The kinase Rio1 and a ribosome collision-dependent decay pathway survey the integrity of 18S rRNA cleavage.

The 18S rRNA sequence is highly conserved, particularly at its 3'-end, which is formed by the endonuclease Nob1. How Nob1 identifies its target sequence is not known, and in vitro experiments have shown Nob1 to be error-prone. Moreover, the sequence around the 3'-end is degenerate with similar sites nearby. Here, we used yeast genetics, biochemistry, and next-generation sequencing to investigate a role for the ATPase Rio1 in monitoring the accuracy of the 18S rRNA 3'-end. We demonstrate that Nob1 can miscleave its rRNA substrate and that miscleaved rRNA accumulates upon bypassing the Rio1-mediated quality control (QC) step, but not in healthy cells with intact QC mechanisms. Mechanistically, we show that Rio1 binding to miscleaved rRNA is weaker than its binding to accurately processed 18S rRNA. Accordingly, excess Rio1 results in accumulation of miscleaved rRNA. Ribosomes containing miscleaved rRNA can translate, albeit more slowly, thereby inviting collisions with trailing ribosomes. These collisions result in degradation of the defective ribosomes utilizing parts of the machinery for mRNA QC. Altogether, the data support a model in which Rio1 inspects the 3'-end of the nascent 18S rRNA to prevent miscleaved 18S rRNA-containing ribosomes from erroneously engaging in translation, where they induce ribosome collisions. The data also demonstrate how ribosome collisions purify cells of altered ribosomes with different functionalities, with important implications for the concept of ribosome heterogeneity.

Ribosome Assembly and Repair.

Ribosomes synthesize protein in all cells. Maintaining both the correct number and composition of ribosomes is critical for protein homeostasis. To address this challenge, cells have evolved intricate quality control mechanisms during assembly to ensure that only correctly matured ribosomes are released into the translating pool. However, these assembly-associated quality control mechanisms do not deal with damage that arises during the ribosomes' exceptionally long lifetimes and might equally compromise their function or lead to reduced ribosome numbers. Recent research has revealed that ribosomes with damaged ribosomal proteins can be repaired by the release of the damaged protein, thereby ensuring ribosome integrity at a fraction of the energetic cost of producing new ribosomes, appropriate for stress conditions. In this article, we cover the types of ribosome damage known so far, and then we review the known repair mechanisms before surveying the literature for possible additional instances of repair.

A disease associated mutant reveals how Ltv1 orchestrates RP assembly and rRNA folding of the small ribosomal subunit head.

Ribosomes are complex macromolecules assembled from 4 rRNAs and 79 ribosomal proteins (RPs). Their assembly is organized in a highly hierarchical manner, which is thought to avoid dead-end pathways, thereby enabling efficient assembly of ribosomes in the large quantities needed for healthy cellular growth. Moreover, hierarchical assembly also can help ensure that each RP is included in the mature ribosome. Nonetheless, how this hierarchy is achieved remains unknown, beyond the examples that depend on direct RP-RP interactions, which account for only a fraction of the observed dependencies. Using assembly of the small subunit head and a disease-associated mutation in the assembly factor Ltv1 as a model system, we dissect here how the hierarchy in RP binding is constructed. A combination of data from yeast genetics, mass spectrometry, DMS probing and biochemical experiments demonstrate that the LIPHAK-disease-associated Ltv1 mutation leads to global defects in head assembly, which are explained by direct binding of Ltv1 to 5 out of 15 RPs, and indirect effects that affect 4 additional RPs. These indirect effects are mediated by conformational transitions in the nascent subunit that are regulated by Ltv1. Mechanistically, Ltv1 aids the recruitment of some RPs via direct protein-protein interactions, but surprisingly also delays the recruitment of other RPs. Delayed binding of key RPs also delays the acquisition of RNA structure that is stabilized by these proteins. Finally, our data also indicate direct roles for Ltv1 in chaperoning the folding of a key rRNA structural element, the three-helix junction j34-35-38. Thus, Ltv1 plays critical roles in organizing the order of both RP binding to rRNA and rRNA folding, thereby enabling efficient 40S subunit assembly.

