Rescue of stalled ribosomes at the 3′ end of mRNAs in prokaryotes

In bacteria, truncated mRNAs are produced mainly via exonucleolytic degradation of the 3′-ends of mRNAs or endonucleolytic cleavage by RNases such as RNase III. Truncation of mRNAs within the coding region (ORF) results in stop codon loss and stalling of ribosomes at the 3′ end of the resulting nonstop mRNAs. Accumulation of stalled ribosomes is detrimental to cells, and recycling stalled ribosomes is essential for cell viability (14,15). Three ribosome rescue systems, trans-translation and alternative rescue factors A and B (ArfA and ArfB), are responsible for rescuing stalled ribosomes in prokaryotes (reviewed in (16)) (Figure ​(Figure1,1, Table ​Table1).1). In addition, the bacterium Francisella tularensis uses a fourth mechanism in which ArfT acts as an alternative ribosome rescue factor via a mechanism that is distinct from those of ArfA and ArfB (17).

Trans-translation as a quality control system for nonstop mRNAs

The trans-translation system, which is mediated by tmRNA and its binding partner small protein B (SmpB), recycles stalled ribosomes at the 3′-ends of truncated mRNAs. This is followed by degradation of both the incomplete mRNA and the protein. The tmRNA contains a domain similar to the acceptor arm of tRNA^Ala as well as an mRNA-like domain (14,15). The tmRNA–SmpB complex is delivered to an empty A-site by GTP-bound EF-Tu as a quaternary complex, although it does not decode the Ala codon in the mRNA. Translation elongation factor EF-G then translocates the tmRNA–SmpB complex through the ribosome in a manner similar to the translocation that occurs during canonical translation. The ribosome swaps messages, and the subsequent tmRNA-templated translation results in addition of a polypeptide tag to the nascent chain, which guarantees that the ribosome will terminate at a stop codon and be released. Even though trans-translation efficiently adds a degradation tag to the C-termini of truncated polypeptide chains, thereby marking them for degradation, the inactivation of trans-translation is not lethal. This discrepancy is due to the presence of back-up systems mediated by two alternative rescue factors, ArfA and ArfB. These factors recycle stalled ribosomes but allow synthesis of truncated protein variants encoded by stop-less mRNAs (Table ​(Table1).1). These observations indicate that the essential function of trans-translation is to recycle stalled ribosomes rather than to degrade aberrant products (18,19).

Biochemical and structural analyses have revealed the mechanism underlying the addition of the C-terminal degradation tag to aberrant nonstop products via trans-translation and how the tmRNA moves through the ribosome (reviewed in (16,20)) (Figure ​(Figure2).2). When the 70S ribosome stalls at the 3′-end of a nonstop mRNA, EF-Tu delivers Ala–tmRNA–SmpB to the ribosome, where the C-terminal tail of SmpB forms a α-helix in the downstream mRNA channel to form a pre-accommodation state. EF-Tu then leaves, and tmRNA–SmpB accommodates into the A-site in a conformation such that the SmpB tail remains α-helical. The tRNA^Ala-like domain of tmRNA–SmpB directs the alanine on its 3′-CCA into the peptidyl-transferase center (PTC) to form a peptide bond with the nascent peptide. EF-G then translocates tmRNA–SmpB from the A-site into the P-site to release the original mRNA and tRNA. During the translocation step, the SmpB tail flips to the opposite side of the mRNA channel, where it binds in the E-site. The mRNA-like domain then passes through the A-site latch and moves into the space in the mRNA channel. Ala-tRNA^Ala decodes the first codon of the tmRNA ORF, and a peptidyl-transferase reaction transfers the peptide from tmRNA to tRNA^Ala. EF-G then translocates the peptidyl-tRNA^Ala into the P-site, after which the mRNA-like domain is again loaded into the mRNA channel via a latch, this time in the E-site. Translation of the ORF continues until the tmRNA reaches a stop codon, and the peptide is then targeted for degradation by proteases that recognize the polypeptide tag. Together with EF-G, ribosome recycling factor recycles the 70S ribosome in a post-termination state. Therefore, it is likely that ribosome recycling factor and EF-G also dissociate the stalled ribosome into the 30S and 50S subunits and facilitate decay of the nonstop mRNA from the 3′-end. For rapid degradation of the nonstop mRNA, tmRNA–SmpB recruits the highly conserved 3′–5′ exoribonuclease ribonuclease R (RNase R), which is responsible for targeted degradation of nonstop mRNAs after rescue of the stalled ribosome (21,22) (Figure ​(Figure11).

