Good Vibrations: Structural Remodeling of Maturing Yeast Pre-40S Ribosomal Particles Followed by Cryo-Electron Microscopy

Abstract

Assembly of eukaryotic ribosomal subunits is a very complex and sequential process that starts in the nucleolus and finishes in the cytoplasm with the formation of functional ribosomes. Over the past few years, characterization of the many molecular events underlying eukaryotic ribosome biogenesis has been drastically improved by the “resolution revolution” of cryo-electron microscopy (cryo-EM). However, if very early maturation events have been well characterized for both yeast ribosomal subunits, little is known regarding the final maturation steps occurring to the small (40S) ribosomal subunit. To try to bridge this gap, we have used proteomics together with cryo-EM and single particle analysis to characterize yeast pre-40S particles containing the ribosome biogenesis factor Tsr1. Our analyses lead us to refine the timing of the early pre-40S particle maturation steps. Furthermore, we suggest that after an early and structurally stable stage, the beak and platform domains of pre-40S particles enter a “vibrating” or “wriggling” stage, that might be involved in the final maturation of 18S rRNA as well as the fitting of late ribosomal proteins into their mature position.

Altered rRNA processing disrupts nuclear RNA homeostasis via competition for the poly(A)-binding protein Nab2

“…Enp1 is a 40S ribosome biogenesis factor, one of few that is already present in the 90S complex and remains associated until late pre-40S biogenesis in the cytoplasm ( 50 , 51 ). It binds to the 3′end minor domain of the pre-18S rRNA, close to site D in the Internal Transcribed Spacer 1 (ITS1), where, together with Ltv1 and Tsr1, it coordinates stabilization of the pre-40S ribosomal beak structure in its immature conformation, facilitates repositioning of Rps3 (uS3), and late integration of Rps10 (eS10) ( 52–54 , 98 ). Due to its placement on the pre-40S subunit, it has also been suggested to play a role in the maturation of helix (h) 34 and the h34–h35–h38 three-way junction, which contain a central element in the formation of the A-site decoding center (h33/34), key residues for mRNA binding (h29–h32) and accommodation of anticodon-arms for all three tRNAs (h38-h42) ( 53 , 98 , 99 ).…”

“…It binds to the 3′end minor domain of the pre-18S rRNA, close to site D in the Internal Transcribed Spacer 1 (ITS1), where, together with Ltv1 and Tsr1, it coordinates stabilization of the pre-40S ribosomal beak structure in its immature conformation, facilitates repositioning of Rps3 (uS3), and late integration of Rps10 (eS10) ( 52–54 , 98 ). Due to its placement on the pre-40S subunit, it has also been suggested to play a role in the maturation of helix (h) 34 and the h34–h35–h38 three-way junction, which contain a central element in the formation of the A-site decoding center (h33/34), key residues for mRNA binding (h29–h32) and accommodation of anticodon-arms for all three tRNAs (h38-h42) ( 53 , 98 , 99 ). Loss of Enp1 results in early and late pre-rRNA processing and assembly defects causing accumulation of stalled 90S, pre-40S, and pre-60S ribosomes and their aberrant rRNA precursors, several of which become polyadenylated.…”

“…While 23S-containing pre-40S complexes are densely covered in protein ( 103 ), high-throughput rRNA structure probing (ChemModSeq) revealed that pre-40S particles containing Enp1 harbor a 3′ major domain (corresponding to the beak and platform domains of the small ribosomal subunit) with a higher degree of flexibility ( 104 ). Moreover, the three-way junction formed by rRNA helixes h34, h35 and h38 at the base of the head was shown to exist in an immature ‘open’ conformation ( 98 , 105 ). Thus it is conceivable that in the absence of functional Enp1 these open and flexible regions become stabilized, enabling cleavage of 23S at site Q1 (located within the central domain of the pre-18S rRNA) to generate aberrant 17S precursor (which are polyadenylated; Figure 4A ) ( 26 ), but also leave rRNA regions exposed to binding of PABPs.…”

Proteo3Dnet: a web server for the integration of structural information with interactomics data

“…We have queried Proteo3Dnet with a list of interactants obtained by tandem-affinity purification of Saccaromyces cerevisiae precursors to the small ribosomal subunits (hereafter termed pre-40S particles; ( 25 ). These 62 proteins include highly abundant 39 Ribosomal Proteins of the Small ribosomal subunit (RPS) and 8 ribosome biogenesis factors (RBFs) together with 14 much less abundant Ribosomal Proteins of the Large ribosomal subunit (RPL), and the Lrg1 protein.…”

Special Issue: Frontiers in RNA Structure

“…Cryo-EM enables us to visualize dynamic architectures, as portrayed by the Dao Duc laboratory at the University of British Columbia, who review ribosome heterogeneity [ 10 ]. In the same vein, the teams led by Marcoux, Gleizes and Plisson-Chastang at the University of Toulouse in France determine the role of a few of the many ribosome biogenesis factors, demonstrating the power of cryo-EM to analyze conformational heterogeneity [ 11 ].…”