A disease associated mutant reveals how Ltv1 orchestrates RP assembly and rRNA folding of the small ribosomal subunit head.

Ribosomes are complex macromolecules assembled from 4 rRNAs and 79 ribosomal proteins (RPs). Their assembly is organized in a highly hierarchical manner, which is thought to avoid dead-end pathways, thereby enabling efficient assembly of ribosomes in the large quantities needed for healthy cellular growth. Moreover, hierarchical assembly also can help ensure that each RP is included in the mature ribosome. Nonetheless, how this hierarchy is achieved remains unknown, beyond the examples that depend on direct RP-RP interactions, which account for only a fraction of the observed dependencies. Using assembly of the small subunit head and a disease-associated mutation in the assembly factor Ltv1 as a model system, we dissect here how the hierarchy in RP binding is constructed. Our data demonstrate that the LIPHAK-disease-associated Ltv1 mutation leads to global defects in head assembly, which are explained by direct binding of Ltv1 to 5 out of 15 RPs, and indirect effects that affect 4 additional RPs. These indirect effects are mediated by conformational transitions in the nascent subunit that are regulated by Ltv1. Mechanistically, Ltv1 aids the recruitment of some RPs via direct protein-protein interactions, but surprisingly also delays the recruitment of other RPs. Delayed binding of key RPs also delays the acquisition of RNA structure that is stabilized by these proteins. Finally, our data also indicate direct roles for Ltv1 in chaperoning the folding of a key rRNA structural element, the three-helix junction j34-35-38. Thus, Ltv1 plays critical roles in organizing the order of both RP binding to rRNA and rRNA folding, thereby enabling efficient 40S subunit assembly.

Chaperone-directed ribosome repair after oxidative damage.

Because of the central role ribosomes play for protein translation and ribosome-mediated mRNA and protein quality control (RQC), the ribosome pool is surveyed and dysfunctional ribosomes degraded both during assembly, as well as the functional cycle. Oxidative stress downregulates translation and damages mRNAs and ribosomal proteins (RPs). Although damaged mRNAs are detected and degraded via RQC, how cells mitigate damage to RPs is not known. Here, we show that cysteines in Rps26 and Rpl10 are readily oxidized, rendering the proteins non-functional. Oxidized Rps26 and Rpl10 are released from ribosomes by their chaperones, Tsr2 and Sqt1, and the damaged ribosomes are subsequently repaired with newly made proteins. Ablation of this pathway impairs growth, which is exacerbated under oxidative stress. These findings reveal an unanticipated mechanism for chaperone-mediated ribosome repair, augment our understanding of ribosome quality control, and explain previous observations of protein exchange in ribosomes from dendrites, with broad implications for aging and health.

Quality control ensures fidelity in ribosome assembly and cellular health.

The coordinated integration of ribosomal RNA and protein into two functional ribosomal subunits is safeguarded by quality control checkpoints that ensure ribosomes are correctly assembled and functional before they engage in translation. Quality control is critical in maintaining the integrity of ribosomes and necessary to support healthy cell growth and prevent diseases associated with mistakes in ribosome assembly. Its importance is demonstrated by the finding that bypassing quality control leads to misassembled, malfunctioning ribosomes with altered translation fidelity, which change gene expression and disrupt protein homeostasis. In this review, we outline our understanding of quality control within ribosome synthesis and how failure to enforce quality control contributes to human disease. We first provide a definition of quality control to guide our investigation, briefly present the main assembly steps, and then examine stages of assembly that test ribosome function, establish a pass-fail system to evaluate these functions, and contribute to altered ribosome performance when bypassed, and are thus considered "quality control."

Attacking a DEAD problem: The role of DEAD-box ATPases in ribosome assembly and beyond.

DEAD-box proteins are a subfamily of ATPases with similarity to RecA-type helicases that are involved in all aspects of RNA Biology. Despite their potential to regulate these processes via their RNA-dependent ATPase activity, their roles remain poorly characterized. Here I describe a roadmap to study these proteins in the context of ribosome assembly, the process that utilizes more than half of all DEAD-box proteins encoded in the yeast genome.

