RESUMEN
27 years after the yeast genome sequencing, the function of many ORFs remain unknown. Despite the evolutionary distance between human and yeast, homology with the conserved DEAH/DExH-box helicase domains allowed us to list DHX29, DHX36 and DHX57 as three putative homologs of the yeast Ylr419wp. Functional studies first linked the Ylr419w protein to the translating ribosome and cross-linking and analysis of cDNA (CRAC) experiments determined the precise region of Ylr419wp in contact with the ribosome. It corresponds to the loop of the h16 helix in the 18S rRNA designing the translation initiation factor DHX29, as the functional homolog of Ylr419wp.
RESUMEN
Translation initiation is a complex and highly regulated process that represents an important mechanism, controlling gene expression. eIF2A was proposed as an alternative initiation factor, however, its role and biological targets remain to be discovered. To further gain insight into the function of eIF2A in Saccharomyces cerevisiae, we identified mRNAs associated with the eIF2A complex and showed that 24% of the most enriched mRNAs encode proteins related to cell wall biogenesis and maintenance. In agreement with this result, we showed that an eIF2A deletion sensitized cells to cell wall damage induced by calcofluor white. eIF2A overexpression led to a growth defect, correlated with decreased synthesis of several cell wall proteins. In contrast, no changes were observed in the transcriptome, suggesting that eIF2A controls the expression of cell wall-related proteins at a translational level. The biochemical characterization of the eIF2A complex revealed that it strongly interacts with the RNA binding protein, Ssd1, which is a negative translational regulator, controlling the expression of cell wall-related genes. Interestingly, eIF2A and Ssd1 bind several common mRNA targets and we found that the binding of eIF2A to some targets was mediated by Ssd1. Surprisingly, we further showed that eIF2A is physically and functionally associated with the exonuclease Xrn1 and other mRNA degradation factors, suggesting an additional level of regulation. Altogether, our results highlight new aspects of this complex and redundant fine-tuned regulation of proteins expression related to the cell wall, a structure required to maintain cell shape and rigidity, providing protection against harmful environmental stress.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ARN Mensajero/metabolismo , Pared Celular/genética , Pared Celular/metabolismo , Expresión Génica , Regulación Fúngica de la Expresión GénicaRESUMEN
mRNA degradation is one of the main steps of gene expression, and a key player is the 5'-3' exonuclease Xrn1. In Saccharomyces cerevisiae , it was previously shown, by a microscopy approach, that Xrn1 is located to different cellular compartments, depending on physiological state. During exponential growth, Xrn1 is distributed in the cytoplasm, while it co-localizes with eisosomes after the post-diauxic shift (PDS). Here, we biochemically characterize the Xrn1-associated complexes in different cellular states. We demonstrate that, after PDS, Xrn1 but not the decapping nor Lsm1-7/Pat1 complexes associates with eisosomal proteins, strengthening the model that sequestration of Xrn1 in eisosomes preserves mRNAs from degradation during PDS.
RESUMEN
Gene deletion and gene expression alteration can lead to growth defects that are amplified or reduced when a second mutation is present in the same cells. We performed 154 genetic interaction mapping (GIM) screens with query mutants related with RNA metabolism and estimated the growth rates of about 700 000 double mutant Saccharomyces cerevisiae strains. The tested targets included the gene deletion collection and 900 strains in which essential genes were affected by mRNA destabilization (DAmP). To analyze the results, we developed RECAP, a strategy that validates genetic interaction profiles by comparison with gene co-citation frequency, and identified links between 1471 genes and 117 biological processes. In addition to these large-scale results, we validated both enhancement and suppression of slow growth measured for specific RNA-related pathways. Thus, negative genetic interactions identified a role for the OCA inositol polyphosphate hydrolase complex in mRNA translation initiation. By analysis of suppressors, we found that Puf4, a Pumilio family RNA binding protein, inhibits ribosomal protein Rpl9 function, by acting on a conserved UGUAcauUA motif located downstream the stop codon of the RPL9B mRNA. Altogether, the results and their analysis should represent a useful resource for discovery of gene function in yeast.
