Research ArticleCELL BIOLOGY

Fanconi anemia A protein participates in nucleolar homeostasis maintenance and ribosome biogenesis

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Science Advances  01 Jan 2021:
Vol. 7, no. 1, eabb5414
DOI: 10.1126/sciadv.abb5414
  • Fig. 1 Nucleolar abnormalities in FANC pathway–deficient cells.

    (A and C) Wide fields and single nuclei of HeLa (A) or U2OS (C) cells transfected with the indicated siRNAs and stained with anti-NCL (red) and anti-FBL (green) antibodies. DAPI (4′,6-diamidino-2-phenylindole) staining visualizes nuclei. (B and D) Percentage of HeLa (B) or U2OS (D) cells with canonical nucleoli (irregular shape) or a round or cap-like shape. The mean of six (B) or three (D) independent experiments ± SEM is reported. NS, not significant. (E) Electronic micrographs showing nucleoli as observed in the majority of HeLa cells (i) and the disorganized/condensed nucleoli observed after FANCA depletion (ii and iii). (F) Percentage of HeLa cells ± SEM with altered nucleoli 72 hours following transfection with untargeted (n = 6) (siLacZ) or FANCA-targeted (n = 6), FANCC-targeted n = 4), FANCG-targeted (n = 3), or FANCD2-targeted (n = 4) siRNA. (G and H) Percentage of cells with altered nucleoli in (G) WT, FANCA−/−, and FANCA-corrected, FANCC−/−, and FANCG−/− primary fibroblasts and in (H) WT, MRC5, FANCC−/− (GM00449), and FANCG−/− primary fibroblasts (PD352) and their immortalized counterparts (MRC5-SV, GM13136, and GM16335) under basal conditions or following FANCA depletion. The dotted red line represents the mean of the five primary WT cells. (I) Percentage of cells with altered nucleoli in HEK293- and HEK293-FlagFANCA–expressing cells before or after siRNA-mediated FANCA and/or FANCG depletion. Bars represent the mean of three to six independent experiments ± SEM. (J and K) Western blots showing the expression of the indicated proteins in HEK293 and HEK293-FlagFANCA cells. Extracts from independent experiments or transfections in the same experiment (bis) are shown. Statistics were assessed with two-tailed unpaired Student’s t tests (*P < 0.05, **P < 0.01, and ***P < 0.005).

  • Fig. 2 Nucleolar fragility and DNA damage signaling in FANCA-deficient cells.

    (A) Subcellular localization of FANCA, FANCC, and FANCD2. WCE, whole-cell extract. FBL and lamin A/C identify nucleolar and nuclear/nucleoplasmic fractions, respectively. (B) FANCA cellular localization as evaluated by confocal microscopy on cells transiently expressing a YFP-tagged FANCA construct. Cells were counterstained with FBL or UBF to identify nucleoli and DAPI to stain DNA. (C) Percentage of HCT116 cells with γ-H2AX foci–positive nucleoli. Cells were transfected with the indicated siRNAs and analyzed at 72 hours. Bars represent the mean of three independent experiments ± SEM. (D) Genomic PCR analysis of rDNA units 72 hours following FANCA or FANCD2 depletion in HeLa cells by using forward (F)–reverse (R) or reverse-reverse primers (see inset). Red arrows indicate new bands, suggestive of rDNA rearrangements. (E) Percentage of HeLa cells with altered nucleoli following transfection with untargeted (siLacZ) or FANCA-targeted siRNAs and exposure to caffeine, an ATM-specific inhibitor (ATMi), or cotransfected with ATM or ATR siRNA. Bars represent the mean of three independent experiments ± SEM. (F) Images of HeLa cells costained with anti–R-loops (green), anti-FBL (red), and DAPI (blue). Dots in the diagram represent the intensity of R-loop staining measured using CellProfiler software inside the FBL-positive region for each cell transfected with the indicated siRNAs. Cells incubated with RNaseH, which eliminates DNA-RNA hybrids, validate antibody specificity. A representative experiment of three is shown. At least 100 cells were scored for each condition. Statistical significance was assessed with the Z (normal distribution) test (*P < 0.05 and ***P < 0.005). A.U., Arbitrary Unit.

  • Fig. 3 rDNA transcription and rRNA processing in FANCA-deficient cells.