The promises and pitfalls of specialized ribosomes.

The concept of specialized ribosomes has garnered equal amounts of interest and skepticism since it was first introduced. We ask researchers in the field to provide their perspective on the topic and weigh in on the evidence (or lack thereof) and what the future may bring.

Using DMS-MaPseq to uncover the roles of DEAD-box proteins in ribosome assembly.

DMS (dimethylsulfate) is a time-tested chemical probe for nucleic acid secondary structure that has recently re-emerged as a powerful tool to study RNA structure and structural changes, by coupling it to high throughput sequencing techniques. This variant, termed DMS-MaPseq, allows for mapping of all RNAs in a cell at the same time. However, if an RNA adopts different structures, for example during the assembly of an RNA-protein complex, or as part of its functional cycle, then DMS-MaPseq cannot differentiate between these structures, and an ensemble average will be produced. This is especially challenging for long-lived RNAs, such as ribosomes, whose steady-state abundance far exceeds that of any assembly intermediates, rendering those inaccessible to DMS-MaPseq on total RNAs. These challenges can be overcome by purification of assembly intermediates stalled at specific assembly steps (or steps in the functional cycle), via a combination of affinity tags and mutants stalled at defined steps, and subsequent DMS probing of these intermediates. Interpretation of the differences in DMS accessibility is facilitated by additional structural information, e.g. from cryo-EM experiments, available for many functional RNAs. While this approach is generally useful for studying RNA folding or conformational changes within RNA-protein complexes, it can be particularly valuable for studying the role(s) of DEAD-box proteins, as these tend to lead to larger conformational rearrangements, often resulting from the release of an RNA-binding protein from a bound RNA. Here we provide an adaptation of the DMS-MaPseq protocol to study RNA conformational transitions during ribosome assembly, which addresses the challenges arising from the presence of many assembly intermediates, all at concentrations far below that of mature ribosomes. While this protocol was developed for the yeast S. cerevisiae, we anticipate that it should be readily transferable to other model organisms for which affinity purification has been established.

The chaperone Tsr2 regulates Rps26 release and reincorporation from mature ribosomes to enable a reversible, ribosome-mediated response to stress.

Although ribosome assembly is quality controlled to maintain protein homeostasis, different ribosome populations have been described. How these form, especially under stress conditions that affect energy levels and stop the energy-intensive production of ribosomes, remains unknown. Here, we demonstrate how a physiologically relevant ribosome population arises during high Na+, sorbitol, or pH stress via dissociation of Rps26 from fully assembled ribosomes to enable a translational response to these stresses. The chaperone Tsr2 releases Rps26 in the presence of high Na+ or pH in vitro and is required for Rps26 release in vivo. Moreover, Tsr2 stores free Rps26 and promotes reincorporation of the protein, thereby repairing the subunit after the Na+ stress subsides. Our data implicate a residue in Rps26 involved in Diamond Blackfan Anemia in mediating the effects of Na+. These data demonstrate how different ribosome populations can arise rapidly, without major energy input and without bypass of quality control mechanisms.

The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase binding in 40S ribosome assembly.

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AFs) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate "handover" from one highly related AF to another, remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the binding of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1 into its functional site. Inactive Tsr3 blocks Rio1 function, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

Assembly factors chaperone ribosomal RNA folding by isolating helical junctions that are prone to misfolding.

