DENR–MCTS1 heterodimerization and tRNA recruitment are required for translation reinitiation

<div><p>The succession of molecular events leading to eukaryotic translation reinitiation—whereby ribosomes terminate translation of a short open reading frame (ORF), resume scanning, and then translate a second ORF on the same mRNA—is not well understood. Density-regulated reinitiation and release factor (DENR) and multiple copies in T-cell lymphoma-1 (MCTS1) are implicated in promoting translation reinitiation both in vitro in translation extracts and in vivo. We present here the crystal structure of MCTS1 bound to a fragment of DENR. Based on this structure, we identify and experimentally validate that DENR residues Glu42, Tyr43, and Tyr46 are important for MCTS1 binding and that MCTS1 residue Phe104 is important for tRNA binding. Mutation of these residues reveals that DENR-MCTS1 dimerization and tRNA binding are both necessary for DENR and MCTS1 to promote translation reinitiation in human cells. These findings thereby link individual residues of DENR and MCTS1 to specific molecular functions of the complex. Since DENR–MCTS1 can bind tRNA in the absence of the ribosome, this suggests the DENR–MCTS1 complex could recruit tRNA to the ribosome during reinitiation analogously to the eukaryotic initiation factor 2 (eIF2) complex in cap-dependent translation.</p></div

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

High-resolution structure of MCTS1 binding an N-terminal fragment of DENR.

<p>(A) Domain architecture of DENR, MCTS1, and the related eIF2D (ligatin) protein. (B) A DENR truncation series identifies aas 24–51 of DENR as the minimum peptide capable of binding MCTS1 in <i>Escherichia coli</i>. Summary of data presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005160#pbio.2005160.s001" target="_blank">S1 Fig</a>. (C) Crystal structure of the minimal DENR–MCTS1 complex. MCTS1 contains an N-terminal DUF1947 (light green) and a C-terminal PUA domain (green). The DENR peptide (light orange) binds along the interface between the N- and C-terminal MCTS1 domains. Anomalous density (14σ), calculated with ANODE, is shown as a mesh. (D) DENR contains a zinc finger at the N-terminus, comprised of Cys34, 37, and 44. Although our DENR construct lacks a fourth residue (Cys53) to complete the Zn²⁺ coordination, the zinc finger is properly folded, as it is completed by His58 of a crystallographically related MCTS1 molecule. aa, amino acid; DENR, density-regulated reinitiation and release factor; DUF1947, domain of unknown function 1947; eIF1, eukaryotic initiation factor 1; eIF2D, eukaryotic initiation factor 2D; MCTS1, multiple copies in T-cell lymphoma-1; MDM2, mouse double minute 2; PUA, pseudouridine synthase and archaeosine transglycosylase; SUI1, suppressors of initiation codon mutations 1; SWIB <i>SWItch</i>/Sucrose Nonfermentable complex B; WH, winged helix</p

Mpp10 represents a platform for the interaction of multiple factors within the 90S pre-ribosome

<div><p>In eukaryotes, ribosome assembly is a highly complex process that involves more than 200 assembly factors that ensure the folding, modification and processing of the different rRNA species as well as the timely association of ribosomal proteins. One of these factors, Mpp10 associates with Imp3 and Imp4 to form a complex that is essential for the normal production of the 18S rRNA. Here we report the crystal structure of a complex between Imp4 and a short helical element of Mpp10 to a resolution of 1.88 Å. Furthermore, we extend the interaction network of Mpp10 and characterize two novel interactions. Mpp10 is able to bind the ribosome biogenesis factor Utp3/Sas10 through two conserved motifs in its N-terminal region. In addition, Mpp10 interacts with the ribosomal protein S5/uS7 using a short stretch within an acidic loop region. Thus, our findings reveal that Mpp10 provides a platform for the simultaneous interaction with multiple proteins in the 90S pre-ribosome.</p></div

Assembly factor network and reconstitution of the <i>ct</i>Mpp10 module.

