A specialised SKI complex assists the cytoplasmic RNA exosome in the absence of direct association with ribosomes.

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.

Investigation of RNA metabolism through large-scale genetic interaction profiling in yeast.

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.

Nonsense-mediated mRNA decay involves two distinct Upf1-bound complexes.

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.

Dealing with pervasive transcription.

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.

Extensive degradation of RNA precursors by the exosome in wild-type cells.

The exosome is a complex involved in the maturation of rRNA and sn-snoRNA, in the degradation of short-lived noncoding RNAs, and in the quality control of RNAs produced in mutants. It contains two catalytic subunits, Rrp6p and Dis3p, whose specific functions are not fully understood. We analyzed the transcriptome of combinations of Rrp6p and Dis3p catalytic mutants by high-resolution tiling arrays. We show that Dis3p and Rrp6p have both overlapping and specific roles in degrading distinct classes of substrates. We found that transcripts derived from more than half of intron-containing genes are degraded before splicing. Surprisingly, we also show that the exosome degrades large amounts of tRNA precursors despite the absence of processing defects. These results underscore the notion that large amounts of RNAs produced in wild-type cells are discarded before entering functional pathways and suggest that kinetic competition with degradation proofreads the efficiency and accuracy of processing.

Antisense transcriptional interference mediates condition-specific gene repression in budding yeast.

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.

Rqc1 and Ltn1 Prevent C-terminal Alanine-Threonine Tail (CAT-tail)-induced Protein Aggregation by Efficient Recruitment of Cdc48 on Stalled 60S Subunits.

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.

The yeast RPL9B gene is regulated by modulation between two modes of transcription termination.

RNA Pol II transcription termination can occur by at least two alternative pathways. Cleavage and polyadenylation by the CPF/CF complex precedes mRNA transcription termination, while the Nrd1 complex is involved in transcription termination of non-coding RNAs such as sno/snRNAs or cryptic unstable transcripts. Here we show that transcription of RPL9B, one of the two genes coding for the ribosomal protein Rpl9p, terminates by either of these two pathways. The balance between these two pathways is modulated in response to the RPL9 gene copy number, resulting in the autoregulation of RPL9B gene expression. This autoregulation mechanism requires a conserved potential stem-loop structure very close to the polyadenylation sites. We propose a model in which Rpl9p, when in excess, binds this conserved 3'-UTR structure, negatively interfering with cleavage and polyadenylation to the benefit of the Nrd1-dependent termination pathway, which, being coupled to degradation by the nuclear exosome, results in downregulation of RPL9B gene expression.

Futile cycle of transcription initiation and termination modulates the response to nucleotide shortage in S. cerevisiae.

Hidden transcription in eukaryotes carries a large potential of regulatory functions that are only recently beginning to emerge. Cryptic unstable transcripts (CUTs) are generated by RNA polymerase II (Pol II) and rapidly degraded after transcription in wild-type yeast cells. Whether CUTs or the act of transcription without RNA production have a function is presently unclear. We describe here a nonconventional mechanism of transcriptional regulation that relies on the selection of alternative transcription start sites to generate CUTs or mRNAs. Transcription from TATA box proximal start sites generates unstable transcripts and downregulates expression of the URA2 gene under repressing conditions. Uracil deprivation activates selection of distal start sites, leading to the production of stable mRNAs. We describe the elements that govern degradation of the CUT and activation of mRNA production by downstream transcription initiation. Importantly, we show that a similar mechanism applies to other genes in the nucleotides biogenesis pathway.

Cdc48-associated complex bound to 60S particles is required for the clearance of aberrant translation products.

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.

The complex eukaryotic transcriptome: unexpected pervasive transcription and novel small RNAs.

Over the past few years, techniques have been developed that have allowed the study of transcriptomes without bias from previous genome annotations, which has led to the discovery of a plethora of unexpected RNAs that have no obvious coding capacities. There are many different kinds of products that are generated by this pervasive transcription; this Review focuses on small non-coding RNAs (ncRNAs) that have been found to be associated with promoters in eukaryotes from animals to yeast. After comparing the different classes of such ncRNAs described in various studies, the Review discusses how the models proposed for their origins and their possible functions challenge previous views of the basic transcription process and its regulation.

Widespread bidirectional promoters are the major source of cryptic transcripts in yeast.

