RESUMEN
Protein synthesis is a major energy-consuming process of the cell that requires the controlled production1-3 and turnover4,5 of ribosomes. Although the past few years have seen major advances in our understanding of ribosome biogenesis, structural insight into the degradation of ribosomes has been lacking. Here we present native structures of two distinct small ribosomal 30S subunit degradation intermediates associated with the 3' to 5' exonuclease ribonuclease R (RNase R). The structures reveal that RNase R binds at first to the 30S platform to facilitate the degradation of the functionally important anti-Shine-Dalgarno sequence and the decoding-site helix 44. RNase R then encounters a roadblock when it reaches the neck region of the 30S subunit, and this is overcome by a major structural rearrangement of the 30S head, involving the loss of ribosomal proteins. RNase R parallels this movement and relocates to the decoding site by using its N-terminal helix-turn-helix domain as an anchor. In vitro degradation assays suggest that head rearrangement poses a major kinetic barrier for RNase R, but also indicate that the enzyme alone is sufficient for complete degradation of 30S subunits. Collectively, our results provide a mechanistic basis for the degradation of 30S mediated by RNase R, and reveal that RNase R targets orphaned 30S subunits using a dynamic mechanism involving an anchored switching of binding sites.
Asunto(s)
Exorribonucleasas , Proteínas Ribosómicas , Ribosomas , Exorribonucleasas/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Cinética , Sitios de UniónRESUMEN
RNA-binding proteins (RBPs) govern the lifespan of nearly all transcripts and play key roles in adaptive responses in microbes. A robust approach to examine protein-RNA interactions involves irradiating cells with UV light to form covalent adducts between RBPs and their cognate RNAs. Combined with RNA or protein purification, these procedures can provide global RBP censuses or transcriptomic maps for all target sequences of a single protein in living cells. The recent development of novel methods has quickly populated the RBP landscape in microorganisms. Here, we provide an overview of prominent UV cross-linking techniques which have been applied to investigate RNA interactomes in microbes. By assessing their advantages and caveats, this technical evaluation intends to guide the selection of appropriate methods and experimental design as well as to encourage the use of complementary UV-dependent techniques to inspect RNA-binding activity.
Asunto(s)
ARN , Rayos Ultravioleta , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Perfilación de la Expresión Génica/métodos , TranscriptomaRESUMEN
Termination of RNA polymerase II (RNAPII) transcription is associated with RNA 3' end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in Saccharomyces cerevisiae does not rely on RNA cleavage for termination but instead terminates via a pathway that requires the Nrd1-Nab3-Sen1 (NNS) complex. Here we show that the Schizosaccharomyces pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3' end processing factors, is enriched at the 3' end of genes, and binds RNA motifs downstream from cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3' untranslated region (UTR) length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3' end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 cotranscriptionally controls alternative polyadenylation.
Asunto(s)
Poliadenilación/genética , Procesamiento Postranscripcional del ARN/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Secuencias de Aminoácidos , Dominios Proteicos , ARN Polimerasa II , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Elongación de la Transcripción GenéticaRESUMEN
The emergence of new pathogens is a major threat to public and veterinary health. Changes in bacterial habitat such as a switch in host or disease tropism are typically accompanied by genetic diversification. Staphylococcus aureus is a multi-host bacterial species associated with human and livestock infections. A microaerophilic subspecies, Staphylococcus aureus subsp. anaerobius, is responsible for Morel's disease, a lymphadenitis restricted to sheep and goats. However, the evolutionary history of S. aureus subsp. anaerobius and its relatedness to S. aureus are unknown. Population genomic analyses of clinical S. aureus subsp. anaerobius isolates revealed a highly conserved clone that descended from a S. aureus progenitor about 1000 years ago before differentiating into distinct lineages that contain African and European isolates. S. aureus subsp. anaerobius has undergone limited clonal expansion, with a restricted population size, and an evolutionary rate 10-fold slower than S. aureus. The transition to its current restricted ecological niche involved acquisition of a pathogenicity island encoding a ruminant host-specific effector of abscess formation, large chromosomal re-arrangements, and the accumulation of at least 205 pseudogenes, resulting in a highly fastidious metabolism. Importantly, expansion of ~87 insertion sequences (IS) located largely in intergenic regions provided distinct mechanisms for the control of expression of flanking genes, including a novel mechanism associated with IS-mediated anti-anti-sense decoupling of ancestral gene repression. Our findings reveal the remarkable evolutionary trajectory of a host-restricted bacterial pathogen that resulted from extensive remodelling of the S. aureus genome through an array of diverse mechanisms in parallel.
