Symportin 1 chaperones 5S RNP assembly during ribosome biogenesis by occupying an essential rRNA-binding site

During 60S biogenesis, mature 5S RNP consisting of 5S RNA, RpL5 and RpL11, assembles into a pre-60S particle, where docking relies on RpL11 interacting with helix 84 (H84) of the 25S RNA. How 5S RNP is assembled for recruitment into the pre-60S is not known. Here we report the crystal structure of a ternary symportin Syo1–RpL5-N–RpL11 complex and provide biochemical and structural insights into 5S RNP assembly. Syo1 guards the 25S RNA-binding surface on RpL11 and competes with H84 for binding. Pull-down experiments show that H84 releases RpL11 from the ternary complex, but not in the presence of 5S RNA. Crosslinking mass spectrometry visualizes structural rearrangements on incorporation of 5S RNA into the Syo1–RpL5–RpL11 complex supporting the formation of a pre-5S RNP. Our data underline the dual role of Syo1 in ribosomal protein transport and as an assembly platform for 5S RNP

Structural basis for 5'-ETS recognition by Utp4 at the early stages of ribosome biogenesis.

Eukaryotic ribosome biogenesis begins with the co-transcriptional assembly of the 90S pre-ribosome. The 'U three protein' (UTP) complexes and snoRNP particles arrange around the nascent pre-ribosomal RNA chaperoning its folding and further maturation. The earliest event in this hierarchical process is the binding of the UTP-A complex to the 5'-end of the pre-ribosomal RNA (5'-ETS). This oligomeric complex predominantly consists of β-propeller and α-solenoidal proteins. Here we present the structure of the Utp4 subunit from the thermophilic fungus Chaetomium thermophilum at 2.15 Å resolution and analyze its function by UV RNA-crosslinking (CRAC) and in context of a recent cryo-EM structure of the 90S pre-ribosome. Utp4 consists of two orthogonal and highly basic β-propellers that perfectly fit the EM-data. The Utp4 structure highlights an unusual Velcro-closure of its C-terminal β-propeller as relevant for protein integrity and potentially Utp8 recognition in the context of the pre-ribosome. We provide a first model of the 5'-ETS RNA from the internally hidden 5'-end up to the region that hybridizes to the 3'-hinge sequence of U3 snoRNA and validate a specific Utp4/5'-ETS interaction by CRAC analysis

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

Model of co-transcriptional assembly of the 90S pre-ribosome.

<p>The nascent 5'-ETS (black line) recruits the early 90S modules (UTP-A, UTP-B, and U3 snoRNP) in a hierarchical fashion, with the UTP-A complex being the first one that binds to the extreme 5'-end of the pre-rRNA. This early assembly intermediate, together with the subsequently transcribed pre-18S rRNA (yellow line) and additional factors, forms the 90S pre-ribosome. Complexes are labeled accordingly. The 3'-hinge region is highlighted in pink. Figure is adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178752#pone.0178752.ref009" target="_blank">9</a>].</p

Uncommon Velcro-closure of the C-terminal β-propeller 2.

<p><b>(A)</b> Schematic representation of the last blade 14 of Utp4. The four β-strands of the blade are represented as arrows in different colours: 14A and B (C-terminus of β-propeller 2, red), 14C (N-terminus of β-propeller 1, blue), and 14D ((His)₆-TEV-tag, grey). <b>(B)</b> Close-up of blade 14 complemented by the very N-terminus of the polypeptide chain, forming an uncommon parallel β-strand 14C (blue) and the artificial TEV site (grey) forming an antiparallel β-strand 14D. The highly conserved residues and their hydrogen-bonding network stabilizing the blade and therefore the Velcro-closure of β-propeller 2 are represented in sticks. Salt-bridges are indicated by dashed lines.</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

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