RESUMEN
Ribosome production is essential for cell growth. Approximately 200 assembly factors drive this complicated pathway that starts in the nucleolus and ends in the cytoplasm. A large number of structural snapshots of the pre-60S pathway have revealed the principles behind large subunit synthesis. To view this SnapShot, open or download the PDF.
Asunto(s)
Nucléolo Celular , Células Eucariotas , Ribosomas , Nucléolo Celular/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/química , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ribosomas/metabolismo , Células Eucariotas/química , Células Eucariotas/citología , Células Eucariotas/metabolismoRESUMEN
Ribosome production is vital for every cell, and failure causes human diseases. It is driven by â¼200 assembly factors functioning along an ordered pathway from the nucleolus to the cytoplasm. Structural snapshots of biogenesis intermediates from the earliest 90S pre-ribosomes to mature 40S subunits unravel the mechanisms of small ribosome synthesis. To view this SnapShot, open or download the PDF.
Asunto(s)
Células Eucariotas , Ribosomas , Humanos , Nucléolo Celular/metabolismo , Células Eucariotas/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/metabolismoRESUMEN
Ribosomes, which synthesize the proteins of a cell, comprise ribosomal RNA and ribosomal proteins, which coassemble hierarchically during a process termed ribosome biogenesis. Historically, biochemical and molecular biology approaches have revealed how preribosomal particles form and mature in consecutive steps, starting in the nucleolus and terminating after nuclear export into the cytoplasm. However, only recently, due to the revolution in cryo-electron microscopy, could pseudoatomic structures of different preribosomal particles be obtained. Together with in vitro maturation assays, these findings shed light on how nascent ribosomes progress stepwise along a dynamic biogenesis pathway. Preribosomes assemble gradually, chaperoned by a myriad of assembly factors and small nucleolar RNAs, before they reach maturity and enter translation. This information will lead to a better understanding of how ribosome synthesis is linked to other cellular pathways in humans and how it can cause diseases, including cancer, if disturbed.
Asunto(s)
Eucariontes/metabolismo , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Nucléolo Celular/metabolismo , Microscopía por Crioelectrón , Humanos , Biogénesis de Organelos , Multimerización de ProteínaRESUMEN
Eukaryotic 60S ribosomal subunits are comprised of three rRNAs and â¼50 ribosomal proteins. The initial steps of their formation take place in the nucleolus, but, owing to a lack of structural information, this process is poorly understood. Using cryo-EM, we solved structures of early 60S biogenesis intermediates at 3.3 Å to 4.5 Å resolution, thereby providing insights into their sequential folding and assembly pathway. Besides revealing distinct immature rRNA conformations, we map 25 assembly factors in six different assembly states. Notably, the Nsa1-Rrp1-Rpf1-Mak16 module stabilizes the solvent side of the 60S subunit, and the Erb1-Ytm1-Nop7 complex organizes and connects through Erb1's meandering N-terminal extension, eight assembly factors, three ribosomal proteins, and three 25S rRNA domains. Our structural snapshots reveal the order of integration and compaction of the six major 60S domains within early nucleolar 60S particles developing stepwise from the solvent side around the exit tunnel to the central protuberance.
Asunto(s)
Chaetomium/química , Biogénesis de Organelos , Subunidades Ribosómicas Grandes de Eucariotas/química , Chaetomium/citología , Microscopía por Crioelectrón , Redes y Vías Metabólicas , Modelos Moleculares , Pliegue del ARN , Ribonucleoproteínas/químicaRESUMEN
The 90S pre-ribosome is an early biogenesis intermediate formed during co-transcriptional ribosome formation, composed of â¼70 assembly factors and several small nucleolar RNAs (snoRNAs) that associate with nascent pre-rRNA. We report the cryo-EM structure of the Chaetomium thermophilum 90S pre-ribosome, revealing how a network of biogenesis factors including 19 ß-propellers and large α-solenoid proteins engulfs the pre-rRNA. Within the 90S pre-ribosome, we identify the UTP-A, UTP-B, Mpp10-Imp3-Imp4, Bms1-Rcl1, and U3 snoRNP modules, which are organized around 5'-ETS and partially folded 18S rRNA. The U3 snoRNP is strategically positioned at the center of the 90S particle to perform its multiple tasks during pre-rRNA folding and processing. The architecture of the elusive 90S pre-ribosome gives unprecedented structural insight into the early steps of pre-rRNA maturation. Nascent rRNA that is co-transcriptionally folded and given a particular shape by encapsulation within a dedicated mold-like structure is reminiscent of how polypeptides use chaperone chambers for their protein folding.