Asunto(s)
Genes Fúngicos , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/genética , Alelos , Eliminación de Gen , Pleiotropía Genética , Fosfatos de Inositol/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
A large share of the non-coding transcriptome in yeast is controlled by the Nrd1-Nab3-Sen1 (NNS) complex, which promotes transcription termination of non-coding RNA (ncRNA) genes, and by the nuclear exosome, which limits the steady-state levels of the transcripts produced. How unconstrained ncRNA levels affect RNA metabolism and gene expression are long-standing and important questions. Here, we show that degradation of ncRNAs by the exosome is required for freeing Nrd1 and Nab3 from the released transcript after termination. In exosome mutants, these factors are sequestered by ncRNAs and cannot be efficiently recycled to sites of transcription, inducing termination defects at NNS targets. ncRNA-dependent, genome-wide termination defects can be recapitulated by the expression of a degradation-resistant, circular RNA containing a natural NNS target in exosome-proficient cells. Our results have important implications for the mechanism of termination, the general impact of ncRNAs abundance, and the importance of nuclear ncRNA degradation.
Asunto(s)
Estabilidad del ARN/genética , ARN no Traducido/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Terminación de la Transcripción Genética , Núcleo Celular/metabolismo , Exosomas/metabolismo , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Modelos Genéticos , ARN de Hongos/genética , ARN no Traducido/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Transcriptoma/genéticaRESUMEN
The Ski2-Ski3-Ski8 (SKI) complex assists the RNA exosome during the 3' to 5' degradation of cytoplasmic transcripts. Previous reports showed that the SKI complex is involved in the 3' to 5' degradation of mRNAs, including 3' untranslated regions (UTRs) and devoid of ribosomes. Paradoxically, we recently showed that the SKI complex directly interacts with ribosomes during the co-translational mRNA decay and that this interaction is necessary for its RNA degradation promoting activity. Here, we characterised a new SKI-associated factor, Ska1, that associates with a subpopulation of the SKI complex. We showed that Ska1 is specifically involved in the degradation of long 3'UTR-containing mRNAs, poorly translated mRNAs as well as other RNA regions not associated with ribosomes, such as cytoplasmic lncRNAs. We further show that the overexpression of SKA1 antagonises the SKI-ribosome association. We propose that the Ska1-SKI complex assists the cytoplasmic exosome in the absence of direct association of the SKI complex with ribosomes.
Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Citoplasma/genética , Estabilidad del ARN , ARN de Hongos/química , ARN Largo no Codificante/química , ARN Mensajero/química , Saccharomyces cerevisiae/metabolismoRESUMEN
Messenger RNA (mRNA) homeostasis represents an essential part of gene expression, in which the generation of mRNA by RNA polymerase is counter-balanced by its degradation by nucleases. The conserved 5'-to-3' exoribonuclease Xrn1 has a crucial role in eukaryotic mRNA homeostasis by degrading decapped or cleaved mRNAs post-translationally and, more surprisingly, also co-translationally. Here we report that active Xrn1 can directly and specifically interact with the translation machinery. A cryo-electron microscopy structure of a programmed Saccharomyces cerevisiae 80S ribosome-Xrn1 nuclease complex reveals how the conserved core of Xrn1 enables binding at the mRNA exit site of the ribosome. This interface provides a conduit for channelling of the mRNA from the ribosomal decoding site directly into the active center of the nuclease, thus separating mRNA decoding from degradation by only 17 ± 1 nucleotides. These findings explain how rapid 5'-to-3' mRNA degradation is coupled efficiently to its final round of mRNA translation.
Asunto(s)
Exorribonucleasas/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Microscopía por Crioelectrón , Exorribonucleasas/genética , Exorribonucleasas/ultraestructura , ARN Mensajero/metabolismo , Ribosomas/genética , Ribosomas/ultraestructura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructuraRESUMEN
Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway involved in many cellular pathways and crucial for telomere maintenance and embryo development. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals, but a universal NMD model is lacking. We used affinity purification coupled with mass spectrometry and an improved data analysis protocol to characterize the composition and dynamics of yeast NMD complexes in yeast (112 experiments). Unexpectedly, we identified two distinct complexes associated with Upf1: Upf1-23 (Upf1, Upf2, Upf3) and Upf1-decappingUpf1-decapping contained the mRNA decapping enzyme, together with Nmd4 and Ebs1, two proteins that globally affected NMD and were critical for RNA degradation mediated by the Upf1 C-terminal helicase region. The fact that Nmd4 association with RNA was partially dependent on Upf1-23 components and the similarity between Nmd4/Ebs1 and mammalian Smg5-7 proteins suggest that NMD operates through conserved, successive Upf1-23 and Upf1-decapping complexes. This model can be extended to accommodate steps that are missing in yeast, to serve for further mechanistic studies of NMD in eukaryotes.