<p>(A) One of the top fractions derived from 15–40% sucrose gradient ultracentrifugation containing the free pool of the yeast Mpp10 complex and associated factors, analyzed by SDS-PAGE and Coomassie staining. The sample was first tandem affinity-purified via yeast Imp4-FTpA (Mpp10 factor) and subsequently resolved by sucrose gradient ultracentrifugation. The labeled proteins were identified by mass spectrometry. Degradation products of Imp4 and dimeric version of Mpp10 are marked with a hashtag (#). (B) Systematic Yeast-2-hybrid analysis of core and associated <i>ct</i>Mpp10 assembly factors. The indicated constructs were N-terminally fused to either GAL4 activation domain (AD) or GAL4 binding domain (BD). Yeast transformants were spotted onto SDC-Leu-Trp (permissive), SDC-Leu- Trp-His (selective), and SDC-Leu-Trp-Ade plates (selective for only strong interactions) and grown for 3 days at 30°C. (C) Recombinant co-expression and split tandem-affinity-purification (via pA-TEV-<i>ct</i>Mpp10 and Flag-<i>ct</i>Imp3) of the <i>ct</i>Mpp10 core complex with stepwise addition of associated factors Utp3 and Rps5/uS7. The complexes were assembled by over-expression of indicated protein combinations in yeast (pGAL, high-copy plasmids). Shown are the corresponding Flag-peptide eluates analyzed by SDS-PAGE and Coomassie staining and Western blotting labeled bands were identified by mass spectrometry. Tags were fused to the N-terminus of the corresponding proteins. (D) Position of the <i>ct</i>Mpp10 factors <i>ct</i>Imp3 and <i>ct</i>Imp4 within the 90S cryo-EM density revealing their relative position to the U3 snoRNA (orange) and pre-18S rRNA domains (green, cyan, and purple). Density map of the <i>C</i>. <i>thermophilum</i> 90S pre-ribosome (EMDB: EMD-8143) and respective fitted <i>ct</i>Mpp10 complex members (PDB: 5JPQ) are visualized using Chimera. A magnification of the fitted models for <i>ct</i>Imp3 (blue), <i>ct</i>Imp4 (turquois), and <i>ct</i>Rps5/uS7 (yellow) is shown in a circle, illustrating their close neighborhood within the particle.</p

Identification of the minimal Rps5-binding motif within Mpp10.

<p>(A) Yeast 2-hybrid analysis of full-length <i>ct</i>Rps5 tested with four indicated fragments of <i>ct</i>Mpp10. The indicated constructs were N-terminally fused to either GAL4 activation domain (AD) or GAL4 binding domain (BD). Yeast transformants were spotted onto SDC-Leu-Trp, SDC-Leu-Trp-His, and SDC-Leu-Trp-Ade plates and grown for 3 days at 30°C. (B) Size exclusion chromatography (SEC) of the reconstituted GST-<i>ct</i>Mpp10(283–332)-<i>ct</i>Rps5 heterodimer. Based on the elution profile recorded at 280 nm, we analyzed fractions 12–19 by SDS-PAGE and Coomassie staining. The complete elution profile (in ml) using a Superdex 200 10/300 column is shown underneath, with indication of the region corresponding to fractions 12–19. The complex was recombinantly co-expressed in <i>E</i>. <i>coli</i>, purified via GST pull-down and eluted with glutathione (GSH). Labeled bands were verified by mass spectrometry. (C) Assembly of the Mpp10 module by recombinant overexpression of the <i>C</i>. <i>thermophilum</i> counterparts, in <i>S</i>. <i>cerevisiae</i>, followed by split-tag affinity-purification using pA-TEV-<i>ct</i>Mpp10 and Flag-<i>ct</i>Imp3, as first and second bait respectively. The complexes were assembled by over-expression of indicated protein combinations in yeast (pGAL, high-copy plasmids). FLAG eluates were subject to SDS-PAGE followed by Coomassie staining and Western blot analysis using anti-Rps5 and anti-FLAG antibodies.</p

Crystal structure of <i>ct</i>Imp4-<i>ct</i>Mpp10 complex.

<p>(A) Overall structure of the <i>ct</i>Imp4-<i>ct</i>Mpp10 complex. The BRIX domain of <i>ct</i>Imp4 (teal) is completed by helix α2 of <i>ct</i>Mpp10 (grey). Helix α1 of <i>ct</i>Mpp10 interacts via Trp500 with <i>ct</i>Imp4 (inset). For the sake of clarity <i>ct</i>Mpp10 helix α1 is omitted in the other panels. (B) Comparison of <i>ct</i>Imp4-<i>ct</i>Mpp10 with <i>an</i>Rpf2-<i>an</i>Rrs1 (PDB-ID: 5BY8 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183272#pone.0183272.ref030" target="_blank">30</a>]) The overall fold between the BRIX domain proteins <i>ct</i>Imp4 (teal) and <i>an</i>Rpf2 (light-blue) and their ligands <i>ct</i>Mpp10 (grey) and <i>an</i>Rrs1 (orange) is preserved. Beta-augmentation on the C-terminal sub-domain of the BRIX protein is only observed in the <i>an</i>Rpf2-<i>an</i>Rrs1 complex (red circle). (C) Rigid body docking of the <i>ct</i>Imp4-<i>ct</i>Mpp10 complex into the 5.1 Å map of the yeast 90S particle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183272#pone.0183272.ref008" target="_blank">8</a>]. While the BRIX domain of <i>ct</i>Imp4 (teal) and <i>ct</i>Mpp10 helix α2 (grey) are completely covered by electron density, helix α1 of <i>ct</i>Mpp10 is not. This suggests that in context of the 90S, this helix occupies another location. The tip of helix α1 is occupied by a RNA double helix (purple) in the yeast 90S density. (D) Alternative modeling of the termini of <i>ct</i>Imp4 and <i>ct</i>Mpp10 based on the crystal structure and cryo-EM density. In the crystal structure (grey/blue), the N-terminus (residues 515–530) of our <i>ct</i>Mpp10 construct interacts with <i>ct</i>Imp4, whereas when modeled based on the cryo-EM density it extends away (orange) from <i>ct</i>Imp4. Likewise the C-terminus of <i>ct</i>Imp4 can be extended and modeled (red) into the now remaining cavity between <i>ct</i>Mpp10 helix α1 (grey ribbon) and <i>ct</i>Imp4.</p

