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Understanding how RNA structure influences its function has been hampered by a lack of approaches that can accurately quantify RNA structure in vivo ; here, RNA structure is revealed on a global scale and with nucleotide-level resolution, showing that there is less structure within cells than expected from in vitro and in silico analyses. Probing the in vivo RNA structurome Being single-stranded, RNA can adopt a diversity of secondary structures via inter- and intramolecular base-pairing. Three studies published in this issue of Nature provide an in-depth view of the variety, dynamics and functional influence of RNA structures in vivo . Sarah Assmann and colleagues map the in vivo RNA structure of over 10,000 transcripts in the model plant Arabidopsis thaliana . Their struc-seq (structure-seqence) approach incorporates in vivo chemical (DMS) probing and next-generation sequencing to provide single-nucleotide resolution on a genome-wide scale. Distinct patterns of structure are found to be correlated with coding regions, splice sites and polyadenylation sites. Comparison of these results with those obtained by earlier technologies reveals that, although predictions for some classes of genes were fairly accurate, others, such as those involved in stress response, were poorly predicted and may reflect changes that made them more adapted to that condition. Jonathan Weissman and colleagues have also developed a DMS-seq method to globally monitor RNA structure to single-nucleotide precision in yeast and mammalian cells. Comparing their findings with in vitro data, the authors conclude that there is less structure within cells than expected. Even thermostable RNA structures can be denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Howard Chang and colleagues asked a different question: how does RNA secondary structure change on a transcriptome-wide level in related individuals? By calculating the RNA secondary structures of two parents and their child, they find that about 15% of transcribed single-nucleotide variants affect local secondary structure. These 'RiboSNitches' are depleted in certain locations, suggesting that a particular RNA structure at that site is important. This study illustrates that there is much to be learned about how changes in RNA structure, particularly as imparted by genetic variation, can alter gene expression. RNA has a dual role as an informational molecule and a direct effector of biological tasks. The latter function is enabled by RNA’s ability to adopt complex secondary and tertiary folds and thus has motivated extensive computational 1 , 2 and experimental 3 , 4 , 5 , 6 , 7 , 8 efforts for determining RNA structures. Existing approaches for evaluating RNA structure have been largely limited to in vitro systems, yet the thermodynamic forces which drive RNA folding in vitro may not be sufficient to predict stable RNA structures in vivo 5 . Indeed, the presence of RNA-binding proteins and ATP-dependent helicases can influence which structures are present inside cells. Here we present an approach for globally monitoring RNA structure in native conditions in vivo with single-nucleotide precision. This method is based on in vivo modification with dimethyl sulphate (DMS), which reacts with unpaired adenine and cytosine residues 9 , followed by deep sequencing to monitor modifications. Our data from yeast and mammalian cells are in excellent agreement with known messenger RNA structures and with the high-resolution crystal structure of the Saccharomyces cerevisiae ribosome 10 . Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro . Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast shows that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells. Our studies broadly enable the functional analysis of physiological RNA structures and reveal that, in contrast to the Anfinsen view of protein folding whereby the structure formed is the most thermodynamically favourable, thermodynamics have an incomplete role in determining mRNA structure in vivo .

Genome-wide pervasive transcription has been reported in many eukaryotic organisms revealing a highly interleaved transcriptome organization that involves hundreds of previously unknown non-coding RNAs. These recently identified transcripts either exist stably in cells (stable unannotated transcripts, SUTs) or are rapidly degraded by the RNA surveillance pathway (cryptic unstable transcripts, CUTs). One characteristic of pervasive transcription is the extensive overlap of SUTs and CUTs with previously annotated features, which prompts questions regarding how these transcripts are generated, and whether they exert function. Single-gene studies have shown that transcription of SUTs and CUTs can be functional, through mechanisms involving the generated RNAs or their generation itself. So far, a complete transcriptome architecture including SUTs and CUTs has not been described in any organism. Knowledge about the position and genome-wide arrangement of these transcripts will be instrumental in understanding their function. Here we provide a comprehensive analysis of these transcripts in the context of multiple conditions, a mutant of the exosome machinery and different strain backgrounds of Saccharomyces cerevisiae. We show that both SUTs and CUTs display distinct patterns of distribution at specific locations. Most of the newly identified transcripts initiate from nucleosome-free regions (NFRs) associated with the promoters of other transcripts (mostly protein-coding genes), or from NFRs at the 3' ends of protein-coding genes. Likewise, about half of all coding transcripts initiate from NFRs associated with promoters of other transcripts. These data change our view of how a genome is transcribed, indicating that bidirectionality is an inherent feature of promoters. Such an arrangement of divergent and overlapping transcripts may provide a mechanism for local spreading of regulatory signals--that is, coupling the transcriptional regulation of neighbouring genes by means of transcriptional interference or histone modification.

