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Key Points Initial transcriptome analyses were limited to the quantification of the transcripts that correspond to known features, such as mRNA genes or stable non-coding RNAs. Projects aimed at the exhaustive identification of RNAs, unbiased by genome annotations, have uncovered unexpected complexity of the transcriptome in eukaryotes. Several transcripts are generated from genomic regions that were previously thought to be silent or antisense to genes. This widespread genomic transcription is often called 'pervasive transcription'. Advances in understanding the complexity of eukaryotic transcriptomes have been driven by technological breakthroughs. Several distinct approaches have recently evolved to allow rapid, unbiased, genome-wide analyses of transcriptomes. First, DNA microarrays have been developed to the point at which tiled oligonucleotides span whole genomes (genomic tiling microarrays). Second, the development of next-generation sequencing techniques has allowed the efficient and quantitative analysis of sequence tags that either cover whole transcripts or are enriched for the 5′ or 3′ extremities of the RNAs. The new genomic approaches for transcriptome analysis have uncovered a variety of non-coding transcripts. Recently, several classes of small non-coding RNAs have been found to be associated with gene promoters in animals. Although these different classes of RNA differ in their characteristics — such as their modal length — their distribution with respect to gene transcription start sites (TSSs) is remarkably similar. Some of these small RNAs are transcribed in the same orientation as the mRNAs and are usually located a short distance downstream of the gene TSS, and others are transcribed in the opposite direction to the mRNA from upstream of the gene TSS. The origin of these promoter-associated RNAs is unknown. However, several observations point to a possible relationship with the so-called 'paused' polymerases, that is, polymerases that engage in transcription but that pause a few dozens of nucleotides downstream of the TSS. In particular, the distribution of the promoter-associated small RNAs is similar to that of 'engaged' RNA polymerase II, as determined by a novel genome-wide run-on technique. However, how the small RNAs and paused polymerases might be related remains puzzling and is far from established. In yeast, an important part of pervasive transcription gives rise to highly unstable transcripts called cryptic unstable transcripts (CUTs). These transcripts are heterogeneous at their 3′ ends and range in size from ∼200 to ∼600 nucleotides. Another class of more stable transcripts has been distinguished and named stable unannotated transcripts (SUTs), although there is not a clear demarcation between the two classes. Like the promoter-associated small RNAs found in animals, these RNAs are mostly transcribed from nucleosome-free regions, in particular those associated with gene promoters. In addition, they show a divergent distribution profile. However, this profile is not completely equivalent to that observed in animals, as their TSSs are almost exclusively located upstream of the gene TSSs, whether or not they are transcribed in the sense orientation or in divergent orientation relative to the gene. The majority of CUTs and SUTs are divergent from their associated mRNAs. One model for their origin, which is supported by mutational analysis of one example, is that the assembly of pre-initiation complexes (PICs) during transcription initiation is poorly polarized, and so cryptic PICs are often assembled in the wrong orientation relative to the gene. The transcripts they generate are efficiently degraded by an efficient quality control mechanism. The nature of the quality control mechanism that targets CUTs for rapid degradation is well understood. This mechanism is coupled to the peculiar mode of termination of transcription for these RNAs, which resembles the transcription termination of small nucleolar RNAs. This mode of termination is coupled to exonucleolytic degradation by the exosome, assisted by a novel poly(A) polymerase-containing complex called the Trf4–Air2–Mtr4p polyadenylation (TRAMP) complex. Although the role of divergent CUTs in regulation is unknown, several different specific regulation mechanisms have been described that use antisense SUTs or generate sense CUTs. How widespread the use of these unconventional regulation mechanisms is remains to be determined. Likewise, in animals, the general role of promoter-associated transcription remains enigmatic, although several different mechanisms have been described that make use of such transcripts as effectors of gene regulation. Importantly, whatever the precise mechanism is that generates promoter-associated small non-coding RNAs in yeast and animals, these studies indicate that transcription initiation is a poorly polarized process and many, if not most, promoter regions therefore seem to be intrinsically bidirectional. Recent transcriptomic studies have revealed that diverse small RNAs are transcribed from the regions around gene promoters. This Review considers questions prompted by the discovery of these transcripts; for example, what is their origin and are they functional? 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.