While RNAs are known to misfold, the underlying molecular causes have been mainly studied in fragments of biologically relevant larger RNAs. As these small RNAs are dominated by secondary structures, misfolding of these secondary structures remains the most-explored cause for global RNA misfolding. Conversely, how RNA chaperones function in a biological context to promote native folding beyond duplex annealing remains unknown. Here, in a combination of dimethylsulfate mutational profiling with sequencing (DMS-MaPseq), structural analyses, biochemical experiments, and yeast genetics, we show that three-helix junctions are prone to misfolding during assembly of the small ribosomal subunit in vivo. We identify ubiquitous roles for ribosome assembly factors in chaperoning their folding by preventing the formation of premature tertiary interactions, which otherwise kinetically trap misfolded junctions, thereby blocking further progress in the assembly cascade. While these protein chaperones act indirectly by binding the interaction partners of junctions, our analyses also suggest direct roles for small nucleolar RNAs (snoRNAs) in binding and chaperoning helical junctions during transcription. While these assembly factors do not utilize energy to ameliorate misfolding, our data demonstrate how their dissociation renders reversible folding steps irreversible, thereby driving native folding and assembly and setting up a timer that dictates the propensity of misfolded intermediates to escape quality control. Finally, the data demonstrate that RNA chaperones act locally on individual tertiary interactions, in contrast to protein chaperones, which globally unfold misfolded proteins.

An open interface in the pre-80S ribosome coordinated by ribosome assembly factors Tsr1 and Dim1 enables temporal regulation of Fap7.

During their maturation, nascent 40S subunits enter a translation-like quality control cycle, where they are joined by mature 60S subunits to form 80S-like ribosomes. While these assembly intermediates are essential for maturation and quality control, how they form, and how their structure promotes quality control, remains unknown. To address these questions, we determined the structure of an 80S-like ribosome assembly intermediate to an overall resolution of 3.4 Å. The structure, validated by biochemical data, resolves a large body of previously paradoxical data and illustrates how assembly and translation factors cooperate to promote the formation of an interface that lacks many mature subunit contacts but is stabilized by the universally conserved methyltransferase Dim1. We also show how Tsr1 enables this interface by blocking the canonical binding of eIF5B to 40S subunits, while maintaining its binding to 60S. The structure also shows how this interface leads to unfolding of the platform, which allows for temporal regulation of the ATPase Fap7, thus linking 40S maturation to quality control during ribosome assembly.

Quality control of 40S ribosome head assembly ensures scanning competence.

During translation initiation, 40S ribosomes scan the mRNA until they encounter the start codon, where conformational changes produce a translation-competent 80S complex. Destabilizing the scanning complex results in misinitiation at non-AUG codons, demonstrating its importance for fidelity. Here, we use a combination of biochemical and genetic analyses to demonstrate that the ability of the nascent subunit to adopt the scanning complex is tested during assembly via structural mimicry. Specifically, formation of the 80S-like assembly intermediate, which structurally resembles scanning complexes, requires the correct folding of two rRNA elements in the subunit head and the proper positioning of the universally conserved head proteins Rps3, Rps15, Rps20, and Rps29. rRNA misfolding impairs the formation of 80S-like ribosomes, and bypass of individual checkpoints using cancer-associated mutations produces ribosomes defective in accurate start-site selection. Thus, the formation of 80S-like assembly intermediates is a quality control step that ensures scanning competence of the nascent subunit.

Correction: A kinase-dependent checkpoint prevents escape of immature ribosomes into the translating pool.

[This corrects the article DOI: 10.1371/journal.pbio.3000329.].

A kinase-dependent checkpoint prevents escape of immature ribosomes into the translating pool.

Premature release of nascent ribosomes into the translating pool must be prevented because these do not support viability and may be prone to mistakes. Here, we show that the kinase Rio1, the nuclease Nob1, and its binding partner Pno1 cooperate to establish a checkpoint that prevents the escape of immature ribosomes into polysomes. Nob1 blocks mRNA recruitment, and rRNA cleavage is required for its dissociation from nascent 40S subunits, thereby setting up a checkpoint for maturation. Rio1 releases Nob1 and Pno1 from pre-40S ribosomes to discharge nascent 40S into the translating pool. Weak-binding Nob1 and Pno1 mutants can bypass the requirement for Rio1, and Pno1 mutants rescue cell viability. In these strains, immature ribosomes escape into the translating pool, where they cause fidelity defects and perturb protein homeostasis. Thus, the Rio1-Nob1-Pno1 network establishes a checkpoint that safeguards against the release of immature ribosomes into the translating pool.