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Figure 2.

Mechanism of trans-translation. During the first step of trans-translation, the 70S ribosome stalls at the 3′-end of a nonstop mRNA. In step 2, EF-Tu delivers Ala–tmRNA–SmpB to the ribosome, where the C-terminal tail of SmpB forms a α-helix in the downstream mRNA channel to form a pre-accommodation state. In step 3, EF-G translocates tmRNA–SmpB from the A-site into the P-site to release the original mRNA and tRNA. In step 4, the nonstop mRNA is switched for the ORF of the tmRNA and translation is resumed. In step 5, translation of the ORF of the tmRNA results in tagging of the aberrant polypeptide with a degradation tag that is recognized by proteases. In step 6, the ribosome is recycled by conventional translation factors.

Recycling of stalled ribosome complexes in the absence of trans-translation

The two additional rescue systems present in some bacteria, which employ either ArfA and release factor 2 (RF2) or ArfB, rescue stalled ribosomes on truncated mRNAs by hydrolyzing the peptidyl-tRNA (23). ArfA represents a back-up system for trans-translation and is required for cell survival in the absence of tmRNA. ArfA mediates ribosome rescue by recruiting the canonical termination factor RF2 to ribosomes stalled on truncated mRNAs. The C-terminal tail of ArfA probes the vacant mRNA channel and recruits RF2 to the nonstop ribosome to catalyze peptidyl-tRNA hydrolysis. ArfA cooperates with RF2, but does not interact with RF2 in solution (24,25). ArfA initially binds to the 30S subunit in the vicinity of the mRNA channel and recruits RF2 to nonstop ribosomes (25). Analyses of five cryo-electron microscopy (cryo-EM) structures of ArfA-RF2-stalled ribosome complexes revealed that ArfA interacts almost exclusively with the small subunit (16,26–28). In addition, tmRNA represses ArfA expression. A truncated arfA mRNA species is produced via endonucleolytic cleavage of a stem-loop structure in the 3′-end of the mRNA by RNase III (23,29,30). In the presence of tmRNA, the short ArfA protein produced from the truncated arfA mRNA is tagged by tmRNA and targeted for degradation. The full-length ArfA protein is 72 amino acids long and includes a C-terminal hydrophobic region that promotes aggregation of the protein in vivo (23). In the absence of tmRNA, the shorter form of ArfA can be expressed from the truncated arfA mRNA, and, as it lacks the terminal region, it is not degraded. Instead, the short ArfA protein product hydrolyzes the peptidyl-tRNA to rescue and recycle ribosomes stalled on truncated mRNAs (23,24,30,31).

ArfB also probes the vacant mRNA channel of nonstop ribosomes (32). ArfB overexpression can rescue cells lacking the tmRNA and ArfA rescue systems (32). In vivo, ArfB can efficiently catalyze the hydrolysis of peptidyl-tRNAs in ribosomes stalled at the 3′-ends of nonstop mRNAs (31–33). The N-terminal domain of ArfB contains the GGQ motif that catalyzes the hydrolysis of polypeptides from peptidyl-tRNAs. (16,33–36). Mutations in the ArfB GGQ motif impair the rescue activity of ArfB (32,33). Consistently, the N-terminal domain interacts with the large subunit such that the GGQ motif is positioned at the PTC (37). The C-terminal region of ArfB is crucial for ribosome binding and hydrolysis of the peptidyl-tRNA (32–34). The α-helix in the C-terminal region of ArfB binds in the mRNA entry tunnel downstream of the A-site (37), and this interaction distinguishes actively translating ribosomes from those stalled on truncated mRNAs. The N-terminal domain and C-terminal helix are connected by a 12-amino acid flexible linker that adopts an extended conformation on the ribosome. A deletion analysis of the ArfB linker suggested that it is important for positioning the N-terminal domain in the PTC of the ribosome (34).