Pervasive and hidden transcription is widespread in eukaryotes, but its global level, the mechanisms from which it originates and its functional significance are unclear. Cryptic unstable transcripts (CUTs) were recently described as a principal class of RNA polymerase II transcripts in Saccharomyces cerevisiae. These transcripts are targeted for degradation immediately after synthesis by the action of the Nrd1-exosome-TRAMP complexes. Although CUT degradation mechanisms have been analysed in detail, the genome-wide distribution at the nucleotide resolution and the prevalence of CUTs are unknown. Here we report the first high-resolution genomic map of CUTs in yeast, revealing a class of potentially functional CUTs and the intrinsic bidirectional nature of eukaryotic promoters. An RNA fraction highly enriched in CUTs was analysed by a 3' Long-SAGE (serial analysis of gene expression) approach adapted to deep sequencing. The resulting detailed genomic map of CUTs revealed that they derive from extremely widespread and very well defined transcription units and do not result from unspecific transcriptional noise. Moreover, the transcription of CUTs predominantly arises within nucleosome-free regions, most of which correspond to promoter regions of bona fide genes. Some of the CUTs start upstream from messenger RNAs and overlap their 5' end. Our study of glycolysis genes, as well as recent results from the literature, indicate that such concurrent transcription is potentially associated with regulatory mechanisms. Our data reveal numerous new CUTs with such a potential regulatory role. However, most of the identified CUTs corresponded to transcripts divergent from the promoter regions of genes, indicating that they represent by-products of divergent transcription occurring at many and possibly most promoters. Eukaryotic promoter regions are thus intrinsically bidirectional, a fundamental property that escaped previous analyses because in most cases divergent transcription generates short-lived unstable transcripts present at very low steady-state levels.

Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles.

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.

YLR419W is the homolog of the mammalian translation initiation factor DHX29.

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.

Gcn4 misregulation reveals a direct role for the evolutionary conserved EKC/KEOPS in the t6A modification of tRNAs.

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 (t(6)A(37)) 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 t(6)A(37) 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.

Xrn1 biochemically associates with eisosome proteins after the post diauxic shift in yeast.

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.

eIF2A represses cell wall biogenesis gene expression in Saccharomyces cerevisiae.

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.

Ecm1 is a new pre-ribosomal factor involved in pre-60S particle export.

In eukaryotes, ribosome biogenesis is a highly conserved process that starts in the nucleus and ends in the cytoplasm. In actively growing yeast cells, it is estimated that each nuclear pore complex (NPC) contributes to the export of about 25 pre-ribosomal particles per minute. Such an extremely active process requires several redundant export receptors for the pre-60S particles. Here, we report the identification of a novel pre-60S factor, Ecm1, which partially acts like Arx1 and becomes essential when the NPC function is affected. Ecm1 depletion, combined with the deletion of NPC components led to pre-60S retention in the nucleus. Functional links that we identified between Ecm1, 60S biogenesis, pre-60S export, and the NPC were correlated with physical interactions of Ecm1 with pre-60S particles and nucleoporins. These results support that Ecm1 is an additional factor involved in pre-60S export. While Ecm1 and Arx1 have redundant functions, overproduction of either one could not complement the absence of the other, whereas overproduction of Mex67 was able to partially restore the growth defect resulting from the absence of Ecm1 or Arx1. These data highlight the involvement of many factors acting together to export pre-60S particles.

Cordycepin interferes with 3' end formation in yeast independently of its potential to terminate RNA chain elongation.

Cordycepin (3' deoxyadenosine) is a biologically active compound that, when incorporated during RNA synthesis in vitro, provokes chain termination due to the absence of a 3' hydroxyl moiety. We were interested in the effects mediated by this drug in vivo and analyzed its impact on RNA metabolism of yeast. Our results support the view that cordycepin-triphosphate (CoTP) is the toxic component that is limiting cell growth through inhibition of RNA synthesis. Unexpectedly, cordycepin treatment modulated 3' end heterogeneity of ACT1 and ASC1 mRNAs and rapidly induced extended transcripts derived from CYH2 and NEL025c loci. Moreover, cordycepin ameliorated the growth defects of poly(A) polymerase mutants and the pap1-1 mutation neutralized the effects of the drug on gene expression. Our observations are consistent with an epistatic relationship between poly(A) polymerase function and cordycepin action and suggest that a major mode of cordycepin activity reduces 3' end formation efficiency independently of its potential to terminate RNA chain elongation. Finally, chemical-genetic profiling revealed genome-wide pathways linked to cordycepin activity and identified novel genes involved in poly(A) homeostasis.

Physical and genetic interactions of yeast Cwc21p, an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome.

In Saccharomyces cerevisiae, Cwc21p is a protein of unknown function that is associated with the NineTeen Complex (NTC), a group of proteins involved in activating the spliceosome to promote the pre-mRNA splicing reaction. Here, we show that Cwc21p binds directly to two key splicing factors-namely, Prp8p and Snu114p-and becomes the first NTC-related protein known to dock directly to U5 snRNP proteins. Using a combination of proteomic techniques we show that the N-terminus of Prp8p contains an intramolecular fold that is a Snu114p and Cwc21p interacting domain (SCwid). Cwc21p also binds directly to the C-terminus of Snu114p. Complementary chemical cross-linking experiments reveal reciprocal protein footprints between the interacting Prp8 and Cwc21 proteins, identifying the conserved cwf21 domain in Cwc21p as a Prp8p binding site. Genetic and functional interactions between Cwc21p and Isy1p indicate that they have related functions at or prior to the first catalytic step of splicing, and suggest that Cwc21p functions at the catalytic center of the spliceosome, possibly in response to environmental or metabolic changes. We demonstrate that SRm300, the only SR-related protein known to be at the core of human catalytic spliceosomes, is a functional ortholog of Cwc21p, also interacting directly with Prp8p and Snu114p. Thus, the function of Cwc21p is likely conserved from yeast to humans.