RESUMEN
In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery-persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.
Asunto(s)
Daño del ADN , ARN Polimerasa II/metabolismo , Reparación del ADN , Células HEK293 , Humanos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Ubiquitinación , Rayos UltravioletaRESUMEN
The concept of specialized ribosomes has garnered equal amounts of interest and skepticism since it was first introduced. We ask researchers in the field to provide their perspective on the topic and weigh in on the evidence (or lack thereof) and what the future may bring.
Asunto(s)
Biosíntesis de Proteínas , Ribosomas , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
The nucleolus is best known for housing the highly ordered assembly line that produces ribosomal subunits. The >100 ribosome assembly factors in the nucleolus are thought to cycle between two states: an operative state (when integrated into subunit assembly intermediates) and a latent state (upon release from intermediates). Although it has become commonplace to refer to the nucleolus as "being a multilayered condensate," and this may be accurate for latent factors, there is little reason to think that such assertions pertain to the operative state of assembly factors.
Asunto(s)
Nucléolo Celular , ARN RibosómicoRESUMEN
Eukaryotic genomes generate a heterogeneous ensemble of mRNAs and long noncoding RNAs (lncRNAs). LncRNAs and mRNAs are both transcribed by Pol II and acquire 5' caps and poly(A) tails, but only mRNAs are translated into proteins. To address how these classes are distinguished, we identified the transcriptome-wide targets of 13 RNA processing, export, and turnover factors in budding yeast. Comparing the maturation pathways of mRNAs and lncRNAs revealed that transcript fate is largely determined during 3' end formation. Most lncRNAs are targeted for nuclear RNA surveillance, but a subset with 3' cleavage and polyadenylation features resembling the mRNA consensus can be exported to the cytoplasm. The Hrp1 and Nab2 proteins act at this decision point, with dual roles in mRNA cleavage/polyadenylation and lncRNA surveillance. Our data also reveal the dynamic and heterogeneous nature of mRNA maturation, and highlight a subset of "lncRNA-like" mRNAs regulated by the nuclear surveillance machinery.
Asunto(s)
ARN de Hongos/metabolismo , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcriptoma , Procesamiento Postranscripcional del ARN , ARN de Hongos/química , ARN Largo no Codificante/química , ARN Mensajero/química , Ribonucleoproteínas/química , Saccharomyces cerevisiae/genéticaRESUMEN
MicroRNAs (miRNAs) play key roles in gene regulation, but reliable bioinformatic or experimental identification of their targets remains difficult. To provide an unbiased view of human miRNA targets, we developed a technique for ligation and sequencing of miRNA-target RNA duplexes associated with human AGO1. Here, we report data sets of more than 18,000 high-confidence miRNA-mRNA interactions. The binding of most miRNAs includes the 5' seed region, but around 60% of seed interactions are noncanonical, containing bulged or mismatched nucleotides. Moreover, seed interactions are generally accompanied by specific, nonseed base pairing. 18% of miRNA-mRNA interactions involve the miRNA 3' end, with little evidence for 5' contacts, and some of these were functionally validated. Analyses of miRNA:mRNA base pairing showed that miRNA species systematically differ in their target RNA interactions, and strongly overrepresented motifs were found in the interaction sites of several miRNAs. We speculate that these affect the response of RISC to miRNA-target binding.
Asunto(s)
Perfilación de la Expresión Génica , Técnicas Genéticas , MicroARNs/metabolismo , ARN Mensajero/metabolismo , Proteínas Argonautas/genética , Factores Eucarióticos de Iniciación/genética , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , MicroARNs/química , Motivos de Nucleótidos , ARN Mensajero/química , ARN no Traducido/química , ARN no Traducido/metabolismo , Complejo Silenciador Inducido por ARN/metabolismoRESUMEN
Transcription elongation rates influence RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S. cerevisiae. Mapping RNAPI by Miller chromatin spreads or UV crosslinking revealed 5' enrichment and strikingly uneven local polymerase occupancy along the rDNA, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution: folding energy and GC content in the transcription bubble. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high GC in the transcription bubble slows elongation. A mathematical model for RNAPI elongation confirmed the importance of nascent RNA folding in transcription. RNAPI from S. pombe was similarly sensitive to transcript folding, as were S. cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site use, indicating regulatory significance for transcript folding.
