as a positive control for nucleoli. The CSK buffer contains Triton X-100 for pre-extraction before fixation (see Methods). When indicated, the cells were treated with 50 nM ActD for 1 h before pre-extraction, fixation and staining. c, Localization of ectopically expressed GFP-tagged KU70 in mouse ES cells. a–c, n = 3 biologically independent experiments. d, U3 ChIRP-qRT–PCR analysis from HeLa cells. Enrichment levels, relative to input samples, of the U3, 7SK, 18S, and RMRP RNAs were assessed from experimental (−RNase A) or control (+RNase A) ChIRP samples. Data are from two independent biological replicates. e, DNA-PK was also recovered from U3 ChIRP-MS in IMR90 cells. Peptide spectral match (PSM) counts for control (RNase A) and experimental (U3) samples are shown. n = 2 biological replicates.

Article Snippet: Fixed cells were then incubated with primary antibodies in 3% BSA for 1 h at 25 °C, including mouse anti-human KU86 (ThermoFisher, MA5–12933, 1:100), rabbit anti-human DDX21 (Novus, NB100–1718, 1:500) or anti-DNA-PKcs (ThermoFisher, Ab-4(cocktail)), followed by fluorophore-conjugated secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit, Alexa Fluor 594-conjugated anti-rabbit, and cyanine3-conjugated anti-mouse, Invitrogen, 1:500) for 1 h at room temperature.

Techniques: Immunofluorescence, Staining, Positive Control, Extraction, Quantitative RT-PCR, Control

Journal: Nature

Article Title: DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis

doi: 10.1038/s41586-020-2041-2

Figure Lengend Snippet: a, b, irCLIP of DNA-PKcs (a) and KU86 (b) in HeLa cells. RNA (IR800) and protein blots show specific enrichment of RNA UV-crosslinked to DNA-PKcs and KU86. The increased signal in the RNaseA-treated samples corresponds to the accumulation of RNaseA-protected fragments that are directly associated with either KU86 or DNA-PKcs. Results shown are representative of three biologically independent experiments. Vertical line marks the RNA extracted for sequencing. c, e, Fractional distribution of DNA-PKcs (c) and KU86 (e) irCLIP RT stops on major genomic features annotated with HOMER. d, f, Detailed HOMER annotation of DNA-PKcs (d) and KU86 (f) RT stops categorized as intronic and intergenic from c and e. g, h, Genome browser tracks of DNA-PKcs and KU86 irCLIP on the Neat1 (g) and Terc (h) RNA. RT stop values are normalized to 107 mapped reads. i, j, Histograms of DNA-PKcs (i) and KU86 (j) irCLIP RT stops mapping to the 5′ETS, normalized for total reads mapped per experiment. Data from DMSO- (top) or ActD-treated (bottom) HeLa cells are shown. Grey highlighted region is as in Fig. 4a.

Article Snippet: Fixed cells were then incubated with primary antibodies in 3% BSA for 1 h at 25 °C, including mouse anti-human KU86 (ThermoFisher, MA5–12933, 1:100), rabbit anti-human DDX21 (Novus, NB100–1718, 1:500) or anti-DNA-PKcs (ThermoFisher, Ab-4(cocktail)), followed by fluorophore-conjugated secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit, Alexa Fluor 594-conjugated anti-rabbit, and cyanine3-conjugated anti-mouse, Invitrogen, 1:500) for 1 h at room temperature.