Small RNAs make the CUT Two papers in this issue reveal the prevalence of cryptic or hidden transcription in the yeast genome. Cryptic unstable transcripts (CUTs) are a major class of RNA polymerase II transcripts in budding yeast and are degraded immediately after being synthesized. They had therefore escaped detection until recently. In the current papers, high-resolution genome analyses reveal that CUTs arise predominantly from promoter regions and in an antisense direction. There is therefore a widespread occurrence of inherently bidirectional promoters in yeast, which hints at a regulatory function for these non-coding transcripts. One of two papers in this issue that reveal the prevalence of cryptic or hidden transcription in the yeast genome. Cryptic unstable transcripts (CUTs) are a major class of RNA polymerase II transcripts in budding yeast and are degraded immediately after being synthesized. In these papers, high-resolution genome analyses reveal that CUTs arise predominantly from promoter regions and in an antisense direction. There is therefore a widespread occurrence of inherently bidirectional promoters in yeast. Pervasive and hidden transcription is widespread in eukaryotes 1 , 2 , 3 , 4 , 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 5 . These transcripts are targeted for degradation immediately after synthesis by the action of the Nrd1–exosome–TRAMP complexes 6 , 7 . 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 8 , 9 , 10 , 11 , 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.

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.The cryo-EM structure of the Saccharomyces cerevisiae 80S ribosome–Xrn1 nuclease complex reveals how the conserved core of Xrn1 allows binding at the mRNA exit channel of the ribosome, ensuring efficient degradation of mRNA after the final round of translation.

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.

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 endogenousribosome-Skicomplex using cryo-electron microscopy (EM) with a local resolution of the Ski complex ranging from 4 angstroms (Å) in the core to about 10 Â 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.

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. Eukaryotes such as animals, plants and fungi store their DNA within the nucleus of each of their cells. Genes within this DNA contain the instructions needed to make molecules of RNA; some of which can leave the nucleus and be decoded to build proteins. However, not all of the DNA that is copied into RNA actually codes for proteins. Instead, some RNA molecules are important parts of the cell's protein-making machinery in their own right, and others help to regulate the expression of genes as RNAs or proteins. Nevertheless, many non-coding RNAs don't have such clear roles. Often these RNAs—which are called ‘pervasive transcripts’—are quickly destroyed within the nucleus, but it is likely that some molecules will escape this quality-control mechanism. If the cell's protein-making machinery decodes these RNAs, it could lead to the production of faulty or harmful proteins. Recent research suggested that another quality-control mechanism, which typically eradicates incorrectly processed protein-coding RNAs, could also destroy unneeded or harmful pervasive transcripts. But it was not clear how common it was for this process—called ‘nonsense-mediated decay’—to be used for this purpose. Now Malabat, Feuerbach et al. have engineered yeast cells that lacked either the genes required to carry out nonsense-mediated decay or the ability to destroy RNA molecules in the nucleus. Experiments with these yeast cells revealed that about half of all pervasive transcripts can be destroyed via nonsense-mediated decay; this suggests that this mechanism serves as a fail-safe to prevent the build-up of these potentially harmful molecules. Malabat, Feuerbach et al. also revealed that the enzyme complex that copies gene sequences to make RNA molecules will often also copy some extra DNA sequence from before the start of the gene. On the other hand, it is also common for this enzyme complex to miss the start of the gene and produce an RNA molecule that lacks some of the instructions needed to build the correct protein. Further experiments showed that in yeast these two kinds of incorrectly made protein-coding RNAs could both be identified and destroyed by nonsense-mediated decay as well. The next challenge will be to see to what extent these phenomena are conserved in other eukaryotes.

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.

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.

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.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.

Staphylococcus aureus RNAIII is one of the largest regulatory RNAs, which controls several virulence genes encoding exoproteins and cell‐wall‐associated proteins. One of the RNAIII effects is the repression of spa gene (coding for the surface protein A) expression. Here, we show that spa repression occurs not only at the transcriptional level but also by RNAIII‐mediated inhibition of translation and degradation of the stable spa mRNA by the double‐strand‐specific endoribonuclease III (RNase III). The 3′ end domain of RNAIII, partially complementary to the 5′ part of spa mRNA, efficiently anneals to spa mRNA through an initial loop–loop interaction. Although this annealing is sufficient to inhibit in vitro the formation of the translation initiation complex, the coordinated action of RNase III is essential in vivo to degrade the mRNA and irreversibly arrest translation. Our results further suggest that RNase III is recruited for targeting the paired RNAs. These findings add further complexity to the expression of the S. aureus virulon.