    (A) Top, rDNA repeat organization. Yellow boxes indicate regions amplified by ChIP-qPCR analysis: promoter, H42.9; starting codon, H1; and final part, H8, of the RNAPolI-transcribed region, and the inter-rDNA gene sequence H27 (IGS). Bottom, pre-rRNA processing paths. Red boxes indicate probes used in the Northern blot analysis. (B) Experiment showing precursor and mature rRNAs. At 72 hours after transfection, HeLa cells were labeled (20 min) with [32P]orthophosphate and chased with cold orthophosphate for the indicated time. The EtBr-stained gel is shown as a loading control. Right, relative level of different rRNA forms in siFANCA-transfected cells normalized versus the FANC-proficient cells. (C) Northern blot analysis performed with the probe indicated in (A). RNAs were isolated 72 hours after siRNA transfection of HeLa cells. The EtBr-stained gel is shown as a loading control. The quantity measured in FANCA-depleted cells was adjusted to that in FANCA-proficient cells, which was set to 1. Bars represent the mean of four independent experiments ± SEM. Statistical significance was assessed with one-tailed Student’s t tests (*P < 0.05). (D) Ratio WT/siFANCA of the distribution of RNAPolI, H3K9me3, and H3K4me3 on the rDNAs of HeLa cells, as determined by ChIP-qPCR analysis. ChIP was performed 48 hours after siRNA transfection. Bars represent the mean of three experiments ± SEM. (E and F) Cells costained with anti-G4 (green), anti-FBL (red), and DAPI (blue). Dots in the diagram represent the intensity of G4 staining measured using CellProfiler software inside the nucleoli for each cell 48 hours after transfection with the indicated siRNAs. At least 100 cells were scored for each condition. Statistical significance was assessed with a Z (normal distribution) test (***P < 0.005).

  • Fig. 4 FANCA immunoprecipitates with NPM1 and NCL.

    (A) Anti-FANCA rabbit antibodies from Abcam, Bethyl, and Cell Signaling laboratories were used to immunoprecipitate FANCA in cell extracts from exponentially growing human lymphoblasts (HSC93), human bone osteosarcoma cells (U20S), or human embryonic kidney cells (HEK293). Immunoblotting was performed with anti-FANCA Bethyl or Abcam antibodies and antibodies against FANCG, NCL, and NPM1. N.D., Not Done. (B) Immunoprecipitation (IP) with a FANCG antibody followed by immunoblot with antibodies against the indicated proteins. Different quantities of input fractions were analyzed to clearly visualize FANCA and FANCG. (C and D) Immunocomplexes isolated by the FANCA antibody were treated with benzonase [(C), two independent experiments], 150 nM NaCl, or 300 nM NaCl (D) before immunoblot analysis. Different quantities of input fractions were analyzed to clearly visualize FANCA and FANCG. (E) Immunoprecipitation with a FANCA antibody in cell extracts from HSC93 (WT), EGF070 (FANCG−/−), and EGF004 (FANCG−/−) cells followed by immunoblot with antibodies against the indicated proteins. Different quantities of input fractions were loaded. (F) NCL or NPM1 were immunoprecipitated from HEK293 or HSC93 cells. Immunoblot showing the coimmunoprecipitation of the indicated proteins when cell extracts were immunoprecipitated with anti-NCL or anti-NPM1 antibodies.

  • Fig. 5 FANCA deficiency leads to an altered ribosome profile and reduced protein synthesis.

    (A) Time-course incorporation of OP-Puro assessed by FACS in FANCA lymphoblasts (HSC72) and their corrected counterpart (HSC72Corr) cultured in 12 or 15% FCS. (B) Relative levels of OP-Puro incorporation in FANC-proficient (HSC93 and GM3657), FANCA−/− (HSC72 and HSC99), FANCC−/− (HSC536), or FANC-corrected lymphoblasts. Bars represent the mean of three to five independent experiments ± SEM. The value observed in HSC93 cells was set to 1 in each individual experiment. Statistical significance was assessed with a two-tailed Student’s t test against the GM03657 cell line (*P < 0.05 and ***P < 0.005). (C) Western blot showing the expression of FANCA and FANCG in the indicated cell lines. (D) Polysome profiling in FANCA-deficient HSC72 and FANCA-proficient HSC72corr and HSC93 lymphoblasts and quantification of the polysome/monosome ratio in HSC72 relative to HSC72corr cells. Bars represent the mean of four independent experiments ± SEM. The value of the HSC72corr cell was set to 1 in each individual experiment. (E) Western blots showing FANCA presence in the ribosomal-enriched cytoplasmic, 40S, 60S, and monosome (80S) fractions. (F) Histograms representing the mean expression level of the indicated proteins in the cytosol, 80S, and polysomes in FANCA-deficient cells compared with their corrected counterparts. Data are from MS analysis. (G) Western blots showing the expression of the indicated proteins in whole-cell (WCE) and ribosomal-enriched (Tot. Rib.) extracts in HSC72 and HSC7CCorr cells in four experiments. (H) Simplified diagram illustrating the several roles of FANCA in cell physiology.

Supplementary Materials

  • Supplementary Materials

    Fanconi anemia A protein participates in nucleolar homeostasis maintenance and ribosome biogenesis

    Anna Gueiderikh, Frédérique Maczkowiak-Chartois, Guillaume Rouvet, Sylvie Souquére-Besse, Sébastien Apcher, Jean-Jacques Diaz, Filippo Rosselli

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