Asunto(s)
ARN Polimerasa III/genética , ARN Polimerasa II/genética , ARN Polimerasa I/genética , ARN de Hongos/química , Saccharomyces cerevisiae/genética , Elongación de la Transcripción Genética , Composición de Base , Secuencia de Bases , Sitios de Unión , Cromatina/química , Cromatina/metabolismo , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Regulación Fúngica de la Expresión Génica , Unión Proteica , Pliegue del ARN , ARN Polimerasa I/metabolismo , ARN Polimerasa II/metabolismo , ARN Polimerasa III/metabolismo , Sitios de Empalme de ARN , Empalme del ARN , ARN de Hongos/genética , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , TermodinámicaRESUMEN
Cellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNA association. Among translation components, RNA association was most reduced for initiation factors involved in 40S scanning (eukaryotic initiation factor 4A [eIF4A], eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5' ends of mRNAs. Following glucose withdrawal, 5' binding was abolished within 30 s, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5' RNA binding by initiation factors over â¼16 min and provoked mRNA degradation, particularly for translation-related factors, mediated by Xrn1. Taken together, these results reveal mechanisms underlying translational control of gene expression during stress.
Asunto(s)
Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas/fisiología , Estrés Fisiológico/fisiología , Regiones no Traducidas 5' , ARN Helicasas DEAD-box/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Glucosa/metabolismo , Respuesta al Choque Térmico/fisiología , Factores de Iniciación de Péptidos/fisiología , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
In this issue of Molecular Cell, Webster et al. (2018) and Yi et al. (2018) dissect the mechanisms underlying cytoplasmic mRNA deadenylation by the Ccr4-Not (CNOT) complex. Crucial to this process is the poly(A) binding protein Pab1/PABPC1, which both stimulates and suppresses the activity of different deadenylases.
Asunto(s)
Proteínas de Unión a Poli(A) , ARN Mensajero , Proteínas de Unión al ARNRESUMEN
The RNA-binding protein HuD promotes neurogenesis and favors recovery from peripheral axon injury. HuD interacts with many mRNAs, altering both stability and translation efficiency. We generated a nucleotide resolution map of the HuD RNA interactome in motor neuron-like cells, identifying HuD target sites in 1,304 mRNAs, almost exclusively in the 3' UTR. HuD binds many mRNAs encoding mTORC1-responsive ribosomal proteins and translation factors. Altered HuD expression correlates with the translation efficiency of these mRNAs and overall protein synthesis, in a mTORC1-independent fashion. The predominant HuD target is the abundant, small non-coding RNA Y3, amounting to 70% of the HuD interaction signal. Y3 functions as a molecular sponge for HuD, dynamically limiting its recruitment to polysomes and its activity as a translation and neuron differentiation enhancer. These findings uncover an alternative route to the mTORC1 pathway for translational control in motor neurons that is tunable by a small non-coding RNA.
Asunto(s)
Proteína 4 Similar a ELAV/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Neuronas Motoras/fisiología , ARN Pequeño no Traducido/genética , Regiones no Traducidas 3' , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Animales , Línea Celular , Proteína 4 Similar a ELAV/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Neuronas Motoras/metabolismo , Neurogénesis/genética , Polirribosomas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/metabolismoRESUMEN
The RNA-interacting proteome is commonly characterized by UV-crosslinking followed by RNA purification, with protein recovery quantified using SILAC labeling followed by data-dependent acquisition (DDA) of proteomic data. However, the low efficiency of UV-crosslinking, combined with limited sensitivity of the DDA approach often restricts detection to relatively abundant proteins, necessitating multiple mass spec injections of fractionated peptides for each biological sample. Here we report an application of data-independent acquisition (DIA) with SILAC in a total RNA-associated protein purification (TRAPP) UV-crosslinking experiment. This gave 15% greater protein detection and lower inter-replicate variation relative to the same biological materials analyzed using DDA, while allowing single-shot analysis of the sample. As proof of concept, we determined the effects of arsenite treatment on the RNA-bound proteome of HEK293T cells. The DIA dataset yielded similar GO term enrichment for RNA-binding proteins involved in cellular stress responses to the DDA dataset while detecting extra proteins unseen by DDA. Overall, the DIA SILAC approach improved detection of proteins over conventional DDA SILAC for generating RNA-interactome datasets, at a lower cost due to reduced machine time. Analyses are described for TRAPP data, but the approach is suitable for proteomic analyses following essentially any RNA-binding protein enrichment technique.
