Identification of distinct maturation steps involved in human 40S ribosomal subunit biosynthesis

Technical problems intrinsic to the purification of preribosome intermediates have limited our understanding of ribosome biosynthesis in humans. Addressing this issue is important given the implication of this biological process in human disease. Here we report a preribosome purification and tagging strategy that overcomes some of the existing technical difficulties. Using these tools, we find that the pre-40S precursors go through two distinct maturation phases inside the nucleolus and follow a regulatory step that precedes late maturation in the cytoplasm. This regulatory step entails the intertwined actions of both PARN (a metazoan-specific ribonuclease) and RRP12 (a phylogenetically conserved 40S biogenesis factor that has acquired additional functional features in higher eukaryotes). Together, these results demonstrate the usefulness of this purification method for the dissection of ribosome biogenesis in human cells. They also identify distinct maturation stages and metazoan-specific regulatory mechanisms involved in the generation of the human 40S ribosomal subunit.M.D. is supported by grants from the Spanish Ministry of Science, Innovation and Universities (BFU2014-52729-P, BFU2017-88192-P) and the Samuel Solórzano Foundation (FS/36-2017). X.R.B. is supported by grants from the Castilla-León Government (CSI049U16), the Spanish Ministry of Science, Innovation and Universities (SAF2015-64556-R), Worldwide Cancer Research (14-1248), the Ramón Areces Foundation, and the Spanish Association against Cancer (GC16173472GARC). B.N. and G.M. have been supported by predoctoral contracts sponsored by the University of Salamanca and Santander Bank, and S.G.G. by a predoctoral FPU contract of the Spanish Ministry of Education, Culture, and Sports. The Centro de Investigación del Cáncer is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia of the Ministry of Education of the Castilla-León Government (CLC-2017-01). Both Spanish and Castilla-León government-associated funding is partially supported by the European Regional Development Fund.Peer reviewe