Psp2, a novel regulator of autophagy that promotes autophagy-related protein translation.

Macroautophagy/autophagy defines an evolutionarily conserved catabolic process that targets cytoplasmic components for lysosomal degradation. The process of autophagy from initiation to closure is tightly executed and controlled by the concerted action of autophagy-related (Atg) proteins. Although substantial progress has been made in characterizing transcriptional and post-translational regulation of ATG/Atg genes/proteins, little is known about the translational control of autophagy. Here we report that Psp2, an RGG motif protein, positively regulates autophagy through promoting the translation of Atg1 and Atg13, two proteins that are crucial in the initiation of autophagy. During nitrogen starvation conditions, Psp2 interacts with the 5' UTR of ATG1 and ATG13 transcripts in an RGG motif-dependent manner and with eIF4E and eIF4G2, components of the translation initiation machinery, to regulate the translation of these transcripts. Deletion of the PSP2 gene leads to a decrease in the synthesis of Atg1 and Atg13, which correlates with reduced autophagy activity and cell survival. Furthermore, deactivation of the methyltransferase Hmt1 constitutes a molecular switch that regulates Psp2 arginine methylation status as well as its mRNA binding activity in response to starvation. These results reveal a novel mechanism by which Atg proteins become upregulated to fulfill the increased demands of autophagy activity as part of translational reprogramming during stress conditions, and help explain how ATG genes bypass the general block in protein translation that occurs during starvation.

Rrp5 establishes a checkpoint for 60S assembly during 40S maturation.

Even though the RNAs contained in the small (40S) and large (60S) ribosomal subunits are cotranscribed, their assembly proceeds largely separately, involving entirely distinct machineries. Nevertheless, separation of the two subunits, an event that is critical for assembly of the small subunit, is delayed until domain I of the large subunit is transcribed, indicating crosstalk between the two assembly pathways. Here we show that this crosstalk is mediated by the assembly factor Rrp5, one of only three proteins required for assembly of both ribosomal subunits. Quantitative RNA binding and cleavage data demonstrate that early on, Rrp5 blocks separation of the two subunits, and thus 40S maturation by inhibiting the access of Rcl1 to promote cleavage of the nascent rRNA. Upon transcription of domain I of 25S rRNA, the 60S assembly factors Noc1/Noc2 bind both this RNA and Rrp5 to change the Rrp5 RNA binding mode to enable pre-40S rRNA processing. Mutants in the HEAT-repeat domain of Noc1 are deficient in the separation of the subunits, which is rescued by overexpression of wild-type but not inactive Rcl1 in vivo. Thus, Rrp5 establishes a checkpoint for 60S assembly during 40S maturation to ensure balanced levels of the two subunits.

Does functional specialization of ribosomes really exist?

It has recently become clear that ribosomes are much more heterogeneous than previously thought, with diversity arising from rRNA sequence and modifications, ribosomal protein (RP) content and posttranslational modifications (PTMs), as well as bound nonribosomal proteins. In some cases, the existence of these diverse ribosome populations has been verified by biochemical or structural methods. Furthermore, knockout or knockdown of RPs can diversify ribosome populations, while also affecting the translation of some mRNAs (but not others) with biological consequences. However, the effects on translation arising from depletion of diverse proteins can be highly similar, suggesting that there may be a more general defect in ribosome function or stability, perhaps arising from reduced ribosome numbers. Consistently, overall reduced ribosome numbers can differentially affect subclasses of mRNAs, necessitating controls for specificity. Moreover, in order to study the functional consequences of ribosome diversity, perturbations including affinity tags and knockouts are introduced, which can also affect the outcome of the experiment. Here we review the available literature to carefully evaluate whether the published data support functional diversification, defined as diverse ribosome populations differentially affecting translation of distinct mRNA (classes). Based on these observations and the commonly observed cellular responses to perturbations in the system, we suggest a set of important controls to validate functional diversity, which should include gain-of-function assays and the demonstration of inducibility under physiological conditions.