Techniques: Sequencing

Journal: Nature

Article Title: DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis

doi: 10.1038/s41586-020-2041-2

Figure Lengend Snippet: a, KU86 IP-MS and U3 ChIRP-MS overlap. Zhou et al.33 (n = 2 independent MS assays; for each assay, n = 2 technical replicates) identified 292 proteins enriched with KU86 protein. U3 ChIRP-MS identified 483 proteins enriched more than twofold with the U3 snoRNA (Supplementary Table 1). These identified factors were intersected, resulting in 153 proteins in common between the two affinity purification strategies, which is highly significant (hypergeometric P < 6.650 × 10−166). To understand what types of protein were enriched only with KU86, only with U3, or together with both factors, we isolated the enrichment values (−log10(Benjamini)) for GO biological process terms for each of these sets and compared them. Factors commonly bound were biased for rRNA processing, ribosomal terms and SSU biogenesis. U3-specific factors had additional enrichment in these categories and KU86 had a set of unique terms that were not well represented in U3. b, Independent repeat of northern blot analyses of 18S rRNA maturation in v-ABL kinase-transformed B cells from noted genotypes. The probe covers the sequence just after the 18S rRNA (red line). This experiment was repeated independently four times. Another repeat is shown in Fig. 3c. c, d, MS characterization of commercial DNA-PK holoenzyme (Promega) used in EMSA and kinase reactions. A detailed description of proteins and their quality in this mixture has not been published. We subjected the DNA-PK enzyme mix as provided to SDS–PAGE separation followed by LC–MS identification of proteins from mass ranges between 65 and 600 kDa. KU70 and KU86 were clearly present in the gel and via MS. For masses above 130 kDa, DNA-PKcs was the major protein identified. For each of the five slices analysed (coloured regions) we tabulated the starting positions of peptides matching the DNA-PKcs polypeptide and mapped them to the position within DNA-PKcs. As expected, in the highest-molecular-weight slices, we identified peptides across the majority of the length of DNA-PKcs. DNA-PKcs peptides were present in lower slices, but poorer overall coverage was evident, suggesting that these are degradation products. As this was a confirmatory experiment of a validated and commercially available product (see Methods), it was conducted only once.

Article Snippet: Fixed cells were then incubated with primary antibodies in 3% BSA for 1 h at 25 °C, including mouse anti-human KU86 (ThermoFisher, MA5–12933, 1:100), rabbit anti-human DDX21 (Novus, NB100–1718, 1:500) or anti-DNA-PKcs (ThermoFisher, Ab-4(cocktail)), followed by fluorophore-conjugated secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit, Alexa Fluor 594-conjugated anti-rabbit, and cyanine3-conjugated anti-mouse, Invitrogen, 1:500) for 1 h at room temperature.

Techniques: Protein-Protein interactions, Affinity Purification, Isolation, Northern Blot, Transformation Assay, Sequencing, SDS Page, Liquid Chromatography with Mass Spectroscopy, Molecular Weight

Journal: Nature

Article Title: DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis

doi: 10.1038/s41586-020-2041-2

Figure Lengend Snippet: a, DNA-PKcs and KU86 RT stops that map to the four pre-rRNA introns; 5′ETS, ITS1, ITS2, and 3′ETS. irCLIP was performed in biological duplicate. b, Transcript normalized histogram of DNA-PKcs (orange) and KU86 (blue) irCLIP RT stops from DMSO-treated HeLa cells mapping to U3. c, In silico (mFold)-predicted secondary structure of U3. The top three and two peaks of DNA-PKcs and KU86, respectively, are annotated on the secondary structure. The length of the 5′end stem-loop is shown. d, Baculovirus-purified human DNA-PK in vitro kinase phosphorylation assay in the presence of increasing amounts of U3-SL1. Western blots were performed with antibodies against DNA-PKcs phosphorylated at the T2609 cluster (top), total DNA-PK (middle), and KU86 (bottom). e, Baculovirus-purified human DNA-PK in vitro kinase phosphorylation assay against purified human TP53. Activated DNA-PK phosphorylates TP53 on serine 15. An in vitro ATM kinase assay was used as a control. Western blots were performed with antibodies against TP53 phosphorylated on serine 15 (top), total TP53 (middle) and KU86 (bottom). d, e, n = 3 biologically independent experiments.

Article Snippet: Fixed cells were then incubated with primary antibodies in 3% BSA for 1 h at 25 °C, including mouse anti-human KU86 (ThermoFisher, MA5–12933, 1:100), rabbit anti-human DDX21 (Novus, NB100–1718, 1:500) or anti-DNA-PKcs (ThermoFisher, Ab-4(cocktail)), followed by fluorophore-conjugated secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit, Alexa Fluor 594-conjugated anti-rabbit, and cyanine3-conjugated anti-mouse, Invitrogen, 1:500) for 1 h at room temperature.