Asunto(s)
Modelos Biológicos , Complejos Multiproteicos/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , ARN Helicasas/metabolismo , ARN de Hongos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complejos Multiproteicos/genética , ARN Helicasas/genética , ARN de Hongos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Pervasive transcription generates many unstable non-coding transcripts in budding yeast. The transcription of such noncoding RNAs, in particular antisense RNAs (asRNAs), has been shown in a few examples to repress the expression of the associated mRNAs. Yet, such mechanism is not known to commonly contribute to the regulation of a given class of genes. Using a mutant context that stabilized pervasive transcripts, we observed that the least expressed mRNAs during the exponential phase were associated with high levels of asRNAs. These asRNAs also overlapped their corresponding gene promoters with a much higher frequency than average. Interrupting antisense transcription of a subset of genes corresponding to quiescence-enriched mRNAs restored their expression. The underlying mechanism acts in cis and involves several chromatin modifiers. Our results convey that transcription interference represses up to 30% of the 590 least expressed genes, which includes 163 genes with quiescence-enriched mRNAs. We also found that pervasive transcripts constitute a higher fraction of the transcriptome in quiescence relative to the exponential phase, consistent with gene expression itself playing an important role to suppress pervasive transcription. Accordingly, the HIS1 asRNA, normally only present in quiescence, is expressed in exponential phase upon HIS1 mRNA transcription interruption.
Asunto(s)
Regulación Fúngica de la Expresión Génica , ARN sin Sentido/biosíntesis , Eliminación de Gen , ARN Helicasas/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Selenomethionine, a dietary supplement with beneficial health effects, becomes toxic if taken in excess. To gain insight into the mechanisms of action of selenomethionine, we screened a collection of ≈5900 Saccharomyces cerevisiae mutants for sensitivity or resistance to growth-limiting amounts of the compound. Genes involved in protein degradation and synthesis were enriched in the obtained datasets, suggesting that selenomethionine causes a proteotoxic stress. We demonstrate that selenomethionine induces an accumulation of protein aggregates by a mechanism that requires de novo protein synthesis. Reduction of translation rates was accompanied by a decrease of protein aggregation and of selenomethionine toxicity. Protein aggregation was supressed in a ∆cys3 mutant unable to synthetize selenocysteine, suggesting that aggregation results from the metabolization of selenomethionine to selenocysteine followed by translational incorporation in the place of cysteine. In support of this mechanism, we were able to detect random substitutions of cysteinyl residues by selenocysteine in a reporter protein. Our results reveal a novel mechanism of toxicity that may have implications in higher eukaryotes.
Asunto(s)
Agregado de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Selenocisteína/metabolismo , Selenometionina/toxicidad , Secuencia de Aminoácidos , Bases de Datos como Asunto , Eliminación de Gen , Ontología de Genes , Péptidos/química , Péptidos/metabolismo , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismoRESUMEN
Protein quality control mechanisms eliminate defective polypeptides to ensure proteostasis and to avoid the toxicity of protein aggregates. In eukaryotes, the ribosome-bound quality control (RQC) complex detects aberrant nascent peptides that remain stalled in 60S ribosomal particles due to a dysfunction in translation termination. The RQC complex polyubiquitylates aberrant polypeptides and recruits a Cdc48 hexamer to extract them from 60S particles in order to escort them to the proteasome for degradation. Whereas the steps from stalled 60S recognition to aberrant peptide polyubiquitylation by the RQC complex have been described, the mechanism leading to proteasomal degradation of these defective translation products remains unknown. We show here that the RQC complex also exists as a ribosome-unbound complex during the escort of aberrant peptides to the proteasome. In addition, we identify a new partner of this light version of the RQC complex, the E3 ubiquitin ligase Tom1. Tom1 interacts with aberrant nascent peptides and is essential to limit their accumulation and aggregation in the absence of Rqc1; however, its E3 ubiquitin ligase activity is not required. Taken together, these results reveal new roles for Tom1 in protein quality control, aggregate prevention, and, therefore, proteostasis maintenance.