Asunto(s)
Chaetomium/química , Biogénesis de Organelos , Ribosomas/química , Saccharomyces cerevisiae/química , Chaetomium/clasificación , Microscopía por Crioelectrón , Modelos Moleculares , ARN Ribosómico 18S/química , Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Ribosomas/ultraestructuraRESUMEN
The exosome regulates the processing, degradation, and surveillance of a plethora of RNA species. However, little is known about how the exosome recognizes and is recruited to its diverse substrates. We report the identification of adaptor proteins that recruit the exosome-associated helicase, Mtr4, to unique RNA substrates. Nop53, the yeast homolog of the tumor suppressor PICT1, targets Mtr4 to pre-ribosomal particles for exosome-mediated processing, while a second adaptor Utp18 recruits Mtr4 to cleaved rRNA fragments destined for degradation by the exosome. Both Nop53 and Utp18 contain the same consensus motif, through which they dock to the "arch" domain of Mtr4 and target it to specific substrates. These findings show that the exosome employs a general mechanism of recruitment to defined substrates and that this process is regulated through adaptor proteins.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Exosomas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ribosómicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Animales , Ascomicetos/química , Ascomicetos/clasificación , Ascomicetos/genética , ARN Helicasas DEAD-box/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/química , Conformación de Ácido Nucleico , ARN de Hongos/química , ARN de Hongos/metabolismo , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/química , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Alineación de SecuenciaRESUMEN
Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes to form channels across the nuclear envelope. They are large macromolecular assemblies with a complex composition and diverse functions. Apart from facilitating nucleocytoplasmic transport, NPCs are involved in chromatin organization, the regulation of gene expression and DNA repair. Understanding the molecular mechanisms underlying these functions has been hampered by a lack of structural knowledge about the NPC. The recent convergence of crystallographic and biochemical in vitro analysis of nucleoporins (NUPs), the components of the NPC, with cryo-electron microscopic imaging of the entire NPC in situ has provided first pseudo-atomic view of its central core and revealed that an unexpected network of short linear motifs is an important spatial organization principle. These breakthroughs have transformed the way we understand NPC structure, and they provide an important base for functional investigations, including the elucidation of the molecular mechanisms underlying clinically manifested mutations of the nucleocytoplasmic transport system.
Asunto(s)
Proteínas de Complejo Poro Nuclear/genética , Poro Nuclear/química , Poro Nuclear/metabolismo , Transporte Activo de Núcleo Celular , Humanos , Mutación , Proteínas de Complejo Poro Nuclear/metabolismoRESUMEN
Ribosome assembly is catalyzed by numerous trans-acting factors and coupled with irreversible pre-rRNA processing, driving the pathway toward mature ribosomal subunits. One decisive step early in this progression is removal of the 5' external transcribed spacer (5'-ETS), an RNA extension at the 18S rRNA that is integrated into the huge 90S pre-ribosome structure. Upon endo-nucleolytic cleavage at an internal site, A1, the 5'-ETS is separated from the 18S rRNA and degraded. Here we present biochemical and cryo-electron microscopy analyses that depict the RNA exosome, a major 3'-5' exoribonuclease complex, in a super-complex with the 90S pre-ribosome. The exosome is docked to the 90S through its co-factor Mtr4 helicase, a processive RNA duplex-dismantling helicase, which strategically positions the exosome at the base of 5'-ETS helices H9-H9', which are dislodged in our 90S-exosome structures. These findings suggest a direct role of the exosome in structural remodeling of the 90S pre-ribosome to drive eukaryotic ribosome synthesis.
