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.This work was supported by Deutsche Forschungsgemeinschaft (DFG) (SFB638, Z4 to I. S. and HU363/15-1 to E.H. and the Leibniz programme to I.S.); Cluster of Excellence CellNetworks (EcTOP1 to I.S. and E.H.); Funding for open access charge: DFG [Leibniz Programme]. M.K. was funded by a Kekule Fellowship (VCI)

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

Explorando el plegamiento metallo-beta-lactamasa en el genoma de Acinetobacter baumanni

Acinetobacter baumannii fue considerado siempre como un patógeno de relativa baja virulencia, pero durante las dos últimas décadas, este microorganismo oportunista ha emergido como uno de los mayores problemas encarados por el sistema clínico en hospitales de todo el mundo. Como consecuencia inevitable de la presión selectiva impuesta por el uso abusivo de antibióticos en el tratamiento de infecciones, se han descrito cepas clínicas multirresistentes de A. baumannii, con una alta capacidad para desarrollar diferentes mecanismos de resistencia. Atendiendo a los antibióticos β-lactámicos, el principal mecanismo de resistencia es la producción de β-lactamasas, que son enzimas bacterianas capaces de degradar el antibiótico antes de que alcance su diana en la bacteria, permitiéndole a ésta una gran facilidad de adaptación al medio hospitalario y de crecimiento en asociación con su hospedador humano. Estas enzimas pueden ser clasificadas en diferentes grupos en base a su secuencia de amino ácidos o acorde a su perfil hidrolítico de sustratos e inhibidores. Dentro de esa clasificación, en este trabajo nos hemos centrado en la clase B de las β-lactamasas, que son un grupo de proteínas con una conservada estructura tridimensional, pertenecientes a una conocida superfamilia de proteínas, las metallo-β-lactamasas. Esta superfamilia de las metallo-β-lactamasas fue por primera vez descrita por Neuwald en 1997. Sus miembros presentan una ubicua distribución a través de los tres dominios biológicos, lo cual sugiere una gran importancia funcional así como un origen ancestral. Son proteínas que se caracterizan por compartir un estable plegamiento tridimensional compuesto por cuatro regiones bien definidas, que son dos grupos centrales de láminas-beta rodeados a ambos lados por αlfa-hélices que quedan expuestas al solvente. En todas las estructuras conocidas hasta el momento, el sitio activo se sitúa entre los dos grupos de láminas beta enfrentadas, dónde uno o dos átomos metálicos, preferencialmente zinc, queda unido por una fuerte interacción con los residuos altamente conservados que confeccionan el motivo característico de esta superfamilia en su sitio activo. Este canónico plegamiento representa el mínimo dominio necesario para definir a una proteína funcional dentro de esta superfamilia, aunque gracias a diversos dominios adicionales el rango de reacciones químicas que estas proteínas pueden catalizar ha sido ampliamente extendido. Los diversos miembros fueron clasificados en 16 grupos, describiendo su variedad estructural adicional al plegamiento canónico y sus distintas funciones biológicas. Recientemente dentro de esta clasificación, un nuevo grupo 0 fue definido, el cual incluye dos enzimas sin actividad conocida por el momento, pero con un característico dominio adicional al canónico de metalo-β-lactamasa. Con el presente estudio, quisimos identificar nuevos posibles miembros de esta superfamilia dentro del genoma de A. baumannii, que a su vez pudieran intervenir de algún modo en el mecanismo general de resistencia de este conocido patógeno. A través de un meticuloso análisis de su genoma, dos genes fueron identificados, ABAYE3862 y ABAYE0164, codificando para dos proteínas putativas pertenecientes a la superfamilia de las metalo-β-actamasas. La predicción de su estructura secundaria sugirió el canónico plegamiento en αββα de esta superfamilia, llamando nuestra atención el que ninguna de ellas presentase el conservado motivo (HXHXD/H) de unión a zinc en el sitio activo. Estas dos proteínas fueron bautizadas como aMBL-1 y aMBL-2, respectivamente, de acinetobacter Metallo-Beta-Lactamase. Esos dos genes, ABAYE3862 y ABAYE0164, fueron clonados a partir del ADN genómico de A.baumannii y las respectivas proteínas a las que codifican, aMBL-1 y aMBL-2, fueron caracterizadas mediante las correspondientes técnicas biofísicas y bioquímicas. Además, su estructura tridimensional fue resuelta mediante cristalografía de rayos X, haciendo uso para ello de técnicas de faseo experimental, habiendo marcado previamente ambas proteínas con átomos pesados. Estas estructuras representan los primeros datos estructurales de dos nuevos miembros de esta superfamilia ya que ambas proteínas muestran el típico plegamiento metalo-beta-lactamasa aunque con ciertas particularidades. Ambas exhiben un estado de ligomerización mayoritariamente dimérico en solución, con un alongado C-terminal. En el caso de aMBL-1 representa un dominio adicional que por su similitud con los restantes miembros del grupo 0 de la MBL superfamilia podría darnos algún indicio del posible papel de aMBL-1 como transportador de señales extracelulares dentro de la bacteria. En cambio para aMBL-2, esa extensión del C-terminal es la que facilita la formación del dímero dejando los dos sitios activos de cada monómero enfrentados dentro de la U que forman ambas cadenas. Además, los residuos del presunto sitio activo de aMBL-1 difieren ampliamente del canónico, mientras que los de aMBL-2 se asemejan bastante al motivo (HXHXD/H) de unión a zinc e incluso un átomo del metal pudo ser definido (aunque con baja ocupación) en su estructura. Con aMBL-2 creemos haber contribuido en la comprensión de la evolución de la diversidad molecular que caracteriza a esta superfamilia. La proteína aMBL-2 podría definirse como una beta-lactamasa de clase B ancestral que Acinetobacter aún conservar en su genoma, con un sitio activo inmaduro y una modesta actividad hidrolítica frente a los antibióticos beta-lactámicos

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