Techniques: In Silico, Purification, In Vitro, Phospho-proteomics, Western Blot, Kinase Assay, Control

Journal: Nature

Article Title: DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis

doi: 10.1038/s41586-020-2041-2

Figure Lengend Snippet: a, b, Correlation analysis of total RT stops mapping to non-repeat snoRNA transcripts from DDX2135 compared to DNA-PKcs (a) or KU86 (b) irCLIP experiments from DMSO-treated HeLa cells. Correlation analysis was performed using Pearson’s correlation coefficient. n denotes number of snoRNA transcripts bound by each protein. c, EMSA of purified human DNA-PK and in vitro transcribed U3-SL1. Lane 1 contains only Cy7-labelled U3-SL1. Lanes 2–4 show that KU assembles with U3-SL1 at a 1:3 molar ratio, while DNA-PK holoenzyme assembly occurs at a 1:25 molar ratio. Lanes 5 and 6 show that unlabelled U3-SL1 RNA competes away bound labelled U3-SL1 in a dose-dependent manner. d, Supershift EMSA of DNA-PK and U3-SL1 RNA with KU86 antibody. The addition of anti-KU86 confirms the identity of the KU–U3-SL1 band and also shifts up the complex to higher molecular weights. e, A structural mutant of U3-SL1 was generated by introducing point mutations predicted to disrupt the stem-loop structure. This mutant was unable to compete away wildtype U3-SL1 for binding to the KU complex, while unlabelled wild-type U3-SL1 competed efficiently. f, DNA-PK in vitro kinase phosphorylation assay in the presence of increasing amounts of U3-SL1 or DNA. Western blot was performed with an antibody recognizing DNA-PKcs phosphorylated at the T2609 cluster. Asterisks denote cross-reactive fragments that probably include phosphorylated DNA-PKcs fragments, on the basis of MS analyses of the DNA-PK complex (Extended Data Fig. 9c, ​,d).d). g, As in f, but using an antibody recognizing DNA-PKcs phosphorylated at the S2056 cluster. h–j, As in f with the following changes. h, dsDNA was used to activate DNA-PK, NU7441 was included to inhibit specific DNA-PK activity, and western blot analysis monitored the total DNA-PK (total DNA-PKcs) or phosphorylated DNA-PK (DNA-PKcs phoT2609). i, U3-SL1 RNA was used to activate DNA-PK in the absence or presence of the DNA-PK inhibitor NU7441. j, U3-SL1 RNA was used to activate DNA-PK, hydrolysable (ATP) or non-hydrolysable (AppCp) ATP was provided, and western blot analysis monitored KU86 (loading control) or phosphorylated DNA-PK (phoT2609). k, Baculovirus-purified human DNA-PK in vitro kinase phosphorylation assay in the presence of increasing amounts of U3-SL1 or DNA. Western blot was performed with antibodies recognizing DNA-PKcs phosphorylated at the S2056 cluster (top), total DNA-PK (middle) and KU86 (bottom). All EMSA and western blots presented here are representative of three biologically independent experiments.

Article Snippet: Fixed cells were then incubated with primary antibodies in 3% BSA for 1 h at 25 °C, including mouse anti-human KU86 (ThermoFisher, MA5–12933, 1:100), rabbit anti-human DDX21 (Novus, NB100–1718, 1:500) or anti-DNA-PKcs (ThermoFisher, Ab-4(cocktail)), followed by fluorophore-conjugated secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit, Alexa Fluor 594-conjugated anti-rabbit, and cyanine3-conjugated anti-mouse, Invitrogen, 1:500) for 1 h at room temperature.

Techniques: Purification, In Vitro, Mutagenesis, Generated, Binding Assay, Phospho-proteomics, Western Blot, Activity Assay, Control