Asunto(s)
ARN Helicasas DEAD-box/química , Endorribonucleasas/química , Exonucleasas/química , Complejo Multienzimático de Ribonucleasas del Exosoma/ultraestructura , ARN Ribosómico 18S/química , Ribosomas/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Sitios de Unión , Microscopía por Crioelectrón , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Exonucleasas/genética , Exonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Modelos Moleculares , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estabilidad del ARN , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Ribosome assembly is driven by numerous assembly factors, including the Rix1 complex and the AAA ATPase Rea1. These two assembly factors catalyze 60S maturation at two distinct states, triggering poorly understood large-scale structural transitions that we analyzed by cryo-electron microscopy. Two nuclear pre-60S intermediates were discovered that represent previously unknown states after Rea1-mediated removal of the Ytm1-Erb1 complex and reveal how the L1 stalk develops from a pre-mature nucleolar to a mature-like nucleoplasmic state. A later pre-60S intermediate shows how the central protuberance arises, assisted by the nearby Rix1-Rea1 machinery, which was solved in its pre-ribosomal context to molecular resolution. This revealed a Rix12-Ipi32 tetramer anchored to the pre-60S via Ipi1, strategically positioned to monitor this decisive remodeling. These results are consistent with a general underlying principle that temporarily stabilized immature RNA domains are successively remodeled by assembly factors, thereby ensuring failsafe assembly progression.
Asunto(s)
Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Microscopía por Crioelectrón , Escherichia coli/genética , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Eukaryotic ribosome biogenesis involves RNA folding and processing that depend on assembly factors and small nucleolar RNAs (snoRNAs). The 90S (SSU-processome) is the earliest pre-ribosome structurally analyzed, which was suggested to assemble stepwise along the growing pre-rRNA from 5' > 3', but this directionality may not be accurate. Here, by analyzing the structure of a series of 90S assembly intermediates from Chaetomium thermophilum, we discover a reverse order of 18S rRNA subdomain incorporation. Large parts of the 18S rRNA 3' and central domains assemble first into the 90S before the 5' domain is integrated. This final incorporation depends on a contact between a heterotrimer Enp2-Bfr2-Lcp5 recruited to the flexible 5' domain and Kre33, which reconstitutes the Kre33-Enp-Brf2-Lcp5 module on the compacted 90S. Keeping the 5' domain temporarily segregated from the 90S scaffold could provide extra time to complete the multifaceted 5' domain folding, which depends on a distinct set of snoRNAs and processing factors.
Asunto(s)
Chaetomium/metabolismo , Proteínas Fúngicas/metabolismo , Conformación de Ácido Nucleico , ARN de Hongos/metabolismo , ARN Ribosómico 18S/metabolismo , Ribosomas/metabolismo , Chaetomium/genética , Proteínas Fúngicas/genética , ARN de Hongos/genética , ARN Ribosómico 18S/genética , Ribosomas/genéticaRESUMEN
Despite decades of research, the structure and assembly of the nuclear pore complex (NPC), which is composed of â¼30 nucleoporins (Nups), remain elusive. Here, we report the genome of the thermophilic fungus Chaetomium thermophilum (ct) and identify the complete repertoire of Nups therein. The thermophilic proteins show improved properties for structural and biochemical studies compared to their mesophilic counterparts, and purified ctNups enabled the reconstitution of the inner pore ring module that spans the width of the NPC from the anchoring membrane to the central transport channel. This module is composed of two large Nups, Nup192 and Nup170, which are flexibly bridged by short linear motifs made up of linker Nups, Nic96 and Nup53. This assembly illustrates how Nup interactions can generate structural plasticity within the NPC scaffold. Our findings therefore demonstrate the utility of the genome of a thermophilic eukaryote for studying complex molecular machines.