Asunto(s)
Proteómica , Proteínas de Unión al ARN , Humanos , Células HEK293 , Espectrometría de Masas/métodos , Péptidos/análisis , Proteoma/metabolismo , Proteómica/métodos , Proteínas de Unión al ARN/análisisRESUMEN
In budding yeast, the nuclear RNA surveillance system is active on all pre-mRNA transcripts and modulated by nutrient availability. To test the role of nuclear surveillance in reprogramming gene expression, we identified transcriptome-wide binding sites for RNA polymerase II and the exosome cofactors Mtr4 (TRAMP complex) and Nab3 (NNS complex) by UV crosslinking immediately following glucose withdrawal (0, 4, and 8 min). In glucose, mRNA binding by Nab3 and Mtr4 was mainly restricted to promoter-proximal sites, reflecting early transcription termination. Following glucose withdrawal, many growth-related mRNAs showed reduced transcription but increased Nab3 binding, accompanied by downstream recruitment of Mtr4, and oligo(A) tailing. We conclude that transcription termination is followed by TRAMP-mediated RNA decay. Upregulated transcripts evaded increased surveillance factor binding following glucose withdrawal. Some upregulated genes showed use of alternative transcription starts to bypass strong NNS binding sites. We conclude that nuclear surveillance pathways regulate both positive and negative responses to glucose availability.
Asunto(s)
Núcleo Celular/metabolismo , Regulación Fúngica de la Expresión Génica , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , ARN Nuclear/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Adaptación Fisiológica , Sitios de Unión , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Glucosa/deficiencia , Glucosa/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN de Hongos/genética , ARN Mensajero/genética , ARN Nuclear/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de TiempoRESUMEN
Wilms' tumor 1 (WT1) is essential for the development and homeostasis of multiple mesodermal tissues. Despite evidence for post-transcriptional roles, no endogenous WT1 target RNAs exist. Using RNA immunoprecipitation and UV cross-linking, we show that WT1 binds preferentially to 3' untranslated regions (UTRs) of developmental targets. These target mRNAs are down-regulated upon WT1 depletion in cell culture and developing kidney mesenchyme. Wt1 deletion leads to rapid turnover of specific mRNAs. WT1 regulates reporter gene expression through interaction with 3' UTR-binding sites. Combining experimental and computational analyses, we propose that WT1 influences key developmental and disease processes in part through regulating mRNA turnover.
Asunto(s)
Regiones no Traducidas 3'/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , ARN Mensajero/genética , Tumor de Wilms/genética , Tumor de Wilms/metabolismo , Animales , Línea Celular , Regulación hacia Abajo , Eliminación de Gen , Riñón/citología , Mesodermo/metabolismo , Ratones , Células Madre Embrionarias de Ratones , ARN Mensajero/metabolismoRESUMEN
From the earliest comparisons of RNA production with steady-state levels, it has been clear that cells transcribe more RNA than they accumulate, implying the existence of active RNA degradation systems. In general, RNA is degraded at the end of its useful life, which is long for a ribosomal RNA but very short for excised introns or spacer fragments, and is closely regulated for most mRNA species. RNA molecules with defects in processing, folding, or assembly with proteins are identified and rapidly degraded by the surveillance machinery. Because RNA degradation is ubiquitous in all cells, it is clear that it must be carefully controlled to accurately recognize target RNAs. How this is achieved is perhaps the most pressing question in the field.
