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The pseudouridine synthase and archaeosine transglycosylase (PUA) domain is a compact and highly conserved RNA-binding motif that is widespread among diverse types of proteins from the three kingdoms of life. Its three-dimensional architecture is well established, and the structures of several PUA–RNA complexes reveal a common RNA recognition surface, but also some versatility in the way in which the motif binds to RNA. The PUA domain is often part of RNA modification enzymes and ribonucleoproteins, but it has also been unexpectedly found fused to enzymes involved in proline biosynthesis, where it plays an unknown role. The functional impact of the domain varies with the protein studied, ranging from minor to essential effects. PUA motifs are involved in dyskeratosis congenita and cancer, pointing to links between RNA metabolism and human diseases.