Quantitative mining of compositional heterogeneity in cryo-EM datasets of ribosome assembly intermediates

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Powering through ribosome assembly

Ribosome assembly is required for cell growth in all organisms. Classic in vitro work in bacteria has led to a detailed understanding of the biophysical, thermodynamic, and structural basis for the ordered and correct assembly of ribosomal proteins on ribosomal RNA. Furthermore, it has enabled reconstitution of active subunits from ribosomal RNA and proteins in vitro. Nevertheless, recent work has shown that eukaryotic ribosome assembly requires a large macromolecular machinery in vivo. Many of these assembly factors such as ATPases, GTPases, and kinases hydrolyze nucleotide triphosphates. Because these enzymes are likely regulatory proteins, much work to date has focused on understanding their role in the assembly process. Here, we review these factors, as well as other sources of energy, and their roles in the ribosome assembly process. In addition, we propose roles of energy-releasing enzymes in the assembly process, to explain why energy is used for a process that occurs largely spontaneously in bacteria. Finally, we use literature data to suggest testable models for how these enzymes could be used as targets for regulation of ribosome assembly.     

The RimP Protein Is Important for Maturation of the 30S Ribosomal Subunit

The in vivo assembly of ribosomal subunits requires assistance by auxiliary proteins that are not part of mature ribosomes. More such assembly proteins have been identified for the assembly of the 50S than for the 30S ribosomal subunit. Here, we show that the RimP protein (formerly YhbC or P15a) is important for the maturation of the 30S subunit. A rimP deletion (ΔrimP135) mutant in Escherichia coli showed a temperature-sensitive growth phenotype as demonstrated by a 1.2-, 1.5-, and 2.5-fold lower growth rate at 30, 37, and 44 °C, respectively, compared to a wild-type strain. The mutant had a reduced amount of 70S ribosomes engaged in translation and showed a corresponding increase in the amount of free ribosomal subunits. In addition, the mutant showed a lower ratio of free 30S to 50S subunits as well as an accumulation of immature 16S rRNA compared to a wild-type strain, indicating a deficiency in the maturation of the 30S subunit. All of these effects were more pronounced at higher temperatures. RimP was found to be associated with free 30S subunits but not with free 50S subunits or with 70S ribosomes. The slow growth of the rimP deletion mutant was not suppressed by increased expression of any other known 30S maturation factor.     

Structural changes in the ribosome during the elongation cycle

Ample data on structural changes that arise in the ribosome during translation have been accumulated. The most interesting information on such changes has been obtained by cryoelectron microscopy of ribosome complexes with various ligands and by rRNA site-directed mutagenesis combined with a structural analysis of the ribosome by a chemical modification technique (chemical probing). The review considers the best-known structural changes that arise in the translating ribosome upon its interactions with tRNA and the elongation factors. The changes are discussed in the context of interactions between the functional centers of the ribosome. A universal system of rRNA helices and proteins is described in detail. The system integrates the functional centers of the ribosome and allows transduction of allosteric conformational signals. Biochemical data are considered in terms of the structures and interactions of ribosomal elements, and a hypothesis is advanced that the position of the GTPase-associated center in the ribosome regulates the binding of the elongation factors.     

Structure of the 70S Ribosome from the Human Pathogen Acinetobacter baumannii in Complex with Clinically Relevant Antibiotics

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Deletion of EFL1 results in heterogeneity of the 60 S GTPase-associated rRNA conformation

Previous work suggested that the release of the nucleolar Tif6 from nascent 60 S subunits occurs in the cytoplasm and requires the cytoplasmic EF-2-like GTPase, Efl1. To check whether this release involves an rRNA structural rearrangement mediated by Efl1, we analyzed the rRNA conformation of the GTPase center of 80 S ribosomes in three contexts: wild-type, Deltaefl1 and a dominant suppressor R1 of Deltaefl1. This analysis was restricted to domain II and VI of 25 S rRNA. The rRNA analysis of R1 ribosomes allows us to distinguish the effects due to depletion of Efl1 from the resulting nucleolar deficit of Tif6. Efl1 inhibits the EF-2 GTPase activity, suggesting that the two proteins share a similar ribosome-binding site. The 80 S ribosomes from either type failed to show any difference of conformation in the two rRNA domains analyzed. However, the same analysis performed on the pool of free 60 S subunits reveals several rRNA conformational differences between wild-type and Deltaefl1 subunits, whereas that from the suppressor strain is similar to wild-type. This suggests that the nucleolar deficit of Tif6 during assembly of the 60 S preribosomes is responsible for the changes in rRNA conformation observed in Deltaefl1 60 S subunits. We also purified 60 S preribosomes from the three genetic contexts by TAP-tagging Tif6. The protein content of 60 S preribosomes associated with Tif6p in a Deltaefl1 strain are obtained at a lower yield but have, surprisingly, a protein composition that is a priori similar to that of wild-type and the suppressor strain.     

