Genetics animates structure: leveraging genetic interactions to study the dynamics of ribosome biogenesis

Abstract

The assembly of eukaryotic ribosomes follows an assembly line-like pathway in which numerous trans-acting biogenesis factors act on discrete pre-ribosomal intermediates to progressively shape the nascent subunits into their final functional architecture. Recent advances in cryo-electron microscopy have led to high-resolution structures of many pre-ribosomal intermediates; however, these static snapshots do not capture the dynamic transitions between these intermediates. To this end, molecular genetics can be leveraged to reveal how the biogenesis factors drive these dynamic transitions. Here, we briefly review how we recently used the deletion of BUD23 (bud23∆) to understand its role in the assembly of the ribosomal small subunit. The strong growth defect of bud23∆ mutants places a selective pressure on yeast cells for the occurrence of extragenic suppressors that define a network of functional interactions among biogenesis factors. Mapping these suppressing mutations to recently published structures of pre-ribosomal complexes allowed us to contextualize these suppressing mutations and derive a detailed model in which Bud23 promotes a critical transition event to facilitate folding of the central pseudoknot of the small subunit. This mini-review highlights how genetics can be used to understand the dynamics of complex structures, such as the maturing ribosome.

Keywords: Bms1; Bud23; Dhr1; Ribosome biogenesis; SSU Processome.

Fig. 1. Structural overview of small ribosomal subunit biogenesis in eukaryotes.

Small subunit biogenesis is a multi-step process; several SSU assembly intermediates are shown to highlight the structural rearrangements that occur during SSU biogenesis. The SSU Processome (composite of PDBs 5WLC & 5WYJ) is an early precursor which contains assembly factors (AFs, light gray) and ribosomal proteins (RPs, dark gray) that independently chaperone the four domains: the 5’ major, central, 3’ major, and 3’ minor (respectively colored blue, magenta, yellow, and green). A yet unknown signal triggers progression of the Processome which involves the shedding of the 5’-ETS as well as many AFs and RNA rearrangements (colored arrows) that transform it into a more compact final disassembly complex, termed “Dis-C” (PDB 6ZQG) (Cheng et al. 2020). However, a set of factors retained on Dis-C prevent its final transformation into the earliest pre-40S intermediate (PDB 6G4W). We have proposed that the coordinated efforts of Bud23 and the RNA helicase Dhr1 promote the final transition into the pre-40S, allowing the 3’ domains to adopt their near-mature positions (Black et al. 2020). A series of subsequent, less dramatic maturation events fine-tune the rRNA (colored lines; lower panel) to produce the mature 40S (PDB 4V88). The figure is adapted from (Black et al. 2020) Molecular visualizations were generated in UCSF ChimeraX v0.93 (Goddard et al. 2018).

Fig. 2. Summary of the physical interaction network underlying bud23Δ suppression.

a The extragenic suppressors of bud23Δ form a tight network of physical and functional interactions that includes U3 snoRNA. b Mapping this network to the structure of the SSU Processome reveals physical interactions connecting to U3 snoRNA; Bms1 (green), Imp4 (blue), Rps28 (cyan), Utp2 (orange), and Utp14 (brown). Imp4, Utp2, and Bms1 physically connect the U3 snoRNA (magenta) to the 3’ basal subdomain (dark gray), the future binding site of Bud23. The target base of Bud23 methyltransferase, G1575 (red), is shown for reference. c The majority of the Imp4 mutants (magenta sticks) mapped to its interaction interface with the 3’ basal subdomain of 18S RNA. d The Utp2 mutants (green sticks) mapped to its interface with Imp4 near the 3’ basal subdomain. e F58 of Utp2 fits into a hydrophobic pocket in Imp4 (upper) while V170 and P252 of Imp4 help establish this pocket (lower). Amino acid substitutions of these residues disrupt the interaction between Imp4 and Utp2 (Black et al. 2020). Molecular visualizations were generated in MacPyMOL: PyMOL v1.8.2.1 Enhanced for Mac OS X (Schrödinger LLC) using PDB 5WLC.

Fig. 3. Model for how Bud23 promotes the final disassembly step of the SSU Processome.

a The structures of Bms1, Imp4, Rps28, Utp2, Utp14, and Dhr1 as observed in the final disassembly complex (Dis-C, PDB 6ZQG). Factors are colored as in Fig. 2a. Helix 1 of the 18S rRNA has formed. Nucleotides A1137-U1144 of the 18S rRNA are unresolved but originate from the 3’ basal subdomain at G1146. Molecular visualizations were generated MacPyMOL: PyMOL v1.8.2.1 Enhanced for Mac OS X (Schrödinger LLC). b The model for Bud23 function with proteins and RNAs colored as in panel A. Bud23 binding to the 3’ basal subdomain promotes Bms1 activation (black lines), allowing Imp4 and Utp2 eviction and the rRNA rearrangements (gray arrows) that bring residues A1137-U1144 into proximity with helix 1. Subsequent activation of Dhr1 by Utp14 (green arrow) promotes productive unwinding of U3, which allows the rRNA strand containing residues A1137-U1144 to base-pair with the stem-loop of Helix 1 to form Helix 2, completing the CPK.

Fig. 4. Kinetic proofreading model for how extragenic suppressors bypass bud23Δ.

a In wild-type cells, Bud23 binding to the Dis-C complex leads to release of Imp4 and Utp2 and Bms1-dependent repositioning of residues A1137-U1144 of the 18S rRNA into close proximity of U3. The subsequent unwinding of U3 by Dhr1 allows A1137-U1144 to base pair with the stem-loop of Helix 1 to form Helix 2, thereby completing the CPK. b In cells lacking Bud23, the repositioning of residues A1137-U1144 is hindered and the unwinding of U3 by Dhr1 leads to unproductive folding of the rRNA and failure in CPK formation. However, a low rate of productive folding must occur in bud23Δ cells because they are viable, though very slow growing. c In bud23Δ cells harboring a suppressor mutation, the rate of productive CPK folding is partially restored. This can be due to either (1) amino acid substitutions in Dhr1 or Utp14 that slow the Dhr1 activity to allow more time for residues A1137-U1144 to come into position before U3 unwinding occurs or (2) amino acid substitutions in Bms1, Imp4, or Utp2 that promote the repositioning of residues A1137-U1144.