Fanconi anemia protein FANCI functions in ribosome biogenesis

Abstract

Fanconi anemia (FA) is a disease of DNA repair characterized by bone marrow failure and a reduced ability to remove DNA interstrand cross-links. Here, we provide evidence that the FA protein FANCI also functions in ribosome biogenesis, the process of making ribosomes that initiates in the nucleolus. We show that FANCI localizes to the nucleolus and is functionally and physically tied to the transcription of pre-ribosomal RNA (pre-rRNA) and to large ribosomal subunit (LSU) pre-rRNA processing independent of FANCD2. While FANCI is known to be monoubiquitinated when activated for DNA repair, we find that it is predominantly in the deubiquitinated state in the nucleolus, requiring the nucleoplasmic deubiquitinase (DUB) USP1 and the nucleolar DUB USP36. Our model suggests a possible dual pathophysiology for FA that includes defects in DNA repair and in ribosome biogenesis.

Keywords: FANCI; Fanconi anemia; nucleolus; pre-ribosomal RNA; ribosome.

Fig. 1.

FANCI is a nucleolar and nucleoplasmic protein. (A) Western blots for subcellular fractionation controls indicate productive separation of NP and NO. (B) FANCI and FANCD2 are localized to both NP and NO. Western blots for FANCI and FANCD2 show both monoubiquitinated and deubiquitinated forms, indicated by arrows. (C and D) FANCI, but not FANCD2, is enriched in NO. Densitometric quantitation of Western blots from three biological replicates for NP and NO. FANCI (C) and FANCD2 (D) were measured and set relative to the level present in WCE. Statistical significance was calculated using a two-tailed unpaired Mann–Whitney U test (mean ± SD). ns, not significant, *P ≤ 0.05. (E) The nucleolar protein, PES1, coimmunoprecipitates FANCI. Shown are Western blots for FANCI and FANCD2 for WCE (1% total; lanes 1, 4, and 7) and α-PES1 immunoprecipitates (α-PES1 IP; lanes 3, 6, and 9). BOP1 is a known direct physical interactor of PES1 and is a positive control; vinculin was used as a negative control. Unconjugated protein A beads (lanes 2, 5, and 8) also served as a negative control. (F–O) FANCI is colocalized with the nucleolar protein fibrillarin by immunofluorescence in fixed HeLa cells. (F and K) DAPI. (G and L) Anti-FANCI. (H and M) Anti-fibrillarin (FIB). (I and N) Merge of anti-FANCI and anti-fibrillarin. (J and O) Merge of DAPI, anti-FANCI, and anti-fibrillarin. (Scale bars: 10 μm.) A single Z plane is shown.

Fig. 2.

FANCI is physically and functionally associated with pre-rRNA transcription. (A) FANCI is required for RNAPI transcription. Cells were transfected with the indicated siRNAs and, after 48 h, transfected again with plasmids containing firefly luciferase (under the control of the rDNA promoter) and Renilla luciferase (under the control of a constitutive promoter). Luminescence was quantitated 24 h later. Statistical significance for nine biological replicates was calculated using a two-tailed Mann–Whitney U test (mean ± SD). All comparisons are relative to siNT. ns, not significant, ***P ≤ 0.001, ****P ≤ 0.0001. (B) Depletion of FANCI decreases the levels of the primary pre-rRNA. qRT-PCR was used to quantitate the levels of the primary pre-rRNA after depletion of the indicated proteins using the 7SL RNA as an internal control. Relative expression values were calculated with the comparative C_T method (mean ± SD). Statistical significance for three biological replicates was calculated using the Wilcoxon signed-rank test with a hypothetical value of 1.0. All comparisons are relative to siNT. ns, not significant, **P ≤ 0.01. (C) FANCI physically interacts with the large subunit of RNAPI (RPA194). HeLa WCEs were immunoprecipitated (IP) with α-RPA194 and α-FANCI antibodies. Unconjugated protein A beads and preimmune sera were used as negative controls. RPA194 and FANCI were detected by Western blotting. (D–M) FANCI colocalizes with RPA194 by immunofluorescence in fixed HeLa cells. (D and I) DAPI. (E and J) Anti-FANCI. (F and K) Anti-RPA194. (G and L) Merge of anti-FANCI and anti-RPA194. (H and M) Merge of DAPI, anti-FANCI, and anti-RPA194. (Scale bars: 10 μm.) A single Z plane is shown.

Fig. 3.

FANCI functions in LSU pre-rRNA processing. (A) Human pre-rRNA processing schematic. The human pre-rRNA is transcribed as a large precursor, the 47S pre-rRNA, which is processed into the 45S pre-rRNA, both of which are detectable as a single band by Northern blotting, which we refer to as the PTP (51). The 41S, 32S, and 12S pre-rRNAs are detected by a probe (P5) that hybridizes with ITS2. (B) FANCI depletion results in LSU pre-rRNA processing defects. HeLa cells were transfected with the indicated siRNAs, and total RNA was harvested after 72 h of depletion. Pre-rRNAs were separated by gel electrophoresis, and Northern blots were performed using radioactively labeled P5 probe. Northern blotting with a probe against the 7SL RNA was used as a loading control. Small illustrations of the pre-rRNAs are to the right of their respective bands. (C–I) RAMP (51) profiles for Northern blotting from three biological replicates for mock (C), siFANCI-pool (D), siFANCI-2 (E), siFANCI-3 (F), siPES1 (G), siNOL11 (H), and siFANCD2 (I). Statistical significance was calculated using a two-way ANOVA with Sidak multiple comparisons test (mean ± SD). All comparisons are relative to siNT. The x axes of all graphs were equalized, except for siPES1 in G, which has larger RAMP values. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Unmarked bars are not significant. (J) FANCI depletion results in decreased mature 28S rRNA relative to 18S. The ratio of 28S/18S was calculated using an Agilent 2100 Bioanalyzer. Statistical significance was calculated using a one-tailed unpaired Mann–Whitney U test for three biological replicates (mean ± SD). All comparisons are relative to siNT.