Asunto(s)
Genoma Bacteriano/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus/genética , Animales , Evolución Biológica , Ecosistema , Genómica , Humanos , Ganado , Filogenia , Transcriptoma , Secuenciación Completa del GenomaRESUMEN
In bacteria, Hfq is a core RNA chaperone that catalyzes the interaction of mRNAs with regulatory small RNAs (sRNAs). To determine in vivo RNA sequence requirements for Hfq interactions, and to study riboregulation in a bacterial pathogen, Hfq was UV crosslinked to RNAs in enterohemorrhagic Escherichia coli (EHEC). Hfq bound repeated trinucleotide motifs of A-R-N (A-A/G-any nucleotide) often associated with the Shine-Dalgarno translation initiation sequence in mRNAs. These motifs overlapped or were adjacent to the mRNA sequences bound by sRNAs. In consequence, sRNA-mRNA duplex formation will displace Hfq, promoting recycling. Fifty-five sRNAs were identified within bacteriophage-derived regions of the EHEC genome, including some of the most abundant Hfq-interacting sRNAs. One of these (AgvB) antagonized the function of the core genome regulatory sRNA, GcvB, by mimicking its mRNA substrate sequence. This bacteriophage-encoded "anti-sRNA" provided EHEC with a growth advantage specifically in bovine rectal mucus recovered from its primary colonization site in cattle.
Asunto(s)
Escherichia coli O157/virología , Profagos/genética , ARN Pequeño no Traducido/metabolismo , ARN Viral/genética , Animales , Secuencia de Bases , Sitios de Unión , Bovinos , Secuencia de Consenso , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/metabolismo , Proteína de Factor 1 del Huésped/metabolismo , Datos de Secuencia Molecular , Moco/microbiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/genética , ARN Viral/metabolismoRESUMEN
Previous high-throughput studies in Gram-negative bacteria identified a large number of 3'UTR fragments that potentially function as sRNAs. Here we extensively characterize the MalH sRNA. We show that MalH is a stable degradation intermediate derived from the 3' end of malG, which is part of the maltose uptake operon transcript malEFG. Unlike the majority of bacterial sRNAs, MalH is transiently expressed during the transition from the exponential to the stationary growth phase, suggesting that it contributes to adaptation to changes in nutrient availability. Over-expression of MalH reduces expression of general outer membrane porins and MicA, a repressor of the high-affinity maltose/maltodextrin transporter LamB. Disrupting MalH production and function significantly reduces lamB accumulation when maltose is the only available carbon source, presumably due to the accumulation of the MicA repressor. We propose that MalH is part of a regulatory network that, during the transition phase, directly or indirectly promotes accumulation of high-affinity maltose transporters in the outer membrane by dampening competing pathways.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Carbono/metabolismo , Proteínas de Escherichia coli/genética , Porinas/genética , ARN Bacteriano/genética , ARN Mensajero/genética , ARN Pequeño no Traducido/genética , Receptores Virales/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Bacteriana de la Expresión Génica , Maltosa/metabolismo , Operón/genética , Porinas/metabolismo , Unión Proteica , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/metabolismo , RNA-Seq/métodos , Receptores Virales/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
In vivo UV crosslinking identified numerous preribosomal RNA (pre-rRNA) binding sites for the large, highly conserved ribosome synthesis factor Rrp5. Intramolecular complementation has shown that the C-terminal domain (CTD) of Rrp5 is required for pre-rRNA cleavage at sites A0-A2 on the pathway of 18S rRNA synthesis, whereas the N-terminal domain (NTD) is required for A3 cleavage on the pathway of 5.8S/25S rRNA synthesis. The CTD was crosslinked to sequences flanking A2 and to the snoRNAs U3, U14, snR30, and snR10, which are required for cleavage at A0-A2. The NTD was crosslinked to sequences flanking A3 and to the RNA component of ribonuclease MRP, which cleaves site A3. Rrp5 could also be directly crosslinked to several large structural proteins and nucleoside triphosphatases. A key role in coordinating preribosomal assembly and processing was confirmed by chromatin spreads. Following depletion of Rrp5, cotranscriptional cleavage was lost and preribosome compaction greatly reduced.
