TFIIH mutations can impact on translational fidelity of the ribosome

**Background:** Trichothiodystrophy (TTD) is a developmental disorder characterized by brittle hair, ichthyosis, microcephaly, impaired intelligence, recurrent infections, retarded growth, and cataracts. It shares features with Cockayne syndrome (CS), a progeroid disorder. Mutations in the TFIIH complex, which is essential for transcription by RNA polymerases I and II and for DNA repair, cause TTD, CS, and xeroderma pigmentosum (XP). While XP is a cancer-prone DNA repair disorder, TTD and CS are cancer-free but show severe developmental and neurodegenerative symptoms. Previous work suggested that TFIIH mutations disrupt ribosomal biogenesis and proteostasis in CS and TFIIE-mutant TTD cells. This study investigates whether TFIIH mutations in TTD also impair ribosomal function and protein homeostasis.

**Methods:** The study used five TTD patient-derived fibroblast strains with mutations in different TFIIH subunits (p8, XPD, XPB), including both SV40-transformed and primary cells. Two XP patient-derived strains with XPD mutations served as controls. Proteome stability was assessed by heat sensitivity (percentage of pelleted proteins after 15 min at 99°C) and urea-induced unfolding (BisANS fluorescence). Translation activity was measured by O-propargyl-puromycin (OPP) incorporation and 35S-methionine labeling. Translational fidelity was evaluated using a luciferase reporter assay: cells were transfected with a plasmid or in vitro-transcribed mRNA encoding a mutant firefly luciferase (K529N) that is inactive unless misincorporated amino acids restore activity. Pre-rRNA processing was analyzed by Northern blotting with an ITS1 probe. Ribosome composition was examined by mass spectrometry and western blotting of purified ribosomes. Reactive oxygen species (ROS) levels were measured by DCF fluorescence. In vitro translation assays used ribosomes isolated from patient cells combined with ribosome-depleted rabbit reticulocyte lysate, with or without H2O2-oxidized mRNA. Urinary 8-oxo-guanosine was quantified by ELISA. Rescue experiments used N-acetyl cysteine (NAC, 1 mM) and tauroursodeoxycholic acid (TUDCA, 200 μM).

**Key Results:** TTD cells showed reduced proteome stability: heat treatment led to a higher percentage of pelleted proteins in all TTD cells compared to controls (e.g., p8Mut and primary TTD cells), while XP cells did not show this increase. Urea-induced unfolding was elevated in primary TTD cells and p8Mut cells but not in XPDMut cells. OPP incorporation indicated increased translation in all primary TTD cells but not in XP cells. Proliferation was severely retarded in all patient cells. Translational fidelity was significantly impaired in TTD cells: when transfected with mutant luciferase mRNA, all TTD cell lines showed elevated luciferase activity (indicating misincorporation), while XP and control cells did not. siRNA knockdown of the p8 subunit in control fibroblasts induced translational infidelity, confirming a causal role. Pre-rRNA processing was disturbed in most TTD cells, with reduced 41S/47S ratio and accumulation of later intermediates; XP cells showed elevation of all intermediates. Mass spectrometry of purified ribosomes revealed underrepresentation of small subunit ribosomal proteins in p8Mut cells, but not in XPDMut cells. In vitro translation with purified ribosomes did not show elevated error rates under standard conditions, but when mRNA was oxidized with H2O2, ribosomes from TTD cells (but not XP or CS cells) exhibited significantly increased translational infidelity. Endogenous ROS levels were highly elevated in all patient cells, including XP. Urinary 8-oxo-guanosine was significantly elevated in TTD patients compared to relatives. Treatment with NAC (1 mM for 24 h) reduced the translational error rate in TTD cells. TUDCA treatment normalized RNA polymerase I transcription, reduced protein synthesis, and decreased accumulation of processing intermediates in p8Mut and XPDMut cells.

**Clinical Implications:** This study identifies a novel pathomechanism for TTD: TFIIH mutations lead to elevated ROS and production of ribosomes that are unable to accurately translate oxidized mRNA, resulting in proteome instability and loss of proteostasis. This mechanism distinguishes TTD from XP and may underlie the neurodevelopmental and neurodegenerative features of TTD and CS. The finding that NAC and TUDCA can restore translational fidelity and ribosomal biogenesis suggests potential therapeutic strategies for these currently incurable disorders. The use of low-toxicity drugs like NAC and TUDCA, already approved for human use, offers a promising avenue for clinical intervention.