Steps in RQC

RQC is a translation arrest-induced quality control system that leads to degradation of the arrest products via three steps (Figure ​(Figure3):3): recognition of stalled ribosomes, subunit dissociation, and degradation of aberrant nascent polypeptides. In the first step, abnormal stalled ribosomes are recognized and ubiquitinated at one or more specific residues. In yeast, the E3 ubiquitin ligase Hel2 ubiquitinates uS10 at K6/8 and plays a crucial role in RQC (55). Zinc finger protein 598 (ZNF598), a mammalian homolog of Hel2, ubiquitinates the eS10 and uS10 ribosomal proteins at K138/139 and K4/8, respectively, to stimulate translational arrest and RQC triggered by poly-lysine mRNA sequences (56–58) (Table ​(Table2).2). In the second step of RQC, ribosomal ubiquitination induces subunit dissociation, which is a crucial event for Listerin (S. cerevisiae Ltn1)-dependent ubiquitination of the arrest products in the 60S large ribosomal subunit (39–43,53). The RQC-trigger (RQT) complex is required for RQC and is proposed to recognize ubiquitinated stalled ribosomes and induce subunit dissociation in yeast. The RQT complex is composed of three factors: the RNA helicase-family protein Slh1/Rqt2, the ubiquitin-binding protein Cue3/Rqt3, and Rqt4 (Table ​(Table2)2) (55,59). The RQT complex is conserved in mammalian cells; ASCC3 is a homolog of Slh1 (55) and ASCC2 is likely to be a homolog of Cue3. The ATPase activity of Slh1 and the ubiquitin-binding activity of Cue3 are crucial for RQC (55). In the third step of RQC, dissociation of the 40S subunit allows binding of 60S ribosome-nascent chain (RNC) complexes to nuclear export mediator factor (NEMF; S. cerevisiae Rqc2), which then recruits the E3 ubiquitin ligase Listerin (S. cerevisiae Ltn1). Next, Listerin ubiquitinates the nascent chains, thus targeting them for degradation (52,53,60–63). The subsequent proteasomal degradation of a ubiquitinated nascent chain requires its prior release from the 60S subunit. Several lines of evidence indicate that ANKZF1 (Vms1) is responsible for the release of the ubiquitinated polypeptide from the tRNA in the 60S subunit (64–68).

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Figure 3.

Quality control systems induced by ribosome stalling. RQC has three steps. Step 1: The abnormal stalled ribosome is recognized and ubiquitinated at one or more specific residues. Step 2: Ribosome ubiquitination induces subunit dissociation, which is a crucial event for Listerin (S. cerevisiae Ltn1)-dependent ubiquitination of the arrest products on the 60S large ribosomal subunit. The RQT complex is thought to recognize the ubiquitinated stalled ribosome and induce subunit dissociation in yeast. Step 3: Dissociation of the 40S subunits allows binding of 60S RNCs to NEMF (Rqc2), which recruits the E3 ubiquitin ligase Listerin (Ltn1). Subsequently, Listerin ubiquitinates the nascent polypeptide chains, targeting them for degradation. Rqc2 attaches CAT-tails (C-terminal alanyl/threonyl sequences) to stalled polypeptides. The ATPase VCP/p97 (Cdc48) forms a complex with the cofactors UFD1 (Ufd1) and NPLOC4 (Npl4) and unfolds ubiquitinated polypeptides, then extracts the peptidyl-tRNA from the 60S, thereby recruiting it to the 26S proteasome for degradation. Novel roles of ANKZF1 (Vms1)-mediated polypeptide release for proteasomal degradation: Rqc2 catalyzes the C-terminal extension of the stalled tRNA-bound peptides with CAT-tails through a non-canonical elongation reaction without mRNA. CAT-tails functionalize the carboxy termini of stalled polypeptides to drive their degradation on and off the ribosome. Vms1 interacts with the ribosomal 60S subunit to compete with Rqc2 and catalyze peptidyl-tRNA cleavage. Subsequently, tRNA nucleotidyl transferase 1 (TRNT1) is responsible for recycling of ANKZF1-cleaved tRNA fragments.

