RNA helicases are prominent actors in ribosome biogenesis but their molecular targets remain ill defined. We have previously demonstrated that the Prp43p RNA helicase is a central player in the synthesis of both the small and large ribosomal subunits. We identified two co-factors of Prp43p, Pfa1p and Gno1p (PINX1 in humans), that both possess a so-called “G-patch domain”, characterized by the presence of conserved glycine residues. Pfa1p and the human orthologue of Gno1p, PINX1, bind directly to Prp43p and activate the ATPase and helicase activities of Prp43p in vitro. We are investigating precisely when and how the Prp43p/Pfa1p and Prp43p/Gno1p complexes intervene in pre-ribosomal particle maturation and how the G-patch proteins interact with Prp43p and stimulate its ATPase and helicase activities.