Modular assembly of the nucleolar pre-60S ribosomal subunit

Abstract

Early co-transcriptional events during eukaryotic ribosome assembly result in the formation of precursors of the small (40S) and large (60S) ribosomal subunits1. A multitude of transient assembly factors regulate and chaperone the systematic folding of preribosomal RNA subdomains. However, owing to a lack of structural information, the role of these factors during early nucleolar 60S assembly is not fully understood. Here we report cryo-electron microscopy (cryo-EM) reconstructions of the nucleolar pre-60S ribosomal subunit in different conformational states at resolutions of up to 3.4 Å. These reconstructions reveal how steric hindrance and molecular mimicry are used to prevent both premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Among these factors, three Brix-domain proteins and their binding partners form a ring-like structure at ribosomal RNA (rRNA) domain boundaries to support the architecture of the maturing particle. The existence of mutually exclusive conformations of these pre-60S particles suggests that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly. These structures rationalize previous genetic and biochemical data and highlight the mechanisms that drive eukaryotic ribosome assembly in a unidirectional manner.

Assembly and early maturation of large subunit precursors

“…In vivo crosslinking studies of Rrp5 have shown its proximity to several sites on the pre-rRNA, including the central domain of the 18S, ITS1 and domain II of the 25S, consistent with our data (Lebaron et al 2013). Domain II also leads to the association of several factors completing the ring structure around domain I and II which was described in the nucleolar pre-60S structure (Sanghai et al 2018), composed of Nsa1, Rpf1, Mak16, Rrp1, and the already associated Brx1 and Ebp2.…”

“…Fig. 2) (Kater et al 2017;Sanghai et al 2018;Zhou et al 2018). Completion of domain VI induces the incorporation of almost 20 ribosome assembly factors, the 5S rRNA and association of snR10.…”

“…Recent structures of nucleolar pre-60S particles have revealed some of the folding pathways that large subunit pre-rRNA follows (Kater et al 2017;Sanghai et al 2018;Zhou et al 2018) showing that domains I, II and VI of the 25S rRNA, along with the 5.8S rRNA, fold first into a near-mature conformation. Domains III-V remain unfolded in the early stages of large subunit assembly.…”

Two molecules of Has1 RNA helicase function simultaneously in the biogenesis of small and large ribosomal subunits

“…Our data for the wild type Has1 are in a very good agreement with the recently published PAR-CRAC analysis (19). The observed crosslinking sites in the 5.8S/25S region also coincide well with the location of Has1 in several recent cryoEM structures of pre-60S ribosomes (27)(28)(29). The binding sites of the wild type Has1 in the 18S/ITS1 region are in close proximity to the binding sites of the pre-40S assembly factors Enp1, Ltv1 and Tsr1 (22).…”

“…Can Has1 function as a dimer? Has1 was observed to self-interact in the yeast two-hybrid assays (36,37).On the other hand, Has1 identified in the cryoEM structures of pre-60S ribosomes is present as a monomer (27)(28)(29). It is possible that Has1 is recruited as dimer but then separates and each monomer functions separately in the pre-40S and pre-60S pathways.…”