Recognition of aberrant stalled ribosomes: ribosome ubiquitination

Translation of the poly(A) tails of nonstop mRNAs or poly(A) sequences within ORFs results in strong translation arrest and co-translational proteasomal degradation in yeast (69,70), and mammals (4,5,35,42,57,60,71–73). Lysine-encoding poly(A) stretches control translation and induce ribosomes to slide along the mRNA in various organisms (74–76). Specific basic amino acid sequences within ORFs also induce co-translational proteasomal degradation in yeast (70,77), and certain combinations of rare arginine-coding codons are very potent in promoting ribosomal stalling (78) and RQC (55).

An important question in RQC is how ZNF598 (S. cerevisiae Hel2) recognizes and ubiquitinates its specific substrate. The first observation regarding the substrate specificity of Hel2 in yeast was that ribosomes co-purified with tagged Hel2 were mainly in the rotated state with hybrid tRNAs (55). In yeast, translation of tandem rare codons results in formation of di-ribosome (disome) units that consist of the stalled leading ribosome and the following colliding ribosome. In in vitro mammalian translation systems, disomes are formed in the presence of a mutant form of eRF1 that is defective in peptide-releasing activity (73). A cryo-EM analysis of disomes revealed that the colliding ribosomes are positioned in a rotated form (73,79) and recent studies using an in vitro translation system demonstrate that disomes are preferred targets of ZNF598 (Hel2)-dependent ubiquitination (73,79). Recognition of disomes containing colliding ribosomes in the rotated state by ZNF598 (Hel2) (73,79) leads to ubiquitination of uS10 in yeast and uS10, eS10 and uS3 in mammals (Figure ​(Figure3,3, Table ​Table2).2). Subsequently, the RQT complex specifically targets the ubiquitinated ribosomes in the hybrid state to allow dissociation into their respective subunits (55).

The potential connection between RQC and translation initiation repression

ZNF598 and Grb10-interacting GYF protein 2 (GIGYF2) are components of the eIF4E homologous protein (4EHP) complex, which is thought to repress translation by competing with eIF4E for binding to the 5′-cap structure of specific mRNAs. Because 4EHP does not interact with eIF4G, it fails to initiate translation (80). GIGYF2 recruits decapping and deadenylation complexes to 4EHP-containing ribonucleoproteins to induce the translational repression and degradation of target mRNAs (81). In addition, 4EHP-GIGYF2 acts as a cofactor of the zinc finger protein tristetraprolin (TTP), which promotes translational repression and degradation of mRNAs containing AU-rich elements (82). Moreovre, ZNF598 interacts with GIGYF1 via three proline-rich motifs and regulates known TTP targets, thereby regulating the inflammatory potential of cytokine-producing cells (83). Finally, a recent study proposed that ZNF598 recognizes ribosome stalling and inhibits translation initiation by recruiting translational repressor GIGYF2 (84).

The role of the RQT complex in subunit dissociation

The RQT complex is proposed to recognize ubiquitinated stalled ribosomes and induce subunit dissociation (55,59) (Figure ​(Figure3,3, Table ​Table2).2). The ubiquitin-binding activity of Cue3 is crucial for RQC but is not required for the interaction between the RQT complex and ubiquitinated ribosomes. The ATPase activity of Slh1 is also crucial for RQC, and the proposed model is that the interaction between the RQT complex and K63-linked polyubiquitinated ribosomes and ATP hydrolysis by Slh1 play essential roles in subunit dissociation (55). Although the mechanism by which the RNA helicase activity of Slh1 induces subunit dissociation remains unknown, the structure of ubiquitinated ribosomes may be modulated by Slh1 in an RNA helicase activity-dependent manner. Biochemical evidence is needed to demonstrate that the RQT complex is responsible for the dissociation of stalled ribosomes into their subunits.