NMR structure of bacterial ribosomal protein l20: implications for ribosome assembly and translational control

L20 is a specific protein of the bacterial ribosome, which is involved in the early assembly steps of the 50S subunit and in the feedback control of the expression of its own gene. This dual function involves specific interactions with either the 23S rRNA or its messenger RNA. The solution structure of the free Aquifex aeolicus L20 has been solved. It is composed of an unstructured N-terminal domain comprising residues 1-58 and a C-terminal alpha-helical domain. This is in contrast with what is observed in the bacterial 50S subunit, where the N-terminal region folds as an elongated alpha-helical region. The solution structure of the C-terminal domain shows that several solvent-accessible, conserved residues are clustered on the surface of the molecule and are probably involved in RNA recognition. In vivo studies show that this domain is sufficient to repress the expression of the cistrons encoding L35 and L20 in the IF3 operon. The ability of L20 C-terminal domain to specifically recognise RNA suggests an assembly mechanism for L20 into the ribosome. The pre-folded C-terminal domain would make a primary interaction with a specific site on the 23S rRNA. The N-terminal domain would then fold within the ribosome, participating in its correct 3D assembly.     

Loc1p is required for efficient assembly and nuclear export of the 60S ribosomal subunit

Loc1p is an exclusively nuclear dsRNA-binding protein that affects the asymmetric sorting of ASH1 mRNA to daughter cells in Saccharomyces cerevisiae. In addition to the role in cytoplasmic RNA localization, Loc1p is a constituent of pre-60S ribosomes. Cells devoid of Loc1p display a defect in the synthesis of 60S ribosomal subunits, resulting in “half-mer” polyribosomes. Previously, we reported that Loc1p is located throughout the entire nucleus; however, upon closer inspection we discovered that Loc1p is enriched in the nucleolus consistent with a role in 60S ribosome biogenesis. Given that Loc1p is an RNA-binding protein and presumably functions in the assembly of 60S ribosomal subunits, we investigated if Loc1p has a role in rRNA processing and nuclear export of 60S subunits. Analysis of pre-rRNA processing revealed that loc1Δ cells exhibit gross defects in 25S rRNA synthesis, specifically a delay in processing at sites A0, A1 and A2 in 35S pre-rRNA. Furthermore, loc1Δ cells exhibit nuclear export defects for 60S ribosomal subunits, again, consistent with a role for Loc1p in the assembly of 60S ribosomal subunits. It is attractive to hypothesize that the two phenotypes associated with loc1Δ cells, namely altered ASH1 mRNA localization and ribosome biogenesis, are not mutually exclusive, but that ribosome biogenesis directly impacts mRNA localization.     

A cluster of ribosome synthesis factors regulate pre-rRNA folding and 5.8S rRNA maturation by the Rat1 exonuclease

The 5'-exonuclease Rat1 degrades pre-rRNA spacer fragments and processes the 5'-ends of the 5.8S and 25S rRNAs. UV crosslinking revealed multiple Rat1-binding sites across the pre-rRNA, consistent with its known functions. The major 5.8S 5'-end is generated by Rat1 digestion of the internal transcribed spacer 1 (ITS1) spacer from cleavage site A(3). Processing from A(3) requires the 'A(3)-cluster' proteins, including Cic1, Erb1, Nop7, Nop12 and Nop15, which show interdependent pre-rRNA binding. Surprisingly, A(3)-cluster factors were not crosslinked close to site A(3), but bound sites around the 5.8S 3'- and 25S 5'-regions, which are base paired in mature ribosomes, and in the ITS2 spacer that separates these rRNAs. In contrast, Nop4, a protein required for endonucleolytic cleavage in ITS1, binds the pre-rRNA near the 5'-end of 5.8S. ITS2 was reported to undergo structural remodelling. In vivo chemical probing indicates that A(3)-cluster binding is required for this reorganization, potentially regulating the timing of processing. We predict that Nop4 and the A(3) cluster establish long-range interactions between the 5.8S and 25S rRNAs, which are subsequently maintained by ribosomal protein binding.     

