Fig 2: CSA interaction with FECH is stabilized upon UV exposure. (A, B) Immunoblot analysis with antibodies specific for FECH, HA or DDB1 of CSAFlag-HA-interacting proteins isolated by TAP experiments in CS3BE-cassette1 (-) or CS3BE-wtCSAFlag-HA (+) cells before and after treatment with different stressing agents. Cells were irradiated with UV-C light (10 J/m2) or treated with menadione (MD, 100 µM) or potassium bromate (KBrO3, 20 mM) and analysed after 1 hr of recovery time (A) or at the indicated hours following UV irradiation (B). The protein levels were quantified and normalized to the corresponding HA amount and expressed as fold increase of the corresponding untreated cells. Proteins fold increase relative to the not treated sample are reported in the diagram below the plots. Reported values represent the mean ± SEM (n = 3), *P < 0.05, ***P < 0.001 (Student's t-test). (C) Sub-cellular localization of wtCSAFlag-HA/FECH interaction by proximity ligation assay (PLA) with anti-HA and anti-FECH antibodies in CS3BE-wtCSAFlag-HA and CS3BE-cassette1 cells before and at 2 h of UV irradiation (10 J/m2). Red fluorescence signal visualizes the CSA/FECH interaction. Nuclei were counterstained with DAPI and cells visualized in bright field. Scale bar: 5 µm. (D) Number of cells containing the PLA spots shown in C and expressed as percentage. In each sample 100 cells have been analyzed. The percentage of nuclear and cytoplasmic spots is indicated.

Fig 3: FECH is a regulator of ribosomal transcription. (A, B) RNAP1 occupancy on 47S, 5.8S and intergenic spacer (IGS) of the rDNA repeat in CS3BE-cassette1 and CS3BE-wtCSAFlag-HA cells (A, left panel), in CS3BE-wtCSAFlag-HA under basal condition and at 2 h after UV irradiation (A, right panel) and in CS3BE-wtCSAFlag-HA cells after treatment for 120 h with control (CTR), FECH or CSA siRNA molecules (B). The occupancy identified by ChIP analysis is expressed as percentage of the input after subtraction of the corresponding background value (IgG antibody). Reported values represent the mean ± SEM (n = 3), *P < 0.05, **P < 0.01, ns = not statistically significant (Student's t-test). On the right of panel B, a schematic representation of the rDNA repeat indicating the position of the ChIP amplicons (bleu), the probe for the northern blot described in (E) and the real time RT-PCR amplicons (black) described in (F); (C, D) Representative images of nucleolar run-on transcription assay in Dichlororibo-furanosyl (DRB)-treated primary fibroblast from normal (C3PV or C5BO), CS-A (CS7PV, CS11PV or CS15PV) or erythropoietic protoporphyria (EPP, GMO5008) cells before (C) or after treatment for 120 h with control (CTR), FECH or CSA siRNA molecules (D). The incorporation of bromodeoxyuridine (BrdU) was visualized by indirect immunofluorescence with anti-BrdU antibodies. Images were obtained by overlaying the DAPI stained nucleus (gray) with the corresponding BrdU fluorescence (green). The nucleolar fluorescence intensity was visualized as intensity profile with the MetaMorph software, measured with the ImageJ software and represented in the diagram on the right (au: arbitrary units). Reported values represent the mean ± SEM (n = 20), **P < 0.01, ***P < 0.001, ns: not significant (Student's t-test). Scale bar: 5µm. (E) Northern blot analysis of CS3BE-wtCSAFlag-HA treated for 72 h with control (CTR), FECH, CSA, RPS10 or RPS15 siRNA and hybridized with the ITS1 probe indicated in (A). The intensity of rRNA precursor bands has been quantified and normalized to the intensity of the corresponding actin band or to the 47S band and reported on the diagrams on the right. Reported values represent the mean ± SEM (n = 3), *P < 0.05, **P < 0.01 (Student's t-test). (F) Real time RT-PCR analysis of 47S and 18S rRNA levels in primary dermal fibroblasts from an healthy individual (C3PV), an EPP (GMO5008) and CS-A patient (CS7PV) before (-) and after 10 J/m2 UV irradiation (+). The 47S transcript levels were normalized to the expression level of the corresponding housekeeping gene GAPDH (top) while the 18S transcript levels were normalized to the expression level of the corresponding 47S (middle and bottom, respectively). Reported values represent the mean ± SEM (n = 4), **P < 0.01, ***P < 0.001 (Student's t-test).

Fig 4: Graphical model illustrating the dissociation from the chromatin of the RNAP1–CSs–FECH–RPs complex stalled by the UV-induced DNA lesion during rRNA transcription. In the absence of CSA the complex maintains its binding to the chromatin.

Fig 5: CSA interaction with FECH occurs mainly in the nucleus. (A) Immunoblot analysis of the mitochondrial fraction (Mit) separated from the remaining portion of cell lysate (Nuc/Cyt) in CS3BE-cassette1 (-) or CS3BE-wtCSAFlag-HA cells (+) with antibodies raised against FECH, HA, the inner membrane mitochondrial marker SDHA and the nuclear/cytosolic markers PCNA or c-JUN. (B) Co-immunofluorescence analysis with antibodies raised against FECH and the mitochondrial protein TOMM20 in CS3BE-wtCSAFlag-HA cells or in primary dermal fibroblasts from the healthy donor C3PV. Nuclei were counterstained with DAPI. Scale bar: 5 µm. (C) Immunoblot analysis with antibodies specific for FECH, HA, the mitochondrial marker SDHA or the nuclear/cytosolic marker PCNA in immunoprecipitations (IP) performed with anti-FECH antibodies in the mitochondrial (Mit) and nuclear/cytosolic (Nuc/Cyt) fractions (input) of CS3BE-wtCSAFlag-HA (+) or CS3BE-cassette1 (-) cells. The intensity of co-immunoprecipitated wtCSAFlag-HA band was quantified and normalized to the amount of immunoprecipitated FECH. Value are reported in the diagram on the right. Reported values represent the mean ± SEM (n = 3), ***P < 0.001 (Student's t-test). (D) Subcellular localization of wtCSAFlag-HA/FECH interaction by proximity ligation assay (PLA) in CS3BE-wtCSAFlag-HA and CS3BE-cassette1 cells by using anti-HA and anti-FECH antibodies. Red fluorescence signal visualizes the wtCSAFlag-HA/FECH interaction. Nuclei were counterstained with DAPI and cells visualized in bright field. Scale bar: 5 µm.