Asunto(s)
Chaetomium/citología , Genoma Fúngico , Poro Nuclear/metabolismo , Secuencia de Aminoácidos , Chaetomium/genética , Proteínas Fúngicas/metabolismo , Datos de Secuencia Molecular , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Filogenia , Alineación de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
During ribosome biogenesis a plethora of assembly factors and essential enzymes drive the unidirectional maturation of nascent pre-ribosomal subunits. The DEAD-box RNA helicase Dbp10 is suggested to restructure pre-ribosomal rRNA of the evolving peptidyl-transferase center (PTC) on nucleolar ribosomal 60S assembly intermediates. Here, we show that point mutations within conserved catalytic helicase-core motifs of Dbp10 yield a dominant-lethal growth phenotype. Such dbp10 mutants, which stably associate with pre-60S intermediates, impair pre-60S biogenesis at a nucleolar stage prior to the release of assembly factor Rrp14 and stable integration of late nucleolar factors such as Noc3. Furthermore, the binding of the GTPase Nug1 to particles isolated directly via mutant Dbp10 bait proteins is specifically inhibited. The N-terminal domain of Nug1 interacts with Dbp10 and the methyltransferase Spb1, whose pre-60S incorporation is also reduced in absence of functional Dbp10 resulting in decreased methylation of 25S rRNA nucleotide G2922. Our data suggest that Dbp10's helicase activity generates the necessary framework for assembly factor docking thereby permitting PTC rRNA methylation and the progression of pre-60S maturation.
Asunto(s)
Peptidil Transferasas , Proteínas de Saccharomyces cerevisiae , Peptidil Transferasas/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ribosomas/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Ribosome biogenesis proceeds along a multifaceted pathway from the nucleolus to the cytoplasm that is extensively coupled to several quality control mechanisms. However, the mode by which 5S ribosomal RNA is incorporated into the developing pre-60S ribosome, which in humans links ribosome biogenesis to cell proliferation by surveillance by factors such as p53-MDM2, is poorly understood. Here, we report nine nucleolar pre-60S cryo-EM structures from Chaetomium thermophilum, one of which clarifies the mechanism of 5S RNP incorporation into the early pre-60S. Successive assembly states then represent how helicases Dbp10 and Spb4, and the Pumilio domain factor Puf6 act in series to surveil the gradual folding of the nearby 25S rRNA domain IV. Finally, the methyltransferase Spb1 methylates a universally conserved guanine nucleotide in the A-loop of the peptidyl transferase center, thereby licensing further maturation. Our findings provide insight into the hierarchical action of helicases in safeguarding rRNA tertiary structure folding and coupling to surveillance mechanisms that culminate in local RNA modification.
