tRNA thiolation defects disrupt cellular proteostasis and tissue homeostasis in mammals

Sulfur modification of tRNA wobble uridines is an evolutionarily conserved mechanism that ensures efficient protein synthesis. In humans, loss of this anticodon modification due to mutations in CTU2 (cytosolic thiouridylase 2) causes DREAM-PL syndrome, a severe congenital disorder often leading to early postnatal death. However, the mechanisms by which loss of tRNA thiolation drives pathology remain unclear. Here, we show that loss of CTU2 triggers significant cellular proteostasis defects in patient cells and model cell lines. Structural and biochemical analyses reveal that the pathogenic CTU2 ^L63P mutation destabilizes the CTU1/CTU2 complex and abolishes tRNA binding and thiolation. Acute loss of CTU2 caused codon-specific ribosome pausing at A-ending codons decoded by thiolated tRNAs, and decreased ribosome occupancy of A-rich transcripts in a dosage-dependent manner. Codon-biased mRNAs transcribed from genes critical for ciliogenesis are predicted to be most affected, linking their reduced translation to DREAM-PL etiology in humans. Surprisingly, Ctu2 ^L63P mice display severe thiolation defects, but develop normally, are viable and fertile. Our findings highlight the importance of functional tRNA thiolation for organismal health in humans and identify species-specific vulnerabilities during embryonic development in mammals.