Asunto(s)
Proteínas Fúngicas/genética , Precursores del ARN/genética , Procesamiento Postranscripcional del ARN , ARN de Hongos/genética , ARN Ribosómico/genética , Ribosomas/genética , Secuencia de Bases , Sitios de Unión , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Proteínas Fúngicas/metabolismo , Datos de Secuencia Molecular , Nucleósido-Trifosfatasa/genética , Nucleósido-Trifosfatasa/metabolismo , Precursores del ARN/metabolismo , ARN Ribosómico/metabolismo , Levaduras/genética , Levaduras/metabolismoRESUMEN
We uncovered a novel role for the spliceosome in regulating mRNA expression levels that involves splicing coupled to RNA decay, which we refer to as spliceosome-mediated decay (SMD). Our transcriptome-wide studies identified numerous transcripts that are not known to have introns but are spliced by the spliceosome at canonical splice sites in Saccharomyces cerevisiae. Products of SMD are primarily degraded by the nuclear RNA surveillance machinery. We demonstrate that SMD can significantly down-regulate mRNA levels; splicing at canonical splice sites in the bromodomain factor 2 (BDF2) transcript reduced transcript levels roughly threefold by generating unstable products that are rapidly degraded by the nuclear surveillance machinery. Regulation of BDF2 mRNA levels by SMD requires Bdf1, a functionally redundant Bdf2 paralog that plays a role in recruiting the spliceosome to the BDF2 mRNA. Interestingly, mutating BDF2 5' splice site and branch point consensus sequences partially suppresses the bdf1Δ temperature-sensitive phenotype, suggesting that maintaining proper levels of Bdf2 via SMD is biologically important. We propose that the spliceosome can also repress protein-coding gene expression by promoting nuclear turnover of spliced RNA products and provide an insight for coordinated regulation of Bdf1 and Bdf2 levels in the cell.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Estabilidad del ARN , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , Mutación , Fenotipo , ARN/genética , Empalme del ARN , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Structure probing coupled with high-throughput sequencing could revolutionize our understanding of the role of RNA structure in regulation of gene expression. Despite recent technological advances, intrinsic noise and high sequence coverage requirements greatly limit the applicability of these techniques. Here we describe a probabilistic modeling pipeline that accounts for biological variability and biases in the data, yielding statistically interpretable scores for the probability of nucleotide modification transcriptome wide. Using two yeast data sets, we demonstrate that our method has increased sensitivity, and thus our pipeline identifies modified regions on many more transcripts than do existing pipelines. Our method also provides confident predictions at much lower sequence coverage levels than those recommended for reliable structural probing. Our results show that statistical modeling extends the scope and potential of transcriptome-wide structure probing experiments.
