The 90S Preribosome Is a Multimodular Structure That Is Assembled through a Hierarchical Mechanism

Abstract

The 90S preribosomal particle is required for the production of the 18S rRNA from a pre-rRNA precursor. Despite the identification of the protein components of this particle, its mechanism of assembly and structural design remain unknown. In this work, we have combined biochemical studies, proteomic techniques, and bioinformatic analyses to shed light into the rules of assembly of the yeast 90S preribosome. Our results indicate that several protein subcomplexes work as discrete assembly subunits that bind in defined steps to the 35S pre-rRNA. The assembly of the t-UTP subunit is an essential step for the engagement of at least five additional subunits in two separate, and mutually independent, assembling routes. One of these routes leads to the formation of an assembly intermediate composed of the U3 snoRNP, the Pwp2p/UTP-B, subunit and the Mpp10p complex. The other assembly route involves the stepwise binding of Rrp5p and the UTP-C subunit. We also report the use of a bioinformatic approach that provides a model for the topological arrangement of protein components within the fully assembled particle. Together, our data identify the mechanism of assembly of the 90S preribosome and offer novel information about its internal architecture.

SUPPLEMENTAL MATERIAL

We thank the Proteomics Unit personnel for their enthusiastic work in the determination of protein complexes by mass spectrometry.

M.D.'s work is supported by the Spanish Ministry of Education and Science (MEC) (BFU2005-06421) and the Castilla-León Autonomous Government (SA040/02). X.R.B.'s work is supported by grants from the U.S. NCI/NIH (5RO1-CA73735-10), MEC (SAF2003-00028), and Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD06/0020/0001, Fondo de Investigaciones Sanitarias [FIS], Carlos III Institute, Spanish Ministry of Health). J.D.L.R.'s work is possible due to a grant from the Spanish Ministry of Health (PI030920). J.P.-F. was supported by a postdoctoral fellowship from the Spanish Cooperative Research Network of Cancer Centers (C03/10, Spanish Ministry of Health) and the RTICC (RD06/0020/0001). All Spanish funding is cosponsored by the European FEDER Program.