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. 2014 Sep 18;55(6):880-890.
doi: 10.1016/j.molcel.2014.07.006. Epub 2014 Aug 14.

Reconstitution of a minimal ribosome-associated ubiquitination pathway with purified factors

Sichen Shao  1 , Ramanujan S Hegde  2
Affiliations

Reconstitution of a minimal ribosome-associated ubiquitination pathway with purified factors

Sichen Shao et al. Mol Cell. .

Abstract

Ribosomes stalled on aberrant mRNAs engage quality control mechanisms that degrade the partially translated nascent polypeptide. Ubiquitination of the nascent protein is mediated by the E3 ligase Listerin via a mechanism involving ribosome subunit dissociation. Here, we reconstitute ribosome-associated ubiquitination with purified factors to define the minimal components and essential steps in this process. We find that the primary role of the ribosome splitting factors Hbs1, Pelota, and ABCE1 is to permit Listerin access to the nascent chain. Listerin alone can discriminate 60S- from 80S-nascent chain complexes to selectively ubiquitinate the former. Splitting factors can be bypassed by artificially removing the 40S subunit, suggesting that mere steric hindrance impedes Listerin recruitment. This was illustrated by a cryo-EM reconstruction of the 60S-Listerin complex that identifies a binding interface that clashes with the 40S ribosomal subunit. These results reveal the mechanistic logic of the core steps in a ribosome-associated quality control pathway.

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Figures

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Graphical abstract
Figure 1
Figure 1
Purified Stalled 80S RNCs Are Ubiquitinated In Vitro (A) Scheme for purifying stalled 80S RNCs housing the model substrate F-VHP-β. (B) F-VHP-β was in vitro translated with 35S-methionine and purified by the scheme in (A). Fractions during the purification were analyzed by SDS-PAGE and autoradiography. The primary product contains an attached tRNA (F-V-β-tRNA), which is hydrolyzed by RNase to F-V-β (lane 7). Lanes 8 and 9 show Coomassie-blue-stained image of lanes 6 and 7, respectively. (C) In vitro translation reaction of F-VHP-β (lane 3) was fractionated using physiologic or high-salt conditions (lanes 1–5) and affinity purified via the FLAG tag (lanes 6 and 7), as shown in (A). All fractions were immunoblotted for Listerin, Hbs1, ABCE1, and the ribosomal proteins L9 and S16. Endogenous Hbs1 and exogenously added Hbs1-DN are labeled. (D) A 10%–30% sucrose gradient analysis of 35S-labeled F-VHP-β RNCs, purified as in (B), with positions of 60S and 80S indicated. (E) Purified 35S-labeled F-VHP-β RNCs were subjected to ubiquitination reactions with or without S-100 derived from RRL. The total reaction products and ubiquitin pull-downs (Ub PD) via the His tag were analyzed by SDS-PAGE and autoradiography. The unmodified substrate (F-V-β-tRNA) and poly-ubiquitinated (poly-Ub) products are indicated. (See also Figure S1.)
Figure 2
Figure 2
Uncoupling Ribosome Splitting and Ubiquitination Activities (A) Coomassie-stained gels of fractions from a high salt extraction of native reticulocyte ribosomes (left) and purified splitting factors (right). (B) Purified 35S-labeled 80S F-VHP-β RNCs were subjected to ubiquitination assays with the indicated components, and the total reaction products were analyzed by autoradiography. (C) Drop-off assay to monitor ribosome splitting using purified 35S-labeled F-β RNCs. The total reaction (T), supernatant (S), and pellet (P) fractions were analyzed by autoradiography, and the relative amount of F-β-tRNA in the supernatant was quantified (% drop off). (D) Purified 35S-labeled 80S F-VHP-β RNCs were subjected to ubiquitination assays with the indicated components, and the total reaction products analyzed by autoradiography. The unmodified substrate (F-V-β-tRNA) and poly-ubiquitinated (poly-Ub) products are indicated. (See also Figure S2.)
Figure 3
Figure 3
Ribosome Splitting Precedes Ubiquitination (A) Purified 35S-labeled 80S F-VHP-β RNCs were subjected to a two-stage ubiquitination reaction with a preincubation (1st) for 10 min with the indicated components, followed by another 10 min incubation (2nd) without or with fraction 2. Total reaction products were analyzed by autoradiography. (B) Schematic diagram illustrating the Mg2+-dependent dissociation of 80S RNC by eIF6. (C) Ubiquitination reactions of 35S-labeled 80S F-VHP-β RNCs at Mg2+ concentrations from 3.4 mM to 14.4 mM. All reactions contained eIF6 and fraction 2 of the ribosome salt wash.
Figure 4
Figure 4
Identification of Ubiquitination Machinery (A) Fractions from the final sucrose gradient step of a purification scheme (see Figure S3A) were analyzed for RNC ubiquitination activity (top), Listerin immunoblot (middle), and Coomassie stain (bottom). (B) Affinity purification of in vitro translated F-VHP-β RNCs via the scheme in Figure S3B. The products purified in the absence or presence of RNase were analyzed by Coomassie staining (left) and immunoblotting (right). (C) Abundance profiles of E3 ubiquitin ligases across gradient fractions 2–7 from (A) identified by mass spectrometry. Fractions 4 and 5, where peak RNC ubiquitination activity was observed in (A), are highlighted in black. (D) Weighted spectral counts of select proteins identified from mass spectrometry analysis of the native RQC purification from panel B (see Table S1 for the complete list). The spectral counts for these same proteins from the sucrose gradient fractions of (A) are also shown. (See also Figure S3 and Table S1.)
Figure 5
Figure 5
Minimal Machinery for Stalled RNC Ubiquitination (A) Ubiquitination reactions of 35S-labeled 80S F-VHP-β RNCs with the indicated components comparing fraction 2 of the ribosome salt wash with purified Listerin at 0.3 nM, a concentration equivalent to that found in fraction 2. (B) 35S-labeled 80S F-VHP-β RNCs were incubated with 75 nM E1, 250 nM UbcH5a, 10 μM ubiquitin, 50 nM splitting factors, 250 nM eIF6, 2.4 nM Listerin, and energy. Aliquots were removed at the indicated time points and analyzed by SDS-PAGE and autoradiography. (C) Ubiquitination reactions of 35S-labeled 80S F-VHP-β RNCs with the indicated purified factors (Listerin at 12 nM). One reaction was pretreated with RNase (lane 6) before the reaction. Shown are the autoradiograph (top) and Coomassie stain (bottom) of the same gel. (See also Figure S4)
Figure 6
Figure 6
Listerin Specifically Recognizes 60S Subunits (A) Coomassie stain of reaction used for cryo-EM analysis containing ∼120 nM F-VHP-β RNCs, equimolar splitting factors (Hbs1, ABCE1, and Pelota), 1 mM ATP, 1 mM GTP, and a ∼10-fold excess of purified Listerin and eIF6. Purified proteins and ribosomal proteins (ribo. prot.) are indicated. (B) Cryo-EM reconstructions of the 60S ribosome subunit (cyan) with eIF6 (gray) and an additional density attributed to Listerin (orange). The ribosomal exit tunnel (asterisk) and L1 stalk are marked. (C) Listerin binding to 60S ribosome subunits clashes (arrows) with where a 40S subunit (yellow) would bind on an 80S ribosome. (D) Fitting of 60S-bound Listerin density into negative stain reconstructions of free Ltn1. (E) Close-up of the region of Listerin interaction with the 60S ribosomal subunit near the exit tunnel with nearby proteins (RPL22, RPL19, and RPL31) indicated. (See also Figure S5.)
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