The pseudouridine synthase PUSL1 modifies U39 of mitochondrial tRNAs and is linked to human neurological phenotypes.

Variants in the mitochondrial and nuclear genomes are linked to a wide range of human disorders marked by impaired mitochondrial function. Among these disorders, there is a growing number of patients with variants affecting mitochondrial RNA biology. Mitochondrial transcripts are pseudouridylated, and some enzymes responsible for this modification-pseudouridine synthases (PUS)-have been identified. Although known as the 'fifth nucleotide' owing to its high abundance in transcripts, the exact cellular role of pseudouridine is still unclear. Here, we expand the group of mitochondrial PUS enzymes by demonstrating that the protein encoded by PUSL1 is an active pseudouridine synthase with mitochondrial localization. Nucleotide-resolution pseudouridine mapping (mito-Ψ-Seq) followed by primer extension analysis showed that PUSL1 selectively modifies universal position 39 of all mitochondrial transfer RNAs (tRNAs) with a uridine residue in this position. Two newly described clinical PUSL1 variants, c.704G > A (p.Arg235Gln) and c.634del, p.Glu212Argfs*26, were functionally studied, presenting defects in pseudouridylation of mt-tRNA position 39 in patient-derived material, corroborating the association of this enzyme with human pathology. Our data show that PUSL1 regulates mitochondrial RNA post-transcriptional processing and its dysfunction and could be associated with neurological phenotypes.