RESEARCH / RQC mechanisms and evolution
Ribosome-associated Quality Control: coming to the rescue of incomplete translation
Ribosome stalling during translation is prevalent and can occur for various reasons, such as due to mRNA damage. Among other undesirable consequences, ribosome stalling produces nascent chains that are incomplete and therefore potentially toxic, so they must be targeted for degradation.
We discovered Listerin/Ltn1 as the E3 ubiquitin ligase that marks aberrant proteins produced by ribosome stalling for degradation. Importantly, we found Listerin to be associated with the large ribosomal subunit, thereby unveiling a novel protein quality control pathway initiated on ribosomes—Ribosome-associated Quality Control (RQC) (Bengtson & Joazeiro 2010). This landmark finding set the RQC field in motion, allowing rapid progress in our understanding of RQC mechanisms.
Among other key contributions, we solved the first-ever structure of Listerin/Ltn1 in complex with the large (60S) ribosomal subunit and the RQC co-factor, Rqc2 (Lyumkis et al 2014). Notably, the structure shed light onto an apparent paradox that is central to cellular quality control in general: it provided a simple explanation for how Listerin can act selectively (in targeting aberrant nascent chains produced by stalled ribosomes while leaving normally elongating nascent chains untouched), yet broadly (in targeting the wide diversity of nascent chains that can be produced by ribosome stalling) (Lyumkis et al 2014).
Discovery of an ancestral RQC pathway in bacteria
More recently, we have discovered that RQC is also active in bacteria (Lytvynenko et al 2019). The finding was unexpected, as SsrA/tmRNA-mediated trans-translation was thought to be the only mechanism targeting ribosome stalling products for proteolysis in bacteria. We found that RqcH, as we named the Bacillus subtilis homolog of yeast Rqc2, interacts genetically with SsrA. Moreover, like Rqc2, RqcH senses nascent chain-obstructed large ribosomal subunits produced by ribosome stalling. Strikingly, with the ubiquitin system being absent in bacteria, RqcH modifies the nascent chains with C-terminal Alanine tails (‘Ala tails’) that act as degradation signals for proteases (Lytvynenko et al 2019).
RqcH, including its residues required for Ala tailing, is also conserved in archaea. Moreover, in several archaeal species, the RqcH homologs are syntenic with factors that act in rescuing stalled ribosomes (Lytvynenko et al 2019). This suggests that organisms in all domains of life have a related RQC mechanism for disposing of nascent-chains produced by incomplete translation, indicating that RQC was already active in the Last Universal Common Ancestor (LUCA) ~3 billion years ago!
How to synthesise protein with only half a ribosome
A major direction of our research continues to be the elucidation of RQC mechanisms. For this purpose, we leverage the various model systems available in the laboratory. For example, materials generated through our studies of bacterial RQC have enabled structural analyses that informed how nascent chains are modified with Ala tails (Filbeck et al 2021).
Ribosome stalling sequesters ribosomal subunits away from the translation-competent pool. To prevent the collapse of translation, dedicated factors sense stalled ribosomes and split them for recycling. It turns out that the splitting reaction leaves large ribosomal subunits still associated with a nascent chain-tRNA conjugate. These obstructed complexes are the substrates of RQC. In bacteria, RqcH senses the obstruction and recruits tRNA-Ala to add Ala residues at the nascent chain C-terminus. In collaboration with the cryo-EM group of Stefan Pfeffer, we have elucidated the structural basis for the Ala tailing reaction, discovered Hsp15/RqcP as an essential factor, and revealed that Ala tailing follows similar principles to canonical translation elongation (Filbeck et al 2021).
The ‘mother of all ribosome collisions’: deep evolutionary conservation of ribosome collision as a stress signal
Another major direction of our research is to identify new components of RQC. For bacterial RQC, we are undertaking both genetic and candidate approaches. For example, since RNA endonucleases containing an SMR domain specifically act on stalled ribosomes in eukaryotes, we tested whether MutS2—the only SMR domain-containing protein in B. subtilis—likewise functions upstream of RQC in bacteria. We found that MutS2 (now renamed RqcU, for RQC-Upstream factor) acts as an unexpected ribosome-binding protein and protects cells against translation elongation inhibitors. Moreover, cryo-EM analyses revealed that RqcU selectively binds to collided disomes. The RqcU ATPase and clamp domains are shaped like a tweezer, which holds to the stalled ribosome adjacent to the ribosomal inter-subunit interface. Following ribosome collision sensing, RqcU splits the subunits of the stalled ribosome, both for recycling the ribosomes and for eliciting RQC (Cerullo et al 2022).
Thus, collided disomes are a conserved mechanism of stress sensing across the kingdoms of life. Moreover, ribosome collisions in eukaryotic and bacterial cells are resolved following a strikingly similar logic, through the action of RNA endonucleases and ATP-dependent ribosome splitting factors, with a key difference being that eukaryotes separated the sensor and effector activities by inserting ubiquitin as an adapter, presumably to allow a higher degree of regulation.
C-terminal alanine tails support proteolysis in mammalian cells
Given the high degree of conservation, it was not surprising to find that the mammalian RqcH homolog, NEMF, also makes Ala tails (Thrun et al 2021). What was striking was to discover that this activity enables NEMF to target RQC substrates for degradation in a Listerin-independent manner: after nascent chains are released from the large ribosomal subunit, the Ala-tail modification is directly sensed by E3 ligases (either CRL2-KLHDC10 or Pirh2/Rchy1) which mark the nascent chains with ubiquitin for proteolysis (Thrun et al 2021). Therefore, the role of Ala tails in protein degradation and quality control is conserved from bacteria to humans.