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Review
. 2021 Sep;46(9):731-743.
doi: 10.1016/j.tibs.2021.03.008. Epub 2021 May 6.

Detecting and Rescuing Stalled Ribosomes

Matthew C J Yip  1 , Sichen Shao  2
Affiliations
Review

Detecting and Rescuing Stalled Ribosomes

Matthew C J Yip et al. Trends Biochem Sci. 2021 Sep.

Abstract

Ribosomes that stall inappropriately during protein synthesis harbor proteotoxic components linked to cellular stress and neurodegenerative diseases. Molecular mechanisms that rescue stalled ribosomes must selectively detect rare aberrant translational complexes and process the heterogeneous components. Ribosome-associated quality control pathways eliminate problematic messenger RNAs and nascent proteins on stalled translational complexes. In addition, recent studies have uncovered general principles of stall recognition upstream of quality control pathways and fail-safe mechanisms that ensure nascent proteome integrity. Here, we discuss developments in our mechanistic understanding of the detection and rescue of stalled ribosomal complexes in eukaryotes.

Keywords: ribosome collisions; ribosome stalling; ribosome-associated quality control (RQC).

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Conflict of interest statement

Declaration of Interests The authors have no interests to declare.

Figures

Figure 1 |
Figure 1 |. Progress on ribosome rescue in eukaryotes.
Recent work has revealed (1) insights into how ribosomes stall while translating specific mRNA sequences (red). The aminoacyl (A), peptidyl (P), and exit (E) tRNA binding sites, decoding center, and peptidyl-transferase center (PTC) on an 80S ribosome are indicated. (2) Ribosome rescue requires factors that specifically detect molecular signatures of ribosome stalling, such as an empty decoding center or ribosome collisions. Ribosome rescue factors activate (3a) local quality control and recycling mechanisms to process individual components of stalled ribosomal complexes and (3b) cellular stress responses to reduce proteotoxic burden.
Figure 2 |
Figure 2 |. Coincidence detection leads to ribosome stalling.
A,In vitro translation of reporters containing 10 lysine residues encoded by different combinations of AAA and AAG codons demonstrate that the simultaneous presence of lysines in the ribosomal exit tunnel and poly(A) mRNA in the decoding center is required to stall mammalian ribosomes [19]. Specifically, ribosomes slow but do not terminally stall when lysines are present in the exit tunnel and AAG codons are in the decoding center (far left; indicated by single !), but strong stalling is detected when lysines occupy the exit tunnel and AAA codons are in the decoding center (far right; indicated by two !). (AAG)10 and (AAA)10 refer to 10 AAG or AAA codons, respectively; (AAA)7(AAG)3 refers to 7 AAA codons followed by 3 AAG codons; (AAG)7(AAA)3 refers to 7 AAG codons followed by 3 AAA codons. B, The coincidence detection model for ribosome stalling suggests that 1) nascent peptide interactions with the ribosomal exit tunnel lead to nonoptimal conformations at the peptidyl transferase center (PTC) that slow translation and allow 2) specific mRNA sequences to adopt intrinsic structures that remodel the decoding center at the A-site of the ribosome, resulting in translational arrest.
Figure 3 |
Figure 3 |. Detecting stalled ribosomes.
A, Two unique molecular signatures of stalled ribosomes are an empty decoding center (top) and ribosome collision interfaces (bottom). Each are recognized by specific ribosome rescue factors that activate molecular pathways leading to dissociation of the stalled ribosome into 60S and 40S ribosomal subunits and activation of ribosome-associated quality control (RQC) pathways. Ribosome collisions also activate cellular pathways that inhibit translation in cis and stress response pathways that downregulate translation (integrated stress response) and regulate cellular survival (ribotoxic stress response). (top) The Hbs1L-Pelota complex engages ribosomes with an empty decoding center, leading to ribosomal subunit dissociation via the ribosome recycling factor ABCE1. (bottom) EDF1 selectively binds collided ribosomes, possibly stabilizing the collision interface to recruit downstream factors. Collision-dependent ribosome rescue factors include the E3 ubiquitin ligase ZNF598, which ubiquitylates small ribosomal subunit proteins leading to ribosomal subunit dissociation by the RQT complex, the translation initiation repressor GIGYF2, the MAP3 kinase ZAKα, and the GCN2 co-activator GCN1. B, Ribosome-protected fragments (RPFs) after nuclease treatment distinguish different ribosomal populations. RPF peaks at 21 and 28 nucleotides correspond to translating ribosomes that lack or contain A-site tRNA respectively (blue) [27]. Ribosomes lacking A-site mRNA protect ~16 nucleotides (teal) [32], and collided ribosomes protect ~60 nucleotides (red) [–43]. C, Effects of translation elongation inhibitor concentrations on ribosome collisions. Treating cells with a high concentration of an elongation inhibitor ‘freezes’ translating ribosomes, preventing collisions. A low concentration of an elongation inhibitor stalls only a subset of ribosomes, resulting in collisions with uninhibited trailing ribosomes [–40].
Figure 4 |
Figure 4 |. Quality control and recycling of peptidyl-tRNAs on stalled ribosomes.
A, Ribosomal subunit dissociation downstream of stall recognition by Hbs1L-Pelota or ZNF598 traps the peptidyl-tRNA on the 60S ribosomal subunit. NEMF (yeast Rqc2) binds the exposed 60S-tRNA interface, which prevents 40S rejoining and facilitates recruitment of the E3 ubiquitin ligase Listerin (yeast Ltn1) to polyubiquitinate the nascent chain. The polyubiquitinated nascent chain may be released by the endonuclease ANKZF1 (yeast Vms1) for proteasomal degradation via a process involving the AAA+ ATPase p97 (yeast Cdc48) and its ubiquitin binding cofactors. B, ANKZF1 cleaves the peptidyl-tRNA just before the invariant 3’CCA nucleotides, generating a CCA-less tRNA intermediate with a terminal 2’,3’-cyclic phosphate (2’,3’>p) that is recycled in a two-step pathway involving the removal of the 2’,3’>p by the phosphatases ELAC1 or ANGEL2, followed by re-addition of the 3’-CCA by TRNT1. TRNT1 also possesses proofreading capabilities that can target defective tRNAs for degradation (QC). C, Rqc2 can catalyze the non-templated addition of C-terminal alanine and possibly threonine residues (CAT tails). CAT tails can push out lysines within the ribosome exit tunnel for ubiquitylation by Listerin. Alternatively, CAT-tailed nascent chains may be released from 60S ribosomal subunits. Off the ribosome, CAT tails may serve as degrons leading to proteasomal degradation, or are otherwise prone to aggregation, which may titrate away chaperones and, in yeast, activate the heat shock response via HSF1.
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