Unfolding machinery for polypeptide ubiquitination and degradation

Stalled ribosomes are ubiquitinated and dissociated into their subunits, yielding 60S RNCs (55,57,58,73,79). The ubiquitin ligase Listerin (S. cerevisiae Ltn1) plays a crucial role in RQC (53,85). A forward genetics approach using a mouse model showed that mutation of the gene encoding Listerin can lead to neurodegeneration (86). NEMF (S. cerevisiae Rqc2) associates with 60S RNCs and recruits Listerin, which ubiquitinates peptidyl-tRNAs on dissociated 60S subunits (53). Rqc2 attaches C-terminal alanyl/threonyl sequences (CAT-tails) to stalled polypeptides (63,87). The ATPase valosin-containing protein (VCP), also known as p97 (S. cerevisiae Cdc48), is the unfolding machinery for ubiquitinated polypeptides. VCP forms a complex with its cofactors UFD1 (S. cerevisiae Ufd1) and NPLOC4 (S. cerevisiae Npl4) to promote the extraction of peptidyl-tRNAs from 60S subunits, and these peptidyl-tRNAs are then recruited to the 26S proteasome for degradation (4,54).

Vms1 connects polypeptide release and proteasomal degradation

In RQC pathways, the mechanism by which tRNA-linked nascent polypeptides are released from stalled 60S subunits prior to proteasomal degradation has been a long-standing puzzle. Several lines of evidence have demonstrated that mammalian ANKZF1 (S. cerevisiae Vms1) releases ubiquitinated nascent proteins from 60S ribosomal subunits for proteasomal degradation (Figure ​(Figure3).3). Analysis of the crystal structure of Vms1 revealed that it is similar to eRF1 (65,88). Vms1 lacks the strict GGQ motif present in eRF1; however, it has a conserved glutamine (Q295) in a GxxQ context that can align with the catalytic glutamine in the eRF1 GGQ motif. The peptide release activity of Vms1 has been shown to depend on Q295 (64,65).

Pestova and co-workers reconstituted the entire mammalian RQC pathway in vitro, from ribosome stalling and recognition and processing of 60S complexes by the RQC ubiquitinating machinery, to ribosomal release of the ubiquitinated nascent polypeptides and their degradation by the 26S proteasome (68). In non-ubiquitinated nascent polypeptides on 60S subunits formed on nonstop mRNAs, the tRNAs are not firmly fixed in the P-site, which allows the peptidyl-tRNA hydrolase Ptrh1 to cleave the nascent peptidyl-tRNA in vitro. The release of non-ubiquitinated nascent polypeptides from the 60S subunit functions as an additional rescue pathway in cases where Listerin-dependent ubiquitination cannot be used. The 60S ribosome-nascent polypeptides are resistant to Ptrh1 due to their association with NEMF (Rqc2), and ubiquitination of nascent polypeptides results in the accommodation of nascent peptidyl-RNAs and susceptibility to ANKZF1. Subsequently, ANKZF1 induces specific cleavage in the tRNA acceptor arm to release ubiquitinated nascent polypeptides, which are then subject to proteasomal degradation. Shao and co-workers also investigated the fate of nascent polypeptide-tRNAs in 60S subunits using a eukaryotic cell-free system and found that ANKZF1 (Vms1) severs peptidyl-tRNAs in RQC complexes by precisely cleaving off the terminal 3′-CCA nucleotides, leaving a 2′,3′-cyclic phosphate (67). After removal of the cyclic phosphate, the sole CCA-adding enzyme tRNA nucleotidyl transferase 1 is responsible for recycling the ANKZF1-cleaved tRNA fragments, while also functioning in tRNA biogenesis in eukaryotes. Further studies may provide evidence whether the recycling of ANKZF1 (Vms1)-cleaved tRNAs is a crucial step to complete the quality control process without leaving behind any remnants. A more recent study by Beckmann and co-workers described the structural basis of the interaction between Vms1 and the ribosomal 60S subunit through which Vms1 competes with Rqc2 and catalyzes peptidyl-tRNA cleavage (66). Cryo-EM structures of native Vms1-60S ribosomal complexes revealed that Vms1 binds to the 60S subunit via the Vms1 release factor-like domain, the N-terminal zinc finger domain, and the ankyrin domain. The Vms1 release factor-like domain interacts with the ribosomal A-site and, like eRF1, projects its GSQ motif toward the PTC. This position overlaps with that observed for Rqc2 aminoacyl-tRNA (aa-tRNA), thus explaining the adversarial activity of Vms1. In the pre-hydrolysis state, the ABCF-type ATPase Arb1 accesses the ribosomal E-site, where it specifically interacts with the peptidyl-tRNA. Arb1 overexpression leads to partial suppression of the phenotypes resulting from Vms1 deletion, including the protein aggregation phenotype. Based on these observations, it is proposed that Arb1 plays a role in positioning peptidyl-tRNAs for cleavage by Vms1 and/or in dislocating peptidyl-tRNAs for tRNA extraction after cleavage (66).