Asunto(s)
Algoritmos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Modelos Estadísticos , ARN/química , ARN/genética , Análisis de Secuencia de ARN/métodos , Transcriptoma/genética , Emparejamiento Base , Secuencia de Bases , Biología Computacional/métodos , Humanos , Conformación de Ácido NucleicoRESUMEN
Eukaryotic ribosomes are assembled by a complex pathway that extends from the nucleolus to the cytoplasm and is powered by many energy-consuming enzymes. Nuclear export is a key, irreversible step in pre-ribosome maturation, but mechanisms underlying the timely acquisition of export competence remain poorly understood. Here we show that a conserved Saccharomyces cerevisiae GTPase Nug2 (also known as Nog2, and as NGP-1, GNL2 or nucleostemin 2 in human) has a key role in the timing of export competence. Nug2 binds the inter-subunit face of maturing, nucleoplasmic pre-60S particles, and the location clashes with the position of Nmd3, a key pre-60S export adaptor. Nug2 and Nmd3 are not present on the same pre-60S particles, with Nug2 binding before Nmd3. Depletion of Nug2 causes premature Nmd3 binding to the pre-60S particles, whereas mutations in the G-domain of Nug2 block Nmd3 recruitment, resulting in severe 60S export defects. Two pre-60S remodelling factors, the Rea1 ATPase and its co-substrate Rsa4, are present on Nug2-associated particles, and both show synthetic lethal interactions with nug2 mutants. Release of Nug2 from pre-60S particles requires both its K(+)-dependent GTPase activity and the remodelling ATPase activity of Rea1. We conclude that Nug2 is a regulatory GTPase that monitors pre-60S maturation, with release from its placeholder site linked to recruitment of the nuclear export machinery.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Núcleo Celular/metabolismo , GTP Fosfohidrolasas/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Citoplasma/metabolismo , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Genes Letales/genética , Modelos Moleculares , Mutación/genética , Potasio/metabolismo , Unión Proteica , Estructura Terciaria de Proteína/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/química , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
In eukaryotes, ribosome biogenesis requires folding and assembly of the precursor rRNA (pre-rRNA) with a large number of proteins and snoRNPs into huge RNA-protein complexes. In spite of intense genetic, biochemical and high-resolution cryo-EM studies in Saccharomyces cerevisiae, information about the structure of the 35S pre-rRNA is limited. To overcome this, we performed high-throughput SHAPE chemical probing on the 35S pre-rRNA within 90S pre-ribosomes. We focused our analyses on external (5'ETS) and internal (ITS1) transcribed spacers as well as the 18S rRNA region. We show that in the 35S pre-rRNA, the central pseudoknot is not formed and the central core of the 18S rRNA is in an open configuration but becomes more constrained in 20S pre-rRNA. The essential ribosome biogenesis protein Mrd1 influences the structure of the 18S rRNA region locally and is involved in organizing the central pseudoknot and surrounding structures. We demonstrate that U3 snoRNA dynamically interacts with the 35S pre-rRNA and that Mrd1 is required for disrupting U3 snoRNA base pairing interactions in the 5'ETS. We propose that the dynamic U3 snoRNA interactions and Mrd1 are essential for establishing the structure of the central core of 18S rRNA that is required for processing and 40S subunit function.
Asunto(s)
Conformación de Ácido Nucleico , ARN Nucleolar Pequeño/genética , Proteínas de Unión al ARN/genética , Ribosomas/genética , Proteínas de Saccharomyces cerevisiae/genética , Sitios de Unión , Nucléolo Celular/química , Nucléolo Celular/genética , Precursores del ARN/genética , ARN Ribosómico 18S/genética , ARN Nucleolar Pequeño/química , Ribosomas/química , Saccharomyces cerevisiae/genéticaRESUMEN
The prokaryotic RNA chaperone Hfq mediates sRNA-mRNA interactions and plays a significant role in post-transcriptional regulation of the type III secretion (T3S) system produced by a range of Escherichia coli pathotypes. UV-crosslinking was used to map Hfq-binding under conditions that promote T3S and multiple interactions were identified within polycistronic transcripts produced from the locus of enterocyte effacement (LEE) that encodes the T3S system. The majority of Hfq binding was within the LEE5 and LEE4 operons, the latter encoding the translocon apparatus (SepL-EspADB) that is positively regulated by the RNA binding protein, CsrA. Using the identified Hfq-binding sites and a series of sRNA deletions, the sRNA Spot42 was shown to directly repress translation of LEE4 at the sepL 5' UTR. In silico and in vivo analyses of the sepL mRNA secondary structure combined with expression studies of truncates indicated that the unbound sepL mRNA is translationally inactive. Based on expression studies with site-directed mutants, an OFF-ON-OFF toggle model is proposed that results in transient translation of SepL and EspA filament assembly. Under this model, the nascent mRNA is translationally off, before being activated by CsrA, and then repressed by Hfq and Spot42.