Asunto(s)
Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Proteínas de Ciclo Celular/metabolismo , Terminación de la Cadena Péptídica Traduccional/fisiología , Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/fisiología , Biosíntesis de Proteínas , Proteolisis , Proteínas de Unión al ARN/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ribosomas/fisiología , Saccharomyces cerevisiae/metabolismo , UbiquitinaciónRESUMEN
Ski2-Ski3-Ski8 (Ski) is a helicase complex functioning with the RNA-degrading exosome to mediate the 3'-5' messenger RNA (mRNA) decay in turnover and quality-control pathways. We report that the Ski complex directly associates with 80S ribosomes presenting a short mRNA 3' overhang. We determined the structure of an endogenous ribosome-Ski complex using cryo-electron microscopy (EM) with a local resolution of the Ski complex ranging from 4 angstroms (Å) in the core to about 10 Å for intrinsically flexible regions. Ribosome binding displaces the autoinhibitory domain of the Ski2 helicase, positioning it in an open conformation near the ribosomal mRNA entry tunnel. We observe that the mRNA 3' overhang is threaded directly from the small ribosomal subunit to the helicase channel of Ski2, primed for ongoing exosome-mediated 3'-5' degradation.
Asunto(s)
ADN Helicasas/ultraestructura , Complejo Multienzimático de Ribonucleasas del Exosoma/ultraestructura , Proteínas Nucleares/ultraestructura , Estabilidad del ARN , Subunidades Ribosómicas Grandes de Eucariotas/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Saccharomyces cerevisiae/enzimología , Microscopía por Crioelectrón , Conformación Proteica , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , ARN Ribosómico/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/enzimologíaRESUMEN
Protein homeostasis is maintained by quality control mechanisms that detect and eliminate deficient translation products. Cytosolic defective proteins can arise from translation of aberrant mRNAs lacking a termination codon (NonStop) or containing a sequence that blocks translation elongation (No-Go), which results in translational arrest. Stalled ribosomes are dissociated, aberrant mRNAs are degraded by the cytoplasmic exosome, and the nascent peptides remaining in stalled 60S exit tunnels are detected by the ribosome-bound quality control complex (RQC) composed of Ltn1, Rqc1, Rqc2, and Cdc48. Whereas Ltn1 polyubiquitylates these nascent peptides, Rqc2 directs the addition of C-terminal alanine-threonine tails (CAT-tails), and a Cdc48 hexamer is recruited to extract the nascent peptides, which are addressed to the proteasome for degradation. Although the functions of most RQC components have been described, the role of Rqc1 in this quality control process remains undetermined. In this article we show that the absence of Rqc1 or Ltn1 results in the aggregation of aberrant proteins, a phenomenon that requires CAT-tail addition to the nascent peptides by Rqc2. Our results suggest that aberrant CAT-tailed protein aggregation results from a defect in Cdc48 recruitment to stalled 60S particles, a process that requires both Rqc1 and Ltn1. These protein aggregates contain Ltn1-dependent polyubiquitin chains and are degraded by the proteasome. Finally, aggregate characterization by proteomics revealed that they contain specific chaperones including Sis1, Sgt2, Ssa1/2, and Hsp82, suggesting that these protein aggregates may be addressed to aggresome-like structures when the RQC complex fails to deliver aberrant nascent peptides to the proteasome for degradation.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Alanina/química , Alanina/genética , Alanina/metabolismo , Western Blotting , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Microscopía Fluorescente , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Agregado de Proteínas , Biosíntesis de Proteínas/genética , Proteolisis , Proteómica/métodos , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Treonina/química , Treonina/genética , Treonina/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Proteína que Contiene ValosinaRESUMEN
Establishing a link between the nonsense-mediated decay pathway and a gene associated with programmed cell death could explain why this pathway is essential in most, but not all, eukaryotes.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido , ARN Mensajero/genética , HumanosRESUMEN
Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA quality-control pathway targeting transcripts such as messenger RNAs harboring premature stop-codons or short upstream open reading frame (uORFs). Our transcription start sites (TSSs) analysis of Saccharomyces cerevisiae cells deficient for RNA degradation pathways revealed that about half of the pervasive transcripts are degraded by NMD, which provides a fail-safe mechanism to remove spurious transcripts that escaped degradation in the nucleus. Moreover, we found that the low specificity of RNA polymerase II TSSs selection generates, for 47% of the expressed genes, NMD-sensitive transcript isoforms carrying uORFs or starting downstream of the ATG START codon. Despite the low abundance of this last category of isoforms, their presence seems to constrain genomic sequences, as suggested by the significant bias against in-frame ATGs specifically found at the beginning of the corresponding genes and reflected by a depletion of methionines in the N-terminus of the encoded proteins.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido , ARN de Hongos/genética , ARN Mensajero/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Codón sin Sentido , Regulación Fúngica de la Expresión Génica , Sistemas de Lectura Abierta , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Nonsense-mediated mRNA decay (NMD) destabilizes eukaryotic transcripts with long 3' UTRs. To investigate whether other transcript features affect NMD, we generated yeast strains expressing chromosomal-derived mRNAs with 979 different promoter and open reading frame (ORF) regions and with the same long, destabilizing 3' UTR. We developed a barcode-based DNA microarray strategy to compare the levels of each reporter mRNA in strains with or without active NMD. The size of the coding region had a significant negative effect on NMD efficiency. This effect was not specific to the tested 3' UTR because two other different NMD reporters became less sensitive to NMD when ORF length was increased. Inefficient NMD was not due to a lack of association of Upf1 to long ORF transcripts. In conclusion, in addition to a long 3' UTR, short translation length is an important feature of NMD substrates in yeast.
Asunto(s)
Regiones no Traducidas 3' , Degradación de ARNm Mediada por Codón sin Sentido , Sistemas de Lectura Abierta , Saccharomyces cerevisiae/metabolismo , Regulación Fúngica de la Expresión Génica , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Eukaryotic genomes are pervasively transcribed. However, it is unclear how many newly found RNAs have functions and how many are byproducts of functional, or spurious, transcription events. Cells control the accumulation of many opportunistic transcripts by limiting their synthesis and by provoking their early transcription termination and decay. In this review, we use S. cerevisiae and mammalian cells as models to discuss the circumstances by which pervasive transcripts are produced and turned over. This ultimately relates to the likelihood, and potential mechanism, of molecular function.
Asunto(s)
ARN no Traducido/genética , Transcripción Genética , Animales , Regulación de la Expresión Génica , Genoma Fúngico , Humanos , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7(L)b within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.
Asunto(s)
ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular , Precursores del ARN/química , Precursores del ARN/metabolismo , ARN Ribosómico/química , ARN Ribosómico 5S/química , ARN Ribosómico 5S/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/químicaRESUMEN
Ribosome stalling on eukaryotic mRNAs triggers cotranslational RNA and protein degradation through conserved mechanisms. For example, mRNAs lacking a stop codon are degraded by the exosome in association with its cofactor, the SKI complex, whereas the corresponding aberrant nascent polypeptides are ubiquitinated by the E3 ligases Ltn1 and Not4 and become proteasome substrates. How translation arrest is linked with polypeptide degradation is still unclear. Genetic screens with SKI and LTN1 mutants allowed us to identify translation-associated element 2 (Tae2) and ribosome quality control 1 (Rqc1), two factors that we found associated, together with Ltn1 and the AAA-ATPase Cdc48, to 60S ribosomal subunits. Translation-associated element 2 (Tae2), Rqc1, and Cdc48 were all required for degradation of polypeptides synthesized from Non-Stop mRNAs (Non-Stop protein decay; NSPD). Both Ltn1 and Rqc1 were essential for the recruitment of Cdc48 to 60S particles. Polysome gradient analyses of mutant strains revealed unique intermediates of this pathway, showing that the polyubiquitination of Non-Stop peptides is a progressive process. We propose that ubiquitination of the nascent peptide starts on the 80S and continues on the 60S, on which Cdc48 is recruited to escort the substrate for proteasomal degradation.