Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Nov 11;111(45):15981-6.
doi: 10.1073/pnas.1413882111. Epub 2014 Oct 27.

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

Dmitry Lyumkis  1 , Dario Oliveira dos Passos  2 , Erich B Tahara  2 , Kristofor Webb  3 , Eric J Bennett  3 , Staal Vinterbo  4 , Clinton S Potter  1 , Bridget Carragher  5 , Claudio A P Joazeiro  6
Affiliations

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

Dmitry Lyumkis et al. Proc Natl Acad Sci U S A. .

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 chain-conjugated 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.

Keywords: Tae2/Nemf; cryo-EM; listerin/Ltn1 E3 ubiquitin ligase; protein quality control; translational surveillance.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
(A) RQC∆R complex structure generated after enriching for nonribosomal density using a likelihood-based classification algorithm (14) (see also Fig. S3), displayed in solid gray, and (B) colored by local resolution. (C) Global Fourier shell correlation curve and nominal resolution value of 9.6 Å for the same map.
Fig. 2.
Fig. 2.
The Ltn1 E3 ubiquitin ligase bridges the ribosomal factor-binding site and the TE. (A) Crown view of the RQC∆R complex structure from the intersubunit surface, filtered to 15 Å to emphasize nonribosomal density that is resolved at lower resolution. Gray, 60S subunit; blue, RQC∆R density. The opening of the nascent chain tunnel (tunnel exit, TE), ribosomal protein Rpl1 (L1), central protuberance (CP), and stalk (St) are indicated. (B) Comparison between the Ltn1 density (yellow) segmented from the RQC∆R complex (Left) and a negative stain reconstruction of purified Ltn1 from the Electron Microscopy Data Bank (EMD 2257) (10) (Right). (C) View of the surface surrounding the TE, with ribosomal proteins and rRNA expansion segment-41 (ES-41) indicated. PDB IDs 3U5D and 3U5E were used for ribosomal RNA and proteins, respectively. Ltn1 is shown in yellow. Additional nonribosomal density is shown in blue. (D) Close-up view of the Ltn1 interaction site on the 60S ribosomal subunit. Ribosomal proteins and RNA in closest proximity are docked into the experimental density (Top); their surfaces within a distance of 15 Å from Ltn1 are shaded yellow (Bottom). (Scale bars, 100 Å.)
Fig. 3.
Fig. 3.
Structural basis for Ltn1’s selective binding to the 60S ribosomal subunit. (A) Overlay of the segmented Ltn1 density from the RQC∆R complex with the 80S ribosome structure as observed in the RQC∆R complex. Ltn1, bright yellow; 60S subunit, gray; 40S subunit, light yellow. (B) The overlap of the Ltn1 and the 40S subunit densities is indicated in red (Ltn1 removed for clarity). (C) SRP-80S complex from Halic et al. (35). SRP is shown in red. (D) Overlay of the segmented Ltn1 structure (yellow) from the RQC∆R complex with the SRP-80S complex from Halic et al. (35).
Fig. 4.
Fig. 4.
Tae2 recognizes the exposed tRNA moiety in a stalled 60S-peptidyl-tRNA structure. (A) Refinement of RQC∆R/tae2∆ after 3D classification and enrichment for nonribosomal density, displayed in solid gray (Top) and colored by local resolution (Middle). The global resolution curve is shown (Bottom) and indicates a nominal value of 11.2 Å. (B) Refinement of RQC∆R (Left) and RQC∆R/tae2∆ (Middle) after enriching for all nonribosomal density, both filtered to 15Å. (Right) comparison between the two structures, with nonribosomal densities present in RQC∆R but not in RQC∆R/tae2∆ shown in red, filtered to 15 Å, and displayed at 5σ above the mean pixel value. (C) Segmented density of the RQC∆R structure in Fig. 1A showing the P-site tRNA (green) and surrounding Tae2-dependent density (orange). Both the tRNA and Tae2 components were segmented using a watershed algorithm implemented in Segger (36). Without atomic resolution, precise localization of Tae2 domain boundaries is not possible. (D) Tae2-dependent density contacts in the vicinity of the ribosomal P site. PDB