The genome of the yeast Saccharomyces cerevisiae is now completely sequenced. Despite successful genetic work in recent years, 60% of yeast genes have no assigned function and half of those encode putative proteins without any homology with known proteins. Genetic analyses, such as suppressor or synthetic lethal screens, have suggested many functional links between gene products, some of which have been confirmed by biochemical means. Altogether, these approaches have led to a fairly extensive knowledge of defined biochemical pathways. However, the integration of these pathways against the background of complexity in a living cell remains to be accomplished. The two-hybrid method applied to the yeast genome might allow the characterization to the network of interactions between yeast proteins, leading to a better understanding of cellular functions. Such an analysis has been performed for the bacteriophage T7 genome that encodes 55 proteins and for Drosophila cell cycle regulators. However, the currently available two-hybrid methodology is not suitable for a large-scale project without specific methodological improvements In particular, the exhaustivity and selectivity of the screens must first be greatly improved. We constructed a new yeast genomic library and developed a highly selective two-hybrid procedure adapted for exhaustive screens of the yeast genome. For each bait we selected a limited set of interacting preys that we classified in categories of distinct heuristic values. Taking into account this classification, new baits were chosen among preys and, in turn, used for second-round screens. Repeating this procedure several times led to the characterization of the network of interactions. Using known pre-mRNA splicing factors as initial baits, we were able to characterize new interactions between known splicing factors, identify new yeast splicing factors, including homologues of human SF1 and SAP49, and reveal novel potential functional links between cellular pathways. Using different cellular pathways as anchor points, this novel strategy allows us to envision the building of an interaction map of the yeast proteome. In addition, this two-hybrid strategy could be applied to other genomes and might help to resolve the human protein linkage map.

No abstract

The molecular mechanism underlying the retention of intron-containing mRNAs in the nucleus is not understood. Here, we show that retention of intron-containing mRNAs in yeast is mediated by perinuclearly located Mlp1. Deletion of MLP1 impairs retention while having no effect on mRNA splicing. The Mlp1-dependent leakage of intron-containing RNAs is increased in presence of ts-prp18 delta, a splicing mutant. When overall pre-mRNA levels are increased by deletion of RRP6, a nuclear exosome component, MLP1 deletion augments leakage of only the intron-containing portion of mRNAs. Our data suggest, moreover, that Mlp1-dependent retention is mediated via the 5' splice site. Intriguingly, we found Mlp-proteins to be present only on sections of the NE adjacent to chromatin. We propose that at this confined site the perinuclear Mlp1 implements a quality control step prior to export, physically retaining faulty pre-mRNAs.

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

The nuclear pore complexes (NPCs) are evolutionarily conserved assemblies that allow traffic between the cytoplasm and the nucleus. In this study, we have identified and characterized a novel human nuclear pore protein, hNup133, through its homology with the Saccharomyces cerevisiae nucleoporin scNup133. Two-hybrid screens and immunoprecipitation experiments revealed a direct and evolutionarily conserved interaction between Nup133 and Nup84/Nup107 and indicated that hNup133 and hNup107 are part of a NPC subcomplex that contains two other nucleoporins (the previously characterized hNup96 and a novel nucleoporin designated as hNup120) homologous to constituents of the scNup84 subcomplex. We further demonstrate that hNup133 and hNup107 are localized on both sides of the NPC to which they are stably associated at interphase, remain associated as part of a NPC subcomplex during mitosis, and are targeted at early stages to the reforming nuclear envelope. Throughout mitosis, a fraction of hNup133 and hNup107 localizes to the kinetochores, thus revealing an unexpected connection between structural NPCs constituents and kinetochores. Photobleaching experiments further showed that the mitotic cytoplasm contains kinetochore-binding competent hNup133 molecules and that in contrast to its stable association with the NPCs the interaction of this nucleoporin with kinetochores is dynamic.

Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.The synthesis of ribosomes is one of the major metabolic pathways of a cell. In Saccharomyces cerevisiae, ribosome assembly begins in the nucleolus after the transcription of two rRNA precursors, the 35S RNA (precursor of the 18S, 5.8S, and 25S rRNAs) and the pre-5S RNA, by RNA polymerases I and III, respectively. The synthesized pre-rRNAs are modified extensively at multiple positions specified by small nucleolar ribonucleoparticles (snoRNPs) or specific enzymes (1,22,33). During rRNA maturation, the 5Ј and 3Ј external transcribed sequences (ETS) and internal transcribed sequence 1 (ITS1) and ITS2 are removed from the 35S precursor RNA by wellordered cleavages and trimming events, which require the enzymatic activities of helicases and endo-and exonucleases (19,37).Cotranscriptional assembly of ribosomal and nonribosomal proteins in the nucleolus gives rise to a large ribonucleoprotein particle corresponding to the 90S preribosomal complexes described more than 20 years ago (35) and recently characterized biochemically (8,14). These early preribosomal complexes are further converted to smaller pre-40S (43S) and pre-60S (66S) particles, precursors of the mature small and large ribosomal subunits. The pre-40S complexes, each containing a precursor of the 18S rRNA, are exported into the cytoplasm, where they give rise to the mature 40S ribosomal particles (36). Most of the large ribosomal subunit proteins are absent from the 90S preribosomes (8,14) and associate in the nucleolus with the pre-rRNA, probably concomitantly with the formation of the pre-60S particles. During pre-60S particle maturation, 27S prerRNA intermediates are converted into 25S and 5.8S mature rRNAs by successive and well-ordered steps. Several pre-60S particles, which differ in their RNA and protein compositions,...

In yeast, the major mRNA degradation pathway is initiated by poly(A) tail shortening that triggers mRNA decapping. The mRNA is then degraded by 5-to-3 exonucleolysis. In mammalian cells, even though poly(A) tail shortening also precedes mRNA degradation, the degradation pathway has not been elucidated. We have used a reverse transcription-PCR approach that relies on mRNA circularization to measure the poly(A) tail length of four mammalian mRNAs. This approach allows for the simultaneous analysis of the 5 and 3 ends of the same mRNA molecule. For all four mRNAs analyzed, this strategy permitted us to demonstrate the existence of small amounts of decapped mRNA species which have a shorter poly(A) tail than their capped counterparts. Kinetic analysis of one of these mRNAs indicates that the decapped species with a short poly(A) tail are mRNA degradation products. Therefore, our results indicate that decapping is preceded by a shortening of the poly(A) tail in mammalian cells, as it is in yeast, suggesting that this mRNA degradation pathway is conserved throughout eukaryotic evolution.Messenger RNA degradation contributes significantly to the regulation of gene expression. In eukaryotes, elucidation of a number of mRNA degradation pathways is under way (for reviews, see refs 1-4). Presently, these pathways are better understood in yeast than in mammalian cells. Both in yeast and in mammalian cells, degradation of most polyadenylylated mRNAs appears to be initiated by poly(A) shortening: transcriptional pulse-chase experiments have shown that shortening of the poly(A) tail precedes mRNA degradation (5, 6). Some regulatory sequence determinants affect mRNA stability by modulating the rate of deadenylylation, whereas others modulate a later degradation step (5-7). This later step has been elucidated in yeast: deadenylylation at the 3Ј end triggers mRNA decapping at the 5Ј end, which is then followed by 5Ј-to-3Ј exonucleolysis (refs. 8 and 9 and references therein). In some specific circumstances, other degradation pathways are observed: exonucleolysis from the 3Ј end can occur when the 5Ј-to-3Ј exonuclease is inactive, and a decapping pathway independent of poly(A) tail shortening is involved in the degradation of mRNAs that show premature translation termination (9-11).In mammalian cells, the degradation pathway that follows deadenylylation is not well understood. Uncapped mRNAs are less stable than their capped counterparts in cell extracts, and enzymatic activities that catalyze mRNA decapping and 5Ј-to-3Ј exonucleolysis have been identified (refs. 1 and 3 and references therein). Furthermore, there is a conservation between yeast and mammalian cells of a functional interaction between the 5Ј and 3Ј ends of mRNAs: in both systems, these two ends contribute to translational control (e.g., see refs. 12 and 13). It is thus tempting to speculate that deadenylylation triggers a decapping-dependent degradation pathway in mammalian cells as well. However, a direct demonstration is lacking.We have developed a revers...

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.