Interaction network of the ribosome assembly machinery from a eukaryotic thermophile

Abstract

Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre-ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre-ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (∼180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in-depth analysis of their protein-protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2-hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein-protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre-ribosome factors forming the ctUTP-A and ctUTP-B modules, and the Brix-domain containing assembly factors of the pre-60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.

A Puzzle of Life: Crafting Ribosomal Subunits

“…Several recent studies have significantly advanced our understanding of these initial assembly events. Biochemical reconstitution and structural approaches revealed the architecture of the UTP-A and UTP-B modules [56][57][58][59][60]. Crosslinking analyses supported the idea that UTP-A is the first module to associate with the 5 0 -external transcribed spacer (ETS) region of the nascent 35S pre-rRNA [27,58] (Figures 1, 2B, and 3 ).…”

“…It would also be interesting to visualize 80S-like ribosomes as they subject SSUs to final quality control before their engagement in translation. We expect that future structural, enzymatic, and reconstitution approaches will continue to benefit from the utilization of thermophilic proteins and preribosomal particles from the eukaryotic organism C. thermophilum [27,59,109]. Further mechanistic and structural insights into ribosome biogenesis may promote the identification and design of small-molecule inhibitors like diazaborine or the ribozinoindoles, which act as specific inhibitors of the AAA-ATPases Drg1 and Rea1, respectively [110,111].…”

Structural basis for 5'-ETS recognition by Utp4 at the early stages of ribosome biogenesis

“…UTP-A was also part of our systematic analysis of ribosome biogenesis factors from Chaetomium thermophilum [12]. The crystal structure of Utp4 was solved at a resolution of 2.15 Å by the single wavelength anomalous dispersion (SAD) method using seleno-methionine labelled protein (Table 1).…”

“…In order to identify direct RNA-protein interactions of Utp4, we performed in vitro RNA-protein UV crosslinking and mapped the binding regions within the target RNA by subsequent cDNA deep sequencing (CRAC) [16]. When we co-expressed the 5'-ETS together with the UTP-A factors in yeast and isolated the UTP-A complex by split-tag tandem affinity purification as reported before [12], we found that the 5'-ETS was co-purifying with this reconstituted 90S module (data not shown). Accordingly, we assembled and purified a similar 5'-ETS/UTP-A RNP carrying His 6 -tagged Utp4, which was used for in vitro UV crosslinking analysis (S3 Fig).…”

“…Although this unusual parallel complementation of the last blade could have been an artefact of our cloning strategy, it apparently represents a specific protein-binding site for Utp8 within the UTP-A complex as deduced from interaction studies [12, 23, 25, 26] and placing the Utp4 structure in a cryo-EM density of the 90S pre-ribosome. Within UTP-A, Utp4 and Utp17 present double-propellers.…”