Ribosome stalling often occurs during the translation of mitochondrially-targeted proteins. Nevertheless, mitochondrial polypeptides on stalled ribosomes are co-translationally imported into the mitochondria and are, therefore, inaccessible to ubiquitination due to the coupling of translation and translocation. Vms1 is a key component in a rescue pathway for ribosome-stalled mitochondrial polypeptides in yeast (89). Ltn1 and Vms1 protect cells against mitochondrial toxicity and maintain cell viability under respiratory conditions (89). These findings indicate that an ANKZF1 (Vms1)-mediated rescue pathway could be more crucial for maintaining protein homeostasis and ensuring cell survival. Under specific stress conditions, such as oxidative stress and rapamycin treatment, the rate of ribosome stalling can increase. Under these conditions, Vms1 accumulates on damaged mitochondria and recruits Cdc48 and Npl4 to facilitate proteasomal protein degradation of mitochondrial outer membrane proteins in yeast (90).

A dual role of Hel2 in two distinct NGD pathways

NGD and RQC require common factors and biochemical events, suggesting that these two quality control pathways are coupled (2,94). Ribosome profiling revealed that endonucleolytic cleavage occurs at sites of premature polyadenylation, and rescue of the ribosomes stalled at these sites is dependent on Dom34, suggesting that translation of poly(A) tails induces NGD as well as RQC (93). Both pathways are initiated by translation arrest and are dependent on 40S subunit-associated Asc1 in yeast (45,79,94,98). Moreover, Hel2-mediated K63-linked polyubiquitination is implicated in RQC after ribosome stalling at polybasic amino acid sequences and tandem CGA codons in yeast (99). Furthermore, ribosomal collisions trigger NGD-induced mRNA cleavage, and it is proposed that colliding ribosomes induce robust uS3 ubiquitination by Hel2 (95). Hel2-mediated uS10 ubiquitination is crucial for RQC; however, Hel2-mediated uS3 ubiquitination at lysine 212 is not essential for RQC or NGD (79); rather, it is involved in quality control for non-functional rRNA decay (100). Therefore, the role of Hel2-mediated uS3 ubiquitination remains to be elucidated.

As mentioned above, a disome unit consists of the leading stalled ribosome and the following colliding ribosome. In a recent study, endonucleolytic cleavage of an NGD-inducing reporter mRNA occurred at sites within disome units and depended on Hel2-mediated K63-linked polyubiquitination of uS10, demonstrating that NGD and RQC are coupled via this ubiquitination event (referred to as NGDRQC+) (79). Moreover, the ATPase activity of the RQT component Slh1 is required for cleavage within disomes, implying that subunit dissociation of stalled ribosomes is required for NGDRQC+. Further experiments revealed the existence of two interdependent branches of the NGD pathway: the RQC-coupled (NGDRQC+) and RQC-uncoupled (NGDRQC–) branches. In the NGDRQC– branch, endonucleolytic mRNA cleavage occurs upstream of stalled disomes when RQC is defective. Cleavage in the NGDRQC– branch requires K63-linked polyubiquitination of the eS7 ribosomal protein; this polyubiquitination also depends on Hel2 but is triggered by monoubiquitination by the E3 ligase Not4. Monoubiquitinated eS7 is a substrate for Hel2-mediated polyubiquitination, suggesting dual roles of Hel2 in two distinct NGD pathways, each of which requires specific ubiquitination events on stalled disomes.

