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 NonStop 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.quality control | Saccharomyces cerevisiae

Eukaryotic pre-ribosomes go through cytoplasmic maturation steps before entering translation. The nucleocytoplasmic proteins participating in these late stages of maturation are reimported to the nucleus. In this study, we describe a functional network focused on Rei1/Ybr267w, a strictly cytoplasmic pre-60S factor indirectly involved in nuclear 27S pre-ribosomal RNA processing. In the absence of Rei1, the nuclear import of at least three other pre-60S factors is impaired. The accumulation in the cytoplasm of a small complex formed by the association of Arx1 with a novel factor, Alb1/Yjl122w, inhibits the release of the putative antiassociation factor Tif6 from the premature large ribosomal subunits and its recycling to the nucleus. We propose a model in which Rei1 is a key factor for the coordinated dissociation and recycling of the last pre-60S factors before newly synthesized large ribosomal subunits enter translation.

Describing at a genomic scale how mutations in different genes influence one another is essential to the understanding of how genotype correlates with phenotype and remains a major challenge in biology. Previous studies pointed out the need for accurate measurements of not only synthetic but also buffering interactions in the characterization of genetic networks and functional modules. We developed a sensitive and efficient method that allows such measurements at a genomic scale in yeast. In a pilot experiment (41 genome-wide screens), we quantified the fitness of 140,000 double deletion strains relative to the corresponding single mutants and identified many genetic interactions. In addition to synthetic growth defects (validated experimentally with factors newly identified as genetically interfering with mRNA degradation), most of the identified genetic interactions measured weak epistatic effects. These weak effects, rarely meaningful when considered individually, were crucial to defining specific signatures for many gene deletions and had a major contribution in defining clusters of functionally related genes.epistasis ͉ functional genomics ͉ genetic screen ͉ mRNA decapping ͉ Saccharomyces cerevisiae F ollowing the completion of the genomic sequence for Saccharomyces cerevisiae, a systematic gene deletion library was built as a tool to link genes to functions and phenotypes. Yet, the phenotypic consequences of single deletions are rarely sufficient to define the function of genes. Moreover, very little is known of the phenotypic influences that different mutations have on each other. A large panel of responses can be observed when combining mutations, from aggravating to neutral, buffering, and even alleviating effects. Several high-throughput genetic screen methods, such as SGA (synthetic genetic array), dSLAM, and SLAM (synthetic lethality analyzed by microarray) (1-3), analyze the growth defect of combining a given query mutation with every gene deletion from the library of tagged nonessential yeast knockouts. These approaches are useful in identifying the strong synthetic defects that are seen for only a minor fraction of all of the possible gene deletion pairs (Ϸ0.5%) (4). However, they are not suited to evaluating more general buffering relationships between genes. Yet, recent studies have demonstrated the importance of accurate measurements of the complete spectrum of genetic interactions to define functional gene modules (5, 6). Broader quantitative measurements of genetic interactions are obtained in epistatic miniarrays (E-MAPs) (5, 7) but at the expense of coverage, because the E-MAP results depend on high-density genetic interaction matrixes made possible by focusing on logically connected gene subsets. Here, we present a method that we call GIM for ''genetic interaction mapping,'' which is not limited to a subset of genes, and allows sensitive and quantitative measurements of the complete spectrum of genetic interactions.Double mutant populations are efficiently obtained by mating and sporulatio...