Fig. 4.

FANCI is required for global protein synthesis. (A) FANCI is required to maintain normal levels of protein synthesis. HeLa cells were transfected with the indicated siRNAs and, after 72 h of depletion, were treated with 1 μM puromycin for 1 h to label nascent peptides. A control treated with 0.5 μM puromycin was included [mock (1/2)]. Nascent peptides labeled with puromycin were detected by Western blotting with an anti-puromycin antibody. Western blotting with an anti–β-actin antibody was used as a loading control. (B) Densitometric quantitation of Western blots from three biological replicates (mean ± SD). Statistical significance for three biological replicates was calculated using the Wilcoxon signed-rank test with a hypothetical value of 1.0. All comparisons are relative to siNT. ns, not significant, **P ≤ 0.01.

Fig. 5.

FANCI is predominantly in the deubiquitinated state in the NO. (A–C) FANCI, but not FANCD2, is more deubiquitinated in the NO than in the NP. (A) Western blots for FANCI and FANCD2 detect both monoubiquitinated and deubiquitinated forms of each protein as indicated by the arrows. Densitometric quantitation of the ratio of monoubiquitinated relative to deubiquitinated FANCI (B) and FANCD2 (C) in untreated cells. Statistical significance for all quantitations in this figure was calculated using a Kruskal–Wallis test with an uncorrected Dunn’s test for multiple comparisons (mean ± SD). ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. All quantitations were performed using the same Western blots as in Fig. 1B. (D–I) Nucleolar FANCI persists predominantly in the deubiquitinated state under DNA damaging conditions. HeLa cells were treated with 2 mM HU (D) or 1 μM MMC (G) for ∼24 h and fractionated into WCE, NP, and NO. For controls, see SI Appendix, Fig. S5 A and B. Western blots for FANCI and FANCD2 detect both monoubiquitinated and deubiquitinated forms of each protein, as indicated by the arrows. (E and F) Quantitation of D. Densitometric quantitation of the ratio of monoubiquitinated relative to deubiquitinated FANCI (E) and FANCD2 (F) in HU-treated cells. (H and I) Quantitation of G. Densitometric quantitation of the ratio of monoubiquitinated relative to deubiquitinated FANCI (H) and FANCD2 (I) in MMC-treated cells. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig. 6.

DNA damage decreases the enrichment of FANCI in the NO. (A and B) HU treatment results in equal distribution of FANCI between NP and NO. Densitometric quantitation of Western blots from three biological replicates for nucleoplasmic and nucleolar FANCI (A) and FANCD2 (B) relative to WCE from cells treated with 2 mM HU for 24 h. All quantitations were performed using the same Western blots as in Fig. 5D. ns, not significant. (C and D) MMC treatment results in increased distribution of FANCI and FANCD2 to the NP relative to the NO. Densitometric quantitation of Western blots from three biological replicates for nucleoplasmic and nucleolar FANCI (C) and FANCD2 (D) relative to WCE from cells treated with 1 μM MMC for 24 h. All quantitations were performed using the same Western blots as in Fig. 5G. Statistical significance for all results in this figure was calculated using a two-tailed unpaired Mann–Whitney U test (mean ± SD). *P ≤ 0.05, **P ≤ 0.01.

Fig. 7.

The DUBs USP1 and USP36 maintain deubiquitinated nucleolar FANCI. (A) FANCI physically interacts with USP1 and USP36. Flp-In T-REx HEK293 cells with tetracycline-inducible expression of either FLAG-EV (empty vector), FLAG-USP1, or FLAG-USP36 were induced for ∼24 h with 1 μg/mL doxycycline hyclate. WCEs were prepared and immunoprecipitated (IP) with preimmune sera (negative control) or anti-FANCI antibody. Proteins with the N-terminal FLAG epitope, FANCI, and vinculin were detected by Western blotting. FANCI is a positive control for immunoprecipitation and vinculin is a negative control. (B) Deubiquitination of nucleolar FANCI requires USP1 and USP36. HeLa cells were transfected with the indicated siRNAs. After 48 h of depletion, cells were treated with 40 nM MMC overnight. After 24 h, cell extracts were prepared and immunoprecipitated with an anti-RPA194 antibody. Unconjugated protein A beads were used as a negative control. FANCI was detected by Western blotting. (C and D) Densitometric quantitation of the ratio of monoubiquitinated relative to deubiquitinated FANCI (FANCI^ub/FANCI) for WCEs (C) and anti-RPA194 immunoprecipitate (D). Statistical significance for all quantitations in this figure was calculated using a Kruskal–Wallis test with an uncorrected Dunn’s test for multiple comparisons (mean ± SD). ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.