Identification of Cue2 as a potential endonuclease responsible for NGD

In a recent study, Green and co-workers used constructs containing 12 tandem CGA rare codon repeats to screen for factors that reduced the mRNA level in a ribosome stalling-dependent manner, and identified the novel factor Cue2 in addition to the RQC factors Slh1 and Hel2 (97). Cue2 contains a ubiquitin-binding Cue domain at its N-terminus and an small MutS-related (SMR) hydrolase domain at its C-terminus. SMR domains show structural similarity to bacterial RNase E and function as DNA-nicking hydrolases (101). In addition, the SMR domain of SOT1 in plants exhibits RNA endonuclease activity (102). When aligned with the structure of the IF3-CTD on the ribosome, the conserved D348, H350 and R402 residues in the SMR domain of Cue2 are positioned along the mRNA channel. These residues are required for Cue2 function, and R402 has previously been implicated in the RNA cleavage activity of SOT1 in plants (102). Ribosome profiling and biochemical analyses have provided strong evidence that Cue2 cleaves mRNAs within the A-site of the colliding ribosome. The Cue2-dependent pathway becomes a major contributor to NGD in cells depleted of factors required for the resolution of stalled ribosome complexes. Further biochemical analyses will reveal the molecular mechanism by which Cue2 is recruited to the ubiquitinated ribosome and induces mRNA cleavage at the vicinity or inside the colliding ribosomes.

Ribosome ubiquitination plays a crucial role in 18S NRD

18S NRD is a quality control mechanism in which the base substitution mutant (A1492C) in the decoding center region of 18S rRNA constituting the 40S subunit is specifically degraded. The mechanisms involved in the 18S NRD pathway are still largely unknown; however, several lines of evidence suggest a connection between 18S NRD and RQC (Figure ​(Figure4).4). For example, Asc1 is involved in 18S NRD (109,110) and is also required for RQC (57,58,94). The C-terminal 212–240 region of uS3 is essential for the 18S NRD pathway (110). The 212th lysine residue (K212) of uS3 is a target of Hel2-dependent ubiquitination (55), and sequential ubiquitination of uS3 at K212 triggers subunit dissociation in the 18S NRD pathway (100). It has been proposed that non-functional 80S ribosomes that stall due to decoding failures are sequentially ubiquitinated at the K212 residue of uS3. Mag2 mediates monoubiquitination of uS3 at K212, followed by Hel2- or Rsp5-mediated polyubiquitination (100). Subsequently, Slh1 stimulates subunit dissociation to promote Xrn1-dependent degradation of the non-functional 18S rRNA in the 40S subunit (100). Asc1 is also involved in uS3 ubiquitination and is thus required for subunit dissociation (100), suggesting that Asc1 is a platform for E3 ubiquitin ligases to recognize the ribosome stalled at the specific structure. Given the role of Slh1 in subunit dissociation in RQC, the crucial roles of ribosome ubiquitination and Slh1 are conserved in quality control pathways induced by ribosome stalling, as depicted in Figure ​Figure44.

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Figure 4.

Ribosome ubiquitination as a trigger of quality control systems induced by ribosome stalling. Left: In RQC, the critical E3 ubiquitin ligase Hel2 and its mammalian homolog ZNF598 recognize disomes containing colliding ribosomes in the rotated state (73,79), leading to ubiquitination of uS10 in yeast and uS10, eS10, and uS3 in mammals. Right: In 18S NRD, non-functional 80S ribosomes containing the A1492C mutation in the decoding center stall due to decoding failures and are sequentially ubiquitinated at the K212 residue of uS3. Mag2 monoubiquitinates uS3 at K212, followed by Hel2-mediated polyubiquitination. Subsequently, the Ski2-like RNA helicase Slh1 in the RQT complex stimulates subunit dissociation to promote Xrn1-dependent degradation of the non-functional 18S rRNA in the 40S subunit (100). In these quality control systems, the ribosome is stalled at the specific conformation, and the specific E3 ligases recognize and ubiquitinate particular sites to induce the subsequent quality control steps.

These findings suggest that the ubiquitination of specific residues of ribosomal proteins is a universal molecular mechanism for determining the fate of ribosomes. Accurate gene expression is guaranteed by the quality control mechanism that recognizes and eliminates ribosomes that are abnormally stalled during translation elongation. Elucidation of the molecular mechanism of 18S NRD, the quality control mechanism of ribosomes lacking function, will form the molecular basis for understanding diseases caused by ribosome defects.