Asunto(s)
ARN Ribosómico , Proteínas de Saccharomyces cerevisiae , Humanos , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Ribosomas/genética , ARN Ribosómico 5S/genética , ARN Ribosómico 5S/metabolismo , ADN Helicasas/metabolismo , Unión Proteica , Proteínas Ribosómicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The rixosome defined in Schizosaccharomyces pombe and humans performs diverse roles in pre-ribosomal RNA processing and gene silencing. Here, we isolate and describe the conserved rixosome from Chaetomium thermophilum, which consists of two sub-modules, the sphere-like Rix1-Ipi3-Ipi1 and the butterfly-like Las1-Grc3 complex, connected by a flexible linker. The Rix1 complex of the rixosome utilizes Sda1 as landing platform on nucleoplasmic pre-60S particles to wedge between the 5S rRNA tip and L1-stalk, thereby facilitating the 180° rotation of the immature 5S RNP towards its mature conformation. Upon rixosome positioning, the other sub-module with Las1 endonuclease and Grc3 polynucleotide-kinase can reach a strategic position at the pre-60S foot to cleave and 5' phosphorylate the nearby ITS2 pre-rRNA. Finally, inward movement of the L1 stalk permits the flexible Nop53 N-terminus with its AIM motif to become positioned at the base of the L1-stalk to facilitate Mtr4 helicase-exosome participation for completing ITS2 removal. Thus, the rixosome structure elucidates the coordination of two central ribosome biogenesis events, but its role in gene silencing may adapt similar strategies.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces , Humanos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Nucleares/metabolismo , Rotación , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas Ribosómicas/genéticaRESUMEN
Deubiquitinating enzymes (DUBs) regulate diverse cellular functions by cleaving ubiquitin from specific protein substrates. How their activities are modulated in various cellular contexts remains poorly understood. The yeast deubiquitinase Ubp8 protein is recruited and activated by the SAGA complex and, together with Sgf11, Sus1, and Sgf73, forms a DUB module responsible for deubiquitinating histone H2B during gene expression. Here, we report the crystal structure of the complete SAGA DUB module, which features two functional lobes structurally coupled by Sgf73. In the "assembly lobe," a long Sgf11 N-terminal helix is clamped onto the Ubp8 ZnF-UBP domain by Sus1. In the "catalytic lobe," an Sgf11 C-terminal zinc-finger domain binds to the Ubp8 catalytic domain next to its active site. Our structural and functional analyses reveal a central role of Sgf11 and Sgf73 in activating Ubp8 for deubiquitinating histone H2B and demonstrate how a DUB can be allosterically regulated by its nonsubstrate partners.
Asunto(s)
Endopeptidasas/química , Endopeptidasas/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Histona Acetiltransferasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Saccharomyces cerevisiae , Alineación de Secuencia , Factores de Transcripción/metabolismo , UbiquitinaRESUMEN
The dynein-related AAA ATPase Rea1 is a preribosomal factor that triggers an unknown maturation step in 60S subunit biogenesis. Using electron microscopy, we show that Rea1's motor domain is docked to the pre-60S particle and its tail-like structure, harboring a metal ion-dependent adhesion site (MIDAS), protrudes from the preribosome. Typically, integrins utilize a MIDAS to bind extracellular ligands, an interaction that is strengthened under applied tensile force. Likewise, the Rea1 MIDAS binds the preribosomal factor Rsa4, which is located on the pre-60S subunit at a site that is contacted by the flexible Rea1 tail. The MIDAS-Rsa4 interaction is essential for ATP-dependent dissociation of a group of non-ribosomal factors from the pre-60S particle. Thus, Rea1 aligns with its interacting partners on the preribosome to effect a necessary step on the path to the export-competent 60S subunit.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/ultraestructura , Adenosina Trifosfato/metabolismo , Citoplasma/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/ultraestructura , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
The transition of the 90S to the pre-40S pre-ribosome is a decisive step in eukaryotic small subunit biogenesis leading to a first pre-40S intermediate (state Dis-C or primordial pre-40S), where the U3 snoRNA keeps the nascent 18S rRNA locally immature. We in vitro reconstitute the ATP-dependent U3 release from this particle, catalyzed by the helicase Dhr1, and follow this process by cryo-EM revealing two successive pre-40S intermediates, Dis-D and Dis-E. The latter has lost not only U3 but all residual 90S factors including the GTPase Bms1. In vitro remodeling likewise induced the formation of the central pseudoknot, a universally conserved tertiary RNA structure that comprises the core of the small subunit decoding center. Thus, we could structurally reveal a key tertiary RNA folding step that is essential to form the active 40S subunit.