A genome-wide screen for synthetic lethal (SL) interactions with loss of the nuclear exosome cofactors Rrp47/Lrp1 or Air1 identified 335 exonucleases, the THO complex required for mRNP assembly, and Ynr024w (Mpp6). SL interactions with mpp6⌬ were confirmed for rrp47⌬ and nuclear exosome component Rrp6. The results of bioinformatic analyses revealed homology between Mpp6 and a human exosome cofactor, underlining the high conservation of the RNA surveillance system. Mpp6 is an RNA binding protein that physically associates with the exosome and was localized throughout the nucleus. The results of functional analyses demonstrated roles for Mpp6 in the surveillance of both pre-rRNA and pre-mRNAs and in the degradation of "cryptic" noncoding RNAs (ncRNAs) derived from intergenic regions and the ribosomal DNA spacer heterochromatin. Strikingly, these ncRNAs are also targeted by other exosome cofactors, including Rrp47, the TRAMP complex (which includes Air1), and the Nrd1/Nab3 complex, and are degraded by both Rrp6 and the core exosome. Heterochromatic transcripts and other ncRNAs are characterized by very rapid degradation, and we predict that functional redundancy is an important feature of ncRNA metabolism.The results of genetic and biochemical analyses indicate that the exosome 3Ј-to-5Ј exonuclease complex has potent in vivo RNA degradation activity, even on highly structured RNAs and large RNA-protein (RNP) complexes. However, the purified eukaryotic exosome shows limited activity in vitro, and this has been interpreted as showing its dependence on cofactors for in vivo activity (reviewed in reference 21). Consistent with this idea, several exosome cofactors have been identified. The Ski2/3/8 complex functions with the cytoplasmic exosome and Ski7 during mRNA turnover and surveillance (3, 53). Known nuclear cofactors include the RNA binding proteins Rrp47 (Lrp1) (31, 34), the Nrd1/Nab3 heterodimer (54), and the TRAMP polyadenylation complexes (16,23,27,51,60). However, analyses of mutations in the exosome complex components have revealed nuclear RNA degradation phenotypes that are not shown by mutations in the TRAMP complexes, Rrp47, or Nrd1/Nab3, strongly suggesting that additional exosome cofactors remained to be identified.Rrp47 is believed to act as a specific cofactor for the nuclear 3Ј exonuclease Rrp6 (31, 44). Although Rrp6 is associated with the nuclear exosome, strains lacking the activity of Rrp6 show RNA processing defects that are distinctly different from those seen following the loss or inactivation of any other exosome component. In contrast, very similar phenotypes are seen in strains with mutations in any of the "core" exosome components that are common to the nuclear and cytoplasmic forms of the complex (reviewed in reference 21). This suggests that the activity of the core exosome requires the intact structure of the complex and is functionally distinct from the activity of Rrp6. The TRAMP complexes appear to function together with both Rrp6 and the core exosome. Saccharomyces cerevisiae has tw...

A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence ofSaccharomyces cerevisiaehas identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (L¯ike Sm¯) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein–protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale.

Ribosome biogenesis on its own consumes up to 80% of a proliferating cell's energy and represents about 95% of total transcription (44). Recent evidence suggests that ribosome biogenesis is required not only for growth but also for regulation of cell growth (51). 25S, 18S, and 5.8S ribosomal RNAs are synthesized by RNA polymerase I (Pol I), while the 5S rRNA is transcribed by RNA Pol III. RNA Pol II produces messenger RNAs encoding 79 ribosomal proteins (RP), expressed from 138 genes in budding yeast (62). To generate mature ribosomal subunits, ribosomal components have to be transported, processed, and assembled, using more than 200 different transacting factors (70). The second group of Pol II-transcribed genes, called Ribi, is coregulated with the RP genes (21, 29).

The EKC/KEOPS complex is universally conserved in Archaea and Eukarya and has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic instability. However, the molecular function of the complex has remained elusive so far. We analyzed the transcriptome of EKC/KEOPS mutants and observed a specific profile that is highly enriched in targets of the Gcn4p transcriptional activator. GCN4 expression was found to be activated at the translational level in mutants via the defective recognition of the inhibitory upstream ORFs (uORFs) present in its leader. We show that EKC/KEOPS mutants are defective for the N6-threonylcarbamoyl adenosine modification at position 37 (t6A37) of tRNAs decoding ANN codons, which affects initiation at the inhibitory uORFs and provokes Gcn4 de-repression. Structural modeling reveals similarities between Kae1 and bacterial enzymes involved in carbamoylation reactions analogous to t6A37 formation, supporting a direct role for the EKC in tRNA modification. These findings are further supported by strong genetic interactions of EKC mutants with a translation initiation factor and with threonine biosynthesis genes. Overall, our data provide a novel twist to understanding the primary function of the EKC/KEOPS and its impact on several essential cellular functions like transcription and telomere homeostasis.

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-decapping. Upf1-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.