Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

Abstract

All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chainconjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes.Tae2/Nemf | translational surveillance | protein quality control | cryo-EM | listerin/Ltn1 E3 ubiquitin ligase D uring the canonical termination and recycling steps of translation, stop codon recognition triggers factor-mediated hydrolysis of the nascent peptidyl-tRNA conjugate, nascent chain release, and ribosome splitting (1-3). Conversely, translation of aberrant mRNA, such as mRNA lacking stop codons ("nonstop mRNA"), renders 80S ribosomes stalled with nascent polypeptides (1-3). Furthermore, "nonstop proteins" cannot be corrected by quality control chaperones and have the potential to interfere with cellular function (3, 4). Not surprisingly, defective termination and recycling are under surveillance by a variety of mechanisms (1-3). In eukaryotes, "rescue factors" homologous to termination factors promote dissociation of translationally halted ribosomes in a stop codon-independent manner (5). However, because rescue factors lack peptidyl-tRNA hydrolase activity, their action results in nascent chains remaining stalled on the released 60S subunit.Ltn1 is the critical E3 ligase mediating ubiquitylation of aberrant proteins that become stalled on ribosomes during translation (4). Mutation of the Ltn1 mouse ortholog, listerin, causes neurodegeneration (6), suggesting an important function for this process. Ltn1 works together with several cofactors as part of the ribosome-associated quality control complex (RQC) (7-9) and appears to first associate with nascent chain-stalled 60S subunits together with two proteins of unknown function, Tae2 and Rqc1 (7, 9). Ltn1-mediated ubiquitylation of t...

Structure and function of the yeast listerin (Ltn1) conserved N-terminal domain in binding to stalled 60S ribosomal subunits

“…The NTD of human and yeast Ltn1 orthologs directly contact components of the RQC complex (19,20,24) and has been shown to be important for proper function of human Ltn1 with respect to stalled polypeptide ubiquitylation (20). The data in Fig.…”

“…Rqc2 increases association of Ltn1 to the RQC complex and enhances ubiquitylation of stalled nascent chains (11,12). In the RQC, Ltn1 and Rqc2 interact with each other near the SRL in a position such that surfaces important for 40S ribosomal subunit association become sterically occluded (19,21). We placed a model of S. cerevisiae Rqc2 [obtained using the coordinates of human Rqc2 (NEMF)] into the yeast RQC maps using rigid body fitting.…”

“…More recently, cryo-EM structures of Ltn1/listerin in complex with Rqc2/NEMF and stalled 60S subunits have been published (19)(20)(21). The structures suggest that Rqc2 recognizes the stalled 60S subunit by simultaneously binding to the tRNA moiety of the stalled peptidyl-tRNA and the 60S intersubunit surface.…”

Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins

“…Recent structural data suggest that the catalytic RING domain of Rkr1 is poised at the opening of the exit tunnel of the dissociated 60S subunit (23,60,61). Nascent polytopic membrane proteins (such as Vma12) are likely to expose amino acids to the cytosol during translocation.…”

Automated data collection in single particle electron microscopy

“…Currently, collection of hundreds of thousands [56] or even millions of particles [4] has become the norm, enabled by automated data collection. This explosion in data collection rates allows the resolution limit to be pushed further [3,[5][6][7] and also permits many more interesting biological questions to be addressed [57], such as the structural analysis of rare complexes and the understanding of minor conformational states [58,59].…”