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

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

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

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

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

Structure of Utp4 from <i>Chaetomium thermophilum</i>.

<p><b>(A)</b> Domain architecture of Utp4. Domains present in the crystal structure are given by residue numbers and are highlighted in colour. The N-terminal β-propeller 1 covers residues from 38 to 381 and is shown in blue and the C-terminal β-propeller 2 (residues 393 to 890) in red. (His)₆-tag and TEV-site are represented in grey. <b>(B)</b> The overall structure of Utp4 presents two 7-bladed β-propellers in tandem. N- and C-termini are indicated and blades are numbered. Each β-blade consists of four β-strands (ABCD). <b>(C)</b> Tertiary interaction of β-propellers. A hairpin between β-strands 2A and 2B of β-propeller 1 packs against an α-helix between β-strands 10D and 11A of β-propeller 2 (view rotated by 90° in respect to <b>A</b>). <b>(D)</b> Surface charge (left panel) and conservation (right panel) of Utp4. The electrostatic surface (red: negative, blue: positive, contoured at ±5 <i>k</i>_<i>B</i>T/e) indicates extended positively charged patches in both β-propellers. Sequence conservation mapped on the molecular surface (magenta: conserved, cyan: variable) is most pronounced around a highly positive charged patch at the N-terminus (indicated with ‘N’).</p

Utp4 in context of the 5'-ETS and 90S pre-ribosome.

<p><b>(A)</b> The crystal structure of Utp4 (rainbow colours) placed into its cryo-EM density (overall 7.3 Å resolution of the particle) in context of the 90S pre-ribosome [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178752#pone.0178752.ref009" target="_blank">9</a>]. The density (grey mesh) is contoured at a 3σ level. The Utp4 structure fits in the EM-density as rigid body validating the relative propeller orientation and loop conformations as seen in the crystal structure in the physiological context. <b>(B)</b> Utp4 (blue: propeller 1 (Utp4-N); red: propeller 2 (Utp4-C)) in context of the entire 5'-ETS <i>de novo</i> modeled (rainbow) in the cryo-EM density. RNA helices and nucleotides at special positions are given. The 5’-end is hidden in the continuous stack of RNA helices 1 and 2. Single stranded RNA-parts are indicated by connecting lines (grey). <b>(C)</b> Utp4/5'-ETS in context of a close-up of the UTP-A complex as part of the entire 90S pre-ribosome complex (grey). The region of U3 snoRNA base-paring at the 3'-end of the 5'-ETS (beyond nucleotide 243) is highlighted in cyan. <b>(D)</b> Utp4/5'-ETS/Utp8 interaction around nucleotide G66 (magenta) identified as major contact point by CRAC analysis. Left panel: Utp4 is indicated by surface potential map (±5 <i>k</i>_<i>B</i>T/e, blue positive). The C-terminus of Utp8 (Utp8-C, end of predicted α-helix and β-strand, no sequence modeled) are given in orange. The α-helix is <i>de novo</i> placed as ideal helix in the cryo-EM density, whereas the β-strand is taken from the artificial strand 14D of the X-ray structure (connection given as dashed lines is unclear). Right panel: Model for the Utp8-C interaction with Utp4. The Velcro-closed ‘2+1+1’ blade 14 is completed <i>in trans</i> by Utp8 and G66 binds to the positive patch (indicated by R³⁴⁴ and K³⁸³) in the Utp4-N/Utp4-C interface.</p

<i>In vitro</i> protein-RNA UV crosslinking analysis of <i>ct</i>Utp4.

<p><b>(A)</b> Hits obtained from deep sequencing analysis of <i>Chaetomium thermophilum</i> His₆-Utp4 (coverage, blue) mapped within the 5'-ETS (nucleotides 1 to 587) after UTP-A/5'-ETS RNP assembly by co-expression in yeast. <b>(B)</b> Mutations (deletions and substitutions) identified after cDNA library synthesis are indicated by red bars. Mutational hot spots observed in the two crosslinked regions are labeled accordingly (G66 and A220). As background control, the UTP-A/5'-ETS complex carrying untagged Utp4 (“no His₆ tag”) was used. The crosslinked region around 5'-ETS bases 100–140, which was found also in the untagged control, is marked with an asterisk. <b>(C)</b> and <b>(D)</b> The two main regions of the 5'-ETS (A53-C96 and A192-C235) that were crosslinked to His₆-Utp4 are shown together the number of mutations per base. The respective 5'-ETS sequence is depicted below. Mutational hot spots G66 and A220 colored in red.</p