Asunto(s)
Precursores del ARN , ARN Ribosómico 18S , ARN Nucleolar Pequeño , Subunidades Ribosómicas Pequeñas de Eucariotas , Precursores del ARN/genética , ARN Ribosómico 18S/genética , ARN Nucleolar Pequeño/genética , Saccharomyces cerevisiae/genética , Conformación de Ácido Nucleico , Subunidades Ribosómicas Pequeñas de Eucariotas/genéticaRESUMEN
Biogenesis of the small ribosomal subunit in eukaryotes starts in the nucleolus with the formation of a 90S precursor and ends in the cytoplasm. Here, we elucidate the enigmatic structural transitions of assembly intermediates from human and yeast cells during the nucleoplasmic maturation phase. After dissociation of all 90S factors, the 40S body adopts a close-to-mature conformation, whereas the 3' major domain, later forming the 40S head, remains entirely immature. A first coordination is facilitated by the assembly factors TSR1 and BUD23-TRMT112, followed by re-positioning of RRP12 that is already recruited early to the 90S for further head rearrangements. Eventually, the uS2 cluster, CK1 (Hrr25 in yeast) and the export factor SLX9 associate with the pre-40S to provide export competence. These exemplary findings reveal the evolutionary conserved mechanism of how yeast and humans assemble the 40S ribosomal subunit, but reveal also a few minor differences.
Asunto(s)
Transporte Activo de Núcleo Celular , Proteínas Ribosómicas , Subunidades Ribosómicas Pequeñas de Eucariotas , Proteínas de Saccharomyces cerevisiae , Humanos , Quinasa de la Caseína I/análisis , Quinasa de la Caseína I/metabolismo , Metiltransferasas/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The thermophilic fungus Chaetomium thermophilum has been used extensively for biochemical and high-resolution structural studies of protein complexes. However, subsequent functional analyses of these assemblies have been hindered owing to the lack of genetic tools compatible with this thermophile, which are typically suited to other mesophilic eukaryotic model organisms, in particular the yeast Saccharomyces cerevisiae. Hence, we aimed to find genes from C. thermophilum that are expressed under the control of different sugars and examine their associated 5' untranslated regions as promoters responsible for sugar-regulated gene expression. To identify sugar-regulated promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes with induced expression in the presence of xylose but repressed expression in glucose-supplemented media. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of a thermostable yellow fluorescent protein (YFP) reporter. With this, we demonstrated xylose-dependent YFP expression by both Western blotting and live-cell imaging fluorescence microscopy. Prompted by these results, we expressed the C. thermophilum orthologue of a well-characterized dominant-negative ribosome assembly factor mutant, under the control of the XDH promoter, which allowed us to induce a nuclear export defect on the pre-60S subunit when C. thermophilum cells were grown in xylose- but not glucose-containing medium. Altogether, our study identified xylose-regulatable promoters in C. thermophilum, which might facilitate functional studies of genes of interest in this thermophilic eukaryotic model organism.
Asunto(s)
Chaetomium , Azúcares , Azúcares/metabolismo , Xilosa/metabolismo , Chaetomium/genética , Chaetomium/metabolismo , Saccharomyces cerevisiae/genética , Glucosa/metabolismoRESUMEN
The rapidly evolving internal transcribed spacer 2 (ITS2) in the pre-ribosomal RNA is one of the most commonly applied phylogenetic markers at species and genus level. Yet, during ribosome biogenesis ITS2 is removed in all eukaryotes by a common, but still unknown, mechanism. Here we describe the existence of an RNA processome, assembled from four conserved subunits, Las1-Grc3-Rat1-Rai1, that carries all the necessary RNA processing enzymes to mediate coordinated ITS2 rRNA removal. Las1 is the long-sought-after endonuclease cleaving 27SB pre-rRNA at site C2 to yield a 5'-OH end at the 26S pre-rRNA and 2',3' cyclic phosphate at the 3' end of 7S pre-rRNA. Subsequently, polynucleotide kinase Grc3 catalyzes ATP-dependent 5'-OH phosphorylation of 26S pre-rRNA, which in turn enables Rat1-Rai1 exonuclease to generate 25S' pre-rRNA. ITS2 processing is reminiscent of tRNA splicing, but instead of subsequent tRNA ligation, the Las1 complex carries along an exonuclease tool to degrade the ITS2 rRNA.