Involvement of the specific nucleolar protein SURF6 in regulation of proliferation and ribosome biogenesis in mouse NIH/3T3 fibroblasts

The nucleolar proteins which link cell proliferation to ribosome biogenesis are regarded to be potentially oncogenic. Here, in order to examine the involvement of an evolutionary conserved nucleolar protein SURF6/Rrp14 in proliferation and ribosome biogenesis in mammalian cells, we established stably transfected mouse NIH/3T3 fibroblasts capable of conditional overexpression of the protein. Cell proliferation was monitored in real-time, and various cell cycle parameters were quantified based on flow cytometry, Br-dU-labeling and conventional microscopy data. We show that overexpression of SURF6 accelerates cell proliferation and promotes transition through all cell cycle phases. The most prominent SURF6 pro-proliferative effects include a significant reduction of the population doubling time, from 19.8 ± 0.7 to 16.2 ± 0.5 hours (t-test, p < 0.001), and of the length of cell division cycle, from 17.6 ± 0.6 to 14.0 ± 0.4 hours (t-test, p < 0.001). The later was due to the shortening of all cell cycle phases but the length of G1 period was reduced most, from 5.7 ± 0.4 to 3.8 ± 0.3 hours, or by ～30%, (t-test, p < 0.05). By Northern blots and qRT-PCR, we further showed that the acceleration of cell proliferation was concomitant with an accumulation of rRNA species along both ribosomal subunit maturation pathways. It is evident, therefore, that like the yeast homologue Rrp14, mammalian SURF6 is involved in various steps of rRNA processing during ribosome biogenesis. We concluded that SURF6 is a novel positive regulator of proliferation and G1/S transition in mammals, implicating that SURF6 is a potential oncogenic protein, which can be further studied as a putative target in anti-cancer therapy.     

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

Studies of the regulation of nucleolar function are critical for ascertaining clearer insights into the basic biological underpinnings of ribosome biogenesis (RB), and for future development of therapeutics to treat cancer and ribosomopathies. A number of high-throughput primary assays based on morphological alterations of the nucleolus can indirectly identify hits affecting RB. However, there is a need for a more direct high-throughput assay for a nucleolar function to further evaluate hits. Previous reports have monitored nucleolar rRNA biogenesis using 5-ethynyl uridine (5-EU) in low-throughput. We report a miniaturized, high-throughput 5-EU assay that enables specific calculation of nucleolar rRNA biogenesis inhibition, based on co-staining of the nucleolar protein fibrillarin (FBL). The assay uses two siRNA controls: a negative non-targeting siRNA control and a positive siRNA control targeting RNA Polymerase 1 (RNAP1; POLR1A), and specifically quantifies median 5-EU signal within nucleoli. Maximum nuclear 5-EU signal can also be used to monitor the effects of putative small-molecule inhibitors of RNAP1, like BMH-21, or other treatment conditions that cause FBL dispersion. We validate the 5-EU assay on 68 predominately nucleolar hits from a high-throughput primary screen, showing that 58/68 hits significantly inhibit nucleolar rRNA biogenesis. Our new method establishes direct quantification of nucleolar function in high-throughput, facilitating closer study of RB in health and disease.     

Understanding ribosome assembly: the structure of in vivo assembled immature 30S subunits revealed by cryo-electron microscopy

Four decades after early in vitro assembly studies demonstrated that ribosome assembly is a controlled process, our understanding of ribosome assembly is still incomplete. Just as structure determination has been so important to understanding ribosome function, so too will it be critical to sorting out the assembly process. Here, we used a viable deletion in the yjeQ gene, a recognized ribosome assembly factor, to isolate and structurally characterize immature 30S subunits assembled in vivo. These small ribosome subunits contained unprocessed 17S rRNA and lacked some late ribosomal proteins. Cryo-electron microscopy reconstructions revealed that the presence of precursor sequences in the rRNA induces a severe distortion in the 3' minor domain of the subunit involved in the decoding of mRNA and interaction with the large ribosome subunit. These findings suggest that rRNA processing events induce key local conformational changes directing the structure toward the mature assembly. We concluded that rRNA processing, folding, and the entry of tertiary r-proteins are interdependent events in the late stages of 30S subunit assembly. In addition, we demonstrate how studies of emerging assembly factors in ribosome biogenesis can help to elucidate the path of subunit assembly in vivo.     

Assembly of Saccharomyces cerevisiae 60S ribosomal subunits: role of factors required for 27S pre-rRNA processing

The precise functions of most of the ～200 assembly factors and 79 ribosomal proteins required to construct yeast ribosomes in vivo remain largely unexplored. To better understand the roles of these proteins and the mechanisms driving ribosome biogenesis, we examined in detail one step in 60S ribosomal subunit assembly-processing of 27SA(3) pre-rRNA. Six of seven assembly factors required for this step (A(3) factors) are mutually interdependent for association with preribosomes. These A(3) factors are required to recruit Rrp17, one of three exonucleases required for this processing step. In the absence of A(3) factors, four ribosomal proteins adjacent to each other, rpL17, rpL26, rpL35, and rpL37, fail to assemble, and preribosomes are turned over by Rat1. We conclude that formation of a neighbourhood in preribosomes containing the A(3) factors establishes and maintains stability of functional preribosomes containing 27S pre-rRNAs. In the absence of these assembly factors, at least one exonuclease can switch from processing to turnover of pre-rRNA.