IDs 3U5D and 3U5E were used for ribosomal RNA and proteins, respectively. P-tRNA (green), ribosomal proteins (teal and blue), and rRNA helices (“H”; violet, red, and purple) in the Tae2-dependent density binding site are indicated (Left); regions within 15 Å of the density are indicated by checkered orange shading (Right). (E) Ltn1-∆R co-IPs with peptidyl-tRNA. Cells expressed Ltn1-∆R-Flag in the presence or absence of a protein A reporter encoded by nonstop mRNA (PtANS). Lysates were used for anti-Flag IP (lanes 4–6), and co-IP’ed PtANS and its tRNA-conjugated form was detected by anti-protein A immunoblot. (Lanes 1–3) Whole cell extract (input). Samples for lanes 3 and 6 were treated with RNase A before loading. (F–I) Steric hindrance prevents simultaneous binding of Tae2 and the 40S ribosomal subunit to the 60S subunit. (F) 80S ribosome structure, with the 60S subunit in gray and the 40S subunit in yellow. (G and H) Only the contour of the 60S subunit is presented. (G) 40S subunit as in F. (H) The Tae2-tRNA density as observed in the RQC∆R complex (Tae2, orange; tRNA, green). (I) The overlap of Tae2 on the 40S subunit density is indicated in red. Tae2 and tRNA were removed for clarity.
Fig. 5.
Fig. 5.
Model for RQC function. (A) Ribosomes can halt translation for a variety of reasons. For example, ribosomes translating aberrant mRNA lacking stop codons (“nonstop mRNA”) eventually reach the poly(A) tail, which encodes poly(Lys); nascent polybasic tracts can cause translation to slow down or pause altogether (4). (B) 80S ribosomes that pause during translation and fail to either resume elongation or terminate translation are rescued by factors that promote splitting of the subunits (e.g., ref. 5). Ribosomal rescue generates free 60S subunits that remain stalled with peptidyl-tRNA (e.g., ref. 5). (C) The Tae2 subunit of the RQC complex recognizes peptidyl-tRNA-stalled 60S subunits via direct interaction with the exposed tRNA. Tae2 also stabilizes the binding of Ltn1, the E3-catalytic subunit of RQC, to the complex. Ltn1 is positioned such that the RING domain sits next to the ribosomal tunnel exit (TE). The RING domain binds to ubiquitin (Ub)-carrying E2 conjugases and stimulates Ub transfer to substrates. The RQC∆R structure implies that the C-terminal poly-Lys tracts characteristic of a subset of Ltn1 substrates would remain buried within the nascent chain exit tunnel, supporting the model that such sequences act as destabilizing motifs (“degrons”) by mediating translational stalling, rather than by acting directly as ubiquitin acceptor sites (4). Ubiquitylation happens while nascent chains are still 60S subunit associated and provides two functions in this pathway. First, it mediates extraction of the stalled protein by recruiting the AAA ATPase Cdc48/VCP/p97. Second, it signals proteasomal degradation of the aberrant polypeptide. (D) In a competing reaction with Tae2 binding, stalled 60S subunits can reassociate with free 40S subunits (17). Because Ltn1 is unable to bind to 80S ribosomes, Tae2-mediated P-tRNA recognition also facilitates Ltn1 binding by preventing ribosomal subunit reassociation.
See this image and copyright information in PMC

References

    1. Graille M, Séraphin B. Surveillance pathways rescuing eukaryotic ribosomes lost in translation. Nat Rev Mol Cell Biol. 2012;13(11):727–735. - PubMed
    1. Petry S, Weixlbaumer A, Ramakrishnan V. The termination of translation. Curr Opin Struct Biol. 2008;18(1):70–77. - PubMed
    1. Moore SD, Sauer RT. The tmRNA system for translational surveillance and ribosome rescue. Annu Rev Biochem. 2007;76:101–124. - PubMed
    1. Bengtson MH, Joazeiro CA. Role of a ribosome-associated E3 ubiquitin ligase in protein quality control. Nature. 2010;467(7314):470–473. - PMC - PubMed
    1. Shoemaker CJ, Eyler DE, Green R. Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay. Science. 2010;330(6002):369–372. - PMC - PubMed

Publication types

MeSH terms

Substances