Asunto(s)
Redes y Vías Metabólicas , Estabilidad del ARN , Animales , Humanos , Ribonucleoproteínas/metabolismo , Transcripción GenéticaRESUMEN
Ssd1, a conserved fungal RNA-binding protein, is important in stress responses, cell division and virulence. Ssd1 is closely related to Dis3L2 of the RNase II family of nucleases, but lacks catalytic activity and likely suppresses translation of bound mRNAs. Previous studies identified RNA motifs enriched in Ssd1-associated transcripts, yet the sequence requirements for Ssd1 binding are not defined. Here, we identify precise binding sites of Ssd1 on RNA using in vivo cross-linking and cDNA analysis. These sites are enriched in 5' untranslated regions of a subset of mRNAs encoding cell wall proteins. We identified a conserved bipartite motif that binds Ssd1 with high affinity in vitro. Active RNase II enzymes have a characteristic, internal RNA binding path; the Ssd1 crystal structure at 1.9 Å resolution shows that remnants of regulatory sequences block this path. Instead, RNA binding activity has relocated to a conserved patch on the surface of the protein. Structure-guided mutations of this surface prevent Ssd1 from binding RNA in vitro and phenocopy Ssd1 deletion in vivo. These studies provide a new framework for understanding the function of a pleiotropic post-transcriptional regulator of gene expression and give insights into the evolution of regulatory and binding elements in the RNase II family.
Asunto(s)
Exorribonucleasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Regiones no Traducidas 5' , Exorribonucleasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Eukaryotic genomes generate vast numbers of non-protein-coding RNAs (ncRNAs) that can inhibit mRNA synthesis through transcription interference, but the mechanisms are unclear. Gill et al. show that transcription of antisense ncRNAs induces 'elongation marks' on histones in promoter regions. These inhibit active nucleosome positioning required to maintain open transcription-initiation sites.
Asunto(s)
Nucleosomas , ARN no Traducido , Histonas/metabolismo , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Systematic analyses, by UV crosslinking, of the precise binding sites for 23 different proteins across the yeast pre-mRNA population have given insights into the in vivo assembly of, and interactions between, pre-mRNA processing, packaging, and transport complexes.
Asunto(s)
Acetaldehído/metabolismo , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Embrión de Mamíferos/metabolismo , Etanol/toxicidad , Proteína del Grupo de Complementación A de la Anemia de Fanconi/genética , Anemia de Fanconi/patología , Animales , Femenino , Humanos , EmbarazoRESUMEN
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
The biogenesis of eukaryotic RNA polymerases is poorly understood. The present study used a combination of genetic and molecular approaches to explore the assembly of RNA polymerase III (Pol III) in yeast. We identified a regulatory link between Rbs1, a Pol III assembly factor, and Rpb10, a small subunit that is common to three RNA polymerases. Overexpression of Rbs1 increased the abundance of both RPB10 mRNA and the Rpb10 protein, which correlated with suppression of Pol III assembly defects. Rbs1 is a poly(A)mRNA-binding protein and mutational analysis identified R3H domain to be required for mRNA interactions and genetic enhancement of Pol III biogenesis. Rbs1 also binds to Upf1 protein, a key component in nonsense-mediated mRNA decay (NMD) and levels of RPB10 mRNA were increased in a upf1Δ strain. Genome-wide RNA binding by Rbs1 was characterized by UV cross-linking based approach. We demonstrated that Rbs1 directly binds to the 3' untranslated regions (3'UTRs) of many mRNAs including transcripts encoding Pol III subunits, Rpb10 and Rpc19. We propose that Rbs1 functions by opposing mRNA degradation, at least in part mediated by NMD pathway. Orthologues of Rbs1 protein are present in other eukaryotes, including humans, suggesting that this is a conserved regulatory mechanism.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Genoma Fúngico , ARN Helicasas/genética , ARN Polimerasa III/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Secuencia Conservada , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Humanos , Degradación de ARNm Mediada por Codón sin Sentido , Unión Proteica/efectos de la radiación , ARN Helicasas/metabolismo , ARN Polimerasa III/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Rayos UltravioletaRESUMEN
Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , Ribonucleótido Reductasas/genética , Ribonucleótidos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Daño del ADN , Desoxirribonucleótidos/metabolismo , Genoma Fúngico , Inestabilidad Genómica , Mutación , Ribonucleasa H/genética , Ribonucleasas/genética , Puntos de Control de la Fase S del Ciclo Celular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Eliminación de SecuenciaRESUMEN
U3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 ß-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 ß-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 ß-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5'-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures.