Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomal particles

ABSTRACT: Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and rRNA remodeling events in the nucleolus, nucleoplasm and cytoplasm. Hundreds of assembly factors (AFs), organized into sequential functional groups, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these AFs function are largely unknown. Here, we use cryo-electron microscopy (cryo-EM), to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged AF Nog2. Our data pinpoint the locations and determine the structures of over 20 AFs, which are enriched in two areas, an arc region extending from the central protuberance (CP) to the polypeptide tunnel exit (PTE), and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S rRNAs. These structural data suggest that the arc-located factors might function to chaperone formation of RNA helices found in the active sites of the subunit, including helices from the CP, peptidyl-transferase center (PTC) and intersubunit bridge. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant AFs and functional rRNA elements, manifesting their critical roles in structural remodeling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodeling events before nuclear export: rotation of the 5S RNP, construction of the active center, and ITS2 removal. Therefore, our structures provide a framework to understand the molecular roles of diverse AFs, and potentially the atomic information therein constitutes a resource to generalize principles governing the elusive functions of nuclear RNA-binding proteins.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Luyao Ma  

LAB HEAD: Meng-Qiu Dong

PROVIDER: PXD003736 | Pride | 2016-06-01

REPOSITORIES: Pride