Asunto(s)
Traslocación Bacteriana/genética , Proteínas de Escherichia coli/genética , Proteína de Factor 1 del Huésped/genética , Fosfoproteínas/genética , Proteínas de Unión al ARN/genética , Proteínas Represoras/genética , Sitios de Unión/genética , Citoesqueleto/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Conformación de Ácido Nucleico/efectos de la radiación , ARN/genética , ARN Mensajero/genética , ARN Mensajero/efectos de la radiación , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/efectos de la radiación , Rayos UltravioletaRESUMEN
Box C/D snoRNAs are known to guide site-specific ribose methylation of ribosomal RNA. Here, we demonstrate a novel and unexpected role for box C/D snoRNAs in guiding 18S rRNA acetylation in yeast. Our results demonstrate, for the first time, that the acetylation of two cytosine residues in 18S rRNA catalyzed by Kre33 is guided by two orphan box C/D snoRNAs-snR4 and snR45 -not known to be involved in methylation in yeast. We identified Kre33 binding sites on these snoRNAs as well as on the 18S rRNA, and demonstrate that both snR4 and snR45 establish extended bipartite complementarity around the cytosines targeted for acetylation, similar to pseudouridylation pocket formation by the H/ACA snoRNPs. We show that base pairing between these snoRNAs and 18S rRNA requires the putative helicase activity of Kre33, which is also needed to aid early pre-rRNA processing. Compared to yeast, the number of orphan box C/D snoRNAs in higher eukaryotes is much larger and we hypothesize that several of these may be involved in base-modifications.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN Ribosómico 18S/metabolismo , ARN Nuclear Pequeño/metabolismo , Acetilación , Acetiltransferasas/química , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Sitios de Unión , Citosina/metabolismo , Unión Proteica , ARN Ribosómico 18S/genética , ARN Nuclear Pequeño/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Regulación Fúngica de la Expresión Génica , Precursores del ARN/metabolismo , ARN Nucleolar Pequeño/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Emparejamiento Base , Secuencia de Bases , Frío , ARN Helicasas DEAD-box/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Mutación Puntual , Biosíntesis de Proteínas , Precursores del ARN/química , Precursores del ARN/genética , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Yeast Npl3 is a highly abundant, nuclear-cytoplasmic shuttling, RNA-binding protein, related to metazoan SR proteins. Reported functions of Npl3 include transcription elongation, splicing and RNA 3' end processing. We used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites, and strand-specific tiling arrays to look at the effects of loss of Npl3 on all transcripts across the genome. We found that Npl3 binds diverse RNA species, both coding and non-coding, at sites indicative of roles in both early pre-mRNA processing and 3' end formation. Tiling arrays and RNAPII mapping data revealed 3' extended RNAPII-transcribed RNAs in the absence of Npl3, suggesting that defects in pre-mRNA packaging events result in termination readthrough. Transcription readthrough was widespread and frequently resulted in down-regulation of neighboring genes. We conclude that the absence of Npl3 results in widespread 3' extension of transcripts with pervasive effects on gene expression.
Asunto(s)
Proteínas Nucleares/genética , Proteínas de Unión al ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Terminación de la Transcripción Genética , Regiones no Traducidas 3' , Proteínas Nucleares/metabolismo , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. However, there is scarce knowledge about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. RESULTS: Here, we reveal that the RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25's endogenous RNA targets and protein binding partners. We demonstrate that TRIM25 controls the levels of Zinc Finger Antiviral Protein (ZAP). Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP. CONCLUSIONS: Our results suggest that many other proteins with the PRY/SPRY domain could have yet uncharacterized RNA-binding potential. Together, our data reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor in their enzymatic activity.