Two hydrophobic patches within the N-terminal region mediate the direct interaction between Mpp10 and Utp3.

<p>(A) Yeast 2-hybrid analysis of full-length <i>ct</i>Utp3 tested with four indicated fragments of <i>ct</i>Mpp10. The indicated constructs were N-terminally fused to either GAL4 activation domain (AD) or GAL4 binding domain (BD). Yeast transformants were spotted onto SDC-Leu-Trp, SDC-Leu-Trp-His, and SDC-Leu-Trp-Ade plates and grown for 3 days at 30°C. (B) Recombinant GST and GST-<i>ct</i>Mpp10 truncations were co-expressed with <i>ct</i>Utp3 in <i>E</i>. <i>coli</i>, and subsequently bound to glutathione resin. GSH-eluates were analyzed by SDS-PAGE followed by Coomassie staining. Labeled bands were identified by mass spectrometry. Lower bands correspond to degradation products and are marked with a hashtag (#). (C) Recombinant overexpression of the <i>C</i>. <i>thermophilum</i> Mpp10 complex components in <i>S</i>. <i>cerevisiae</i>, followed by split-tag affinity-purification using pA-TEV-<i>ct</i>Mpp10 and Flag-<i>ct</i>Imp3, as first and second bait, respectively. The complexes were assembled by over-expression of indicated protein combinations in yeast (pGAL, high-copy plasmids). In this case, we additionally used an N-terminally HA tagged version of <i>ct</i>Utp3. FLAG eluates were subject to SDS-PAGE followed by Coomassie staining and Western blot analysis using anti-HA and anti-FLAG antibodies.</p

Growth analysis of strains with different Mpp10 domain truncations.

<p>(A) Overall domain organization for both <i>ct</i>Mpp10 and <i>sc</i>Mpp10 including the identified minimal binding motifs for <i>ct</i>Utp3, <i>ct</i>Rps5, <i>ct</i>Imp4, and the published <i>ct</i>Imp3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183272#pone.0183272.ref050" target="_blank">50</a>]. (B) Growth analysis of yeast <i>mpp10</i>Δ shuffle strain complemented with the indicated Mpp10 constructs under <i>TEF1</i> promoter control. The <i>mpp10</i>Δ shuffle strain was transformed with <i>HIS3</i> plasmids carrying wild-type <i>MPP10</i> and truncations. Subsequently, the <i>URA3</i>-<i>MPP10</i> shuffle plasmid was shuffled out on SDC+5-FOA plates. Transformants were spotted in 6-fold serial dilutions onto SDC-His or SDC+FOA. Plates were incubated at the indicated temperatures for 3 days.</p

The UTP-A complex from <i>Chaetomium thermophilum</i>.

<p><b>(A)</b> Scheme showing the spatial assembly of the fungal UTP-A complex including the Utp4 X-ray structure and the EM-modeled 5'-ETS. The propellers of remaining UTP-A proteins (Utp8, Utp15, and 2× Utp17) are placed according previous biochemical and EM-studies. The α-solenoidal parts (including whole Utp5) are not included. The entire Utp10 molecule turning around Utp4 is interpreted as also the very C-terminus (atomic model) of Utp8 next to the Velcro-closure of Utp4. The position of the disease-modified arginine in human Utp4 in the interface to Utp10 is highlighted within a red sphere. <b>(B)</b> Comparison of the UTP-A complexes from <i>Chaetomium thermophilum</i> (left panel; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178752#pone.0178752.ref009" target="_blank">9</a>]) and <i>Saccharomyces cerevisiae</i> (right panel; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178752#pone.0178752.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178752#pone.0178752.ref028" target="_blank">28</a>]). While the overall architecture is conserved, the 5'-end of the RNA shows a different arrangement. In addition, the Upt8-Utp4 contact is not visible in the yeast structures.</p