Fig 1.
Identification of Lso2 bound to idle 80S ribosomes.
(A) Left: 3.4 Å resolution cryo-EM map of the in vitro reconstituted yeast Lso2–80S complex; α-helical extra density for Lso2 (red) is found in the intersubunit space. Middle: atomic model of the Lso2–80S complex. Right: 3.5 Å resolution cryo-EM map obtained from a native 80S ribosome preparation from cells grown under nutrient-limiting condition. (B–D): Top views of the Lso2–80S structure superimposed with the structure of a yeast 80S in the nonrotated state (B) (PDB: 6Q8Y [25]) and a yeast 80S in the rotated-2 state (C) (PDB: 5JUP [26]), showing differences between small subunit 18S rRNA in the 2 rotational states. (D) Same view as C, illustrating the position of accommodated A- and P-site tRNAs and mRNA (PDB: 5MC6) [27]. All structures were aligned on the 60S subunit. Hallmark features of the 80S ribosome are labelled. A, acceptor; CP, central protuberance; cryo-EM, cryo-electron microscopy; GAC, GTPase activating center; Lso2, late-annotated short open reading frame 2; P, peptidyl; PDB, Protein Data Bank.
Fig 2.
Lso2 interactions with the ribosome.
(A) Positioning of Lso2 with respect to the small subunit. (B) Zoom on interactions of Lso2 in the P and E sites of the 40S: the Lso2 N-terminus reaches into the cleft between rRNA helices h24, h28, and h44 of the 18S rRNA. All the hydrogen-bond and salt-bridge interactions are indicated with a dashed line. (C) Positioning of Lso2 with respect to the large subunit. (D) Lso2 binds to the large subunit near the CP via the helix-connecting loop. Interactions involve ribosomal proteins uL5 and eL42 as well as H84 and H85 of 25S rRNA. (E) The C-terminal α-helix of Lso2 passes by the major groove of H38A and uL16 towards the stalk base formed by H43 and H44 (GAC) and the P-stalk. The ultimate C-terminus of Lso2 is missing but might be located very close to the P-stalk. BK, beak; CP, central protuberance; DCC, Decoding center; E, exit; GAC, GTPase activating center; Lso2, late-annotated short open reading frame 2; P, peptidyl; PTC, peptidyl transferase center.
Fig 3.
Ribosome binding of Lso2 and CCDC124 is evolutionarily conserved.
(A) 3.0 Å resolution cryo-EM map of the human CCDC124–EBP1–80S ribosome. The reconstruction also contains E-site tRNA. The CCDC124 density is displayed at a different contour level than the ribosome map. (B) Atomic model of the CCDC124–EBP1–80S complex. (C) Conserved residues shown in a sequence alignment of CCDC124 with Lso2. (D) Superposition of Lso2–80S with CCDC124–80S models based on 25S rRNA shows a very similar positioning, indicating a highly conserved binding mode. CCDC124, coiled-coil domain containing short open reading frame 124; CP, central protuberance; cryo-EM, cryo-electron microscopy E, exit; EBP1, ErbB3-binding protein 1; GAC, GTPase activating center; Lso2, late-annotated short open reading frame 2; P, peptidyl; PTC, peptidyl transferase center.
Fig 4.
Hibernating human ribosomes are bound to EBP1 on the peptide exit site.
(A) Side and bottom views of the human eEF2–SERBP1–EBP1–80S model, built from a 2.9 Å resolution cryo-EM map. (B) EBP1 coordinates a part of rRNA expansion segment ES27L below the peptide tunnel exit. A dummy RNA helix was used as a placeholder for ES27L. (C) Zoom on the EBP1 insertion domain reaching into a cleft between uL23 and H59. cryo-EM, cryo-electron microscopy; EBP1, ErbB3-binding protein 1; eEF2, eukaryotic elongation factor 2; SERBP1, SERPINE1 mRNA-binding protein 1.
Fig 5.
In vitro splitting assay with Lso2–80S and Stm1–80S.
(A and B) The Lso2–80S in nonrotated state (displayed as cartoon) was overlaid with ribosome rescue factors Dom34 and Hbs1 (PDB: 3IZQ) [45] or Dom34 and ABCE1 (PDB: 3J16) [46]. In both conformations, Dom34 could accommodate within the A site of the 40S and would not clash with Lso2, leading to the hypothesis that the Dom34 splitting system preferably splits Lso2–80S. (C and D) Representative sucrose gradient profiles from splitting assays carried out without (C) or with (D) splitting factors; raw data can be found in S1 Data. (E) Quantification of relative splitting displayed as fold change over the negative controls (experiments without splitting factors), normalized to the control experiment using puromycin-treated empty 80S (see also S8 and S9 Figs and S2 Data). All experiments were performed in triplicates. A, acceptor; Dom34, duplication of multilocus region 34; Hbs1, Hsp70 subfamily B suppressor; Lso2, late-annotated short open reading frame 2; PDB, Protein Data Bank; Stm1, suppressor of target of Myb protein 1.