Asunto(s)
Dominio B30.2-SPRY , ARN/metabolismo , Factores de Transcripción/genética , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Yeast ribosome synthesis requires 19 different RNA helicases, but none of their pre-rRNA-binding sites were previously known, making their precise functions difficult to determine. Here we identify multiple binding sites for the helicase Prp43 in the 18S and 25S rRNA regions of pre-rRNAs, using UV crosslinking. Binding in 18S was predominantly within helix 44, close to the site of 18S 3' cleavage, in which Prp43 is functionally implicated. Four major binding sites were identified in 25S, including helix 34. In strains depleted of Prp43 or expressing only catalytic point mutants, six snoRNAs that guide modifications close to helix 34 accumulated on preribosomes, implicating Prp43 in their release, whereas other snoRNAs showed reduced preribosome association. Prp43 was crosslinked to snoRNAs that target sequences close to its binding sites, indicating direct interactions. We propose that Prp43 acts on preribosomal regions surrounding each binding site, with distinct functions at different locations.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Precursores del ARN/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Reactivos de Enlaces Cruzados/metabolismo , ARN Helicasas DEAD-box/deficiencia , Datos de Secuencia Molecular , Mutación/genética , Conformación de Ácido Nucleico , Unión Proteica , ARN de Hongos/química , ARN de Hongos/genética , ARN Ribosómico/metabolismo , ARN Nuclear Pequeño/metabolismo , ARN Nucleolar Pequeño/metabolismoRESUMEN
Ribosome assembly in eukaryotes involves the activity of hundreds of assembly factors that direct the hierarchical assembly of ribosomal proteins and numerous ribosomal RNA folding steps. However, detailed insights into the function of assembly factors and ribosomal RNA folding events are lacking. To address this, we have developed ChemModSeq, a method that combines structure probing, high-throughput sequencing and statistical modeling, to quantitatively measure RNA structural rearrangements during the assembly of macromolecular complexes. By applying ChemModSeq to purified 40S assembly intermediates we obtained nucleotide-resolution maps of ribosomal RNA flexibility revealing structurally distinct assembly intermediates and mechanistic insights into assembly dynamics not readily observed in cryo-electron microscopy reconstructions. We show that RNA restructuring events coincide with the release of assembly factors and predict that completion of the head domain is required before the Rio1 kinase enters the assembly pathway. Collectively, our results suggest that 40S assembly factors regulate the timely incorporation of ribosomal proteins by delaying specific folding steps in the 3' major domain of the 20S pre-ribosomal RNA.
Asunto(s)
Precursores del ARN/química , ARN Ribosómico/química , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Secuenciación de Nucleótidos de Alto Rendimiento , Modelos Moleculares , Modelos Estadísticos , Conformación de Ácido Nucleico , Nucleótidos/química , Precursores del ARN/aislamiento & purificación , ARN Ribosómico/aislamiento & purificación , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ARNRESUMEN
During the last step in 40S ribosome subunit biogenesis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously characterized pre-40S components, but retain the endonuclease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in actively growing cells. Nob1 and Pno1 bind overlapping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Modelos Moleculares , Proteínas Nucleares/metabolismo , División del ARN , Precursores del ARN/metabolismo , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismoRESUMEN
The 5'-exonuclease Rat1 degrades pre-rRNA spacer fragments and processes the 5'-ends of the 5.8S and 25S rRNAs. UV crosslinking revealed multiple Rat1-binding sites across the pre-rRNA, consistent with its known functions. The major 5.8S 5'-end is generated by Rat1 digestion of the internal transcribed spacer 1 (ITS1) spacer from cleavage site A(3). Processing from A(3) requires the 'A(3)-cluster' proteins, including Cic1, Erb1, Nop7, Nop12 and Nop15, which show interdependent pre-rRNA binding. Surprisingly, A(3)-cluster factors were not crosslinked close to site A(3), but bound sites around the 5.8S 3'- and 25S 5'-regions, which are base paired in mature ribosomes, and in the ITS2 spacer that separates these rRNAs. In contrast, Nop4, a protein required for endonucleolytic cleavage in ITS1, binds the pre-rRNA near the 5'-end of 5.8S. ITS2 was reported to undergo structural remodelling. In vivo chemical probing indicates that A(3)-cluster binding is required for this reorganization, potentially regulating the timing of processing. We predict that Nop4 and the A(3) cluster establish long-range interactions between the 5.8S and 25S rRNAs, which are subsequently maintained by ribosomal protein binding.