An unexpectedly large fraction of genes in metazoans (human, mouse, zebrafish, worm, fruit fly) express high levels of circularized RNAs containing canonical exons. Here we report that circular RNA isoforms are found in diverse species whose most recent common ancestor existed more than one billion years ago: fungi (Schizosaccharomyces pombe and Saccharomyces cerevisiae), a plant (Arabidopsis thaliana), and protists (Plasmodium falciparum and Dictyostelium discoideum). For all species studied to date, including those in this report, only a small fraction of the theoretically possible circular RNA isoforms from a given gene are actually observed. Unlike metazoans, Arabidopsis, D. discoideum, P. falciparum, S. cerevisiae, and S. pombe have very short introns (∼ 100 nucleotides or shorter), yet they still produce circular RNAs. A minority of genes in S. pombe and P. falciparum have documented examples of canonical alternative splicing, making it unlikely that all circular RNAs are by-products of alternative splicing or 'piggyback' on signals used in alternative RNA processing. In S. pombe, the relative abundance of circular to linear transcript isoforms changed in a gene-specific pattern during nitrogen starvation. Circular RNA may be an ancient, conserved feature of eukaryotic gene expression programs.

Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (>5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes.

Transcription and regulation of genes originate from transcription pre-initiation complexes (PICs). Their structural and positional organization across eukaryotic genomes is unknown. Here we applied lambda exonuclease to chromatin immunoprecipitates (termed ChIP-exo) to examine the precise location of 6,045 PICs in Saccharomyces . PICs, including RNA polymerase II and protein complexes TFIIA, TFIIB, TFIID (or TBP), TFIIE, TFIIF, TFIIH and TFIIK were positioned within promoters and excluded from coding regions. Exonuclease patterns were in agreement with crystallographic models of the PIC, and were sufficiently precise to identify TATA-like elements at so-called TATA-less promoters. These PICs and their transcription start sites were positionally constrained at TFIID-engaged downstream +1 nucleosomes. At TATA-box-containing promoters, which are depleted of TFIID, a +1 nucleosome was positioned to be in competition with the PIC, which may allow greater latitude in start-site selection. Our genomic localization of messenger RNA and non-coding RNA PICs reveals that two PICs, in inverted orientation, may occupy the flanking borders of nucleosome-free regions. Their unambiguous detection may help distinguish bona fide genes from transcriptional noise. Ultra-high-resolution mapping of the eukaryotic transcription machinery across the yeast genome reveals several unifying principles of pre-initiation complexes at coding and non-coding genes. Mapping eukaryotic pre-initiation complexes Assembly of the RNA polymerase II pre-initiation complex (PIC) is a crucial early step in gene transcription. Here, a high-resolution technique termed ChIP-exo is used to map precisely the binding and composition of PICs across the yeast genome. The findings include the presence of two divergently oriented PICs at promoters, and a broader role for TATA-like elements than was previously appreciated. This allows new insights into the mechanism of transcription.

RNA interference (RNAi), a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast Saccharomyces cerevisiae. Here, we show that RNAi is present in other budding yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate small interfering RNAs, which mostly correspond to transposable elements and Y' subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y' messenger RNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a previously unknown class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi.

Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat–intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5′-splice site is cleaved but remains close to the catalytic Mg 2+ site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5′-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2′OH. The 5′-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson–Crick interactions between the branch helix and 5′-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAG AGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation. Cryo-EM reveals the configuration of substrate pre-mRNA within the active spliceosome and suggests how remodelling occurs prior to exon ligation. Structure of the branched splicing complex The excision of introns from RNA is not a concerted process, but is rather an ordered one involving two transesterification reactions by the spliceosome. In the first step, the 5′-splice site is cleaved and the intron end is joined to make a lariat structure. Kiyoshi Nagai and colleagues have captured the Saccharomyces cerevisiae spliceosome stalled immediately after this first transesterification (branching) reaction by cryo-electron microscopy single-particle reconstruction at an overall resolution of 3.8 Å. The configuration of the RNA within the complex suggests that remodelling occurs before the second step, exon ligation.

Heterochromatin is a key architectural feature of eukaryotic chromosomes, which endows particular genomic domains with specific functional properties. The capacity of heterochromatin to restrain the activity of mobile elements, isolate DNA repair in repetitive regions and ensure accurate chromosome segregation is crucial for maintaining genomic stability. Nucleosomes at heterochromatin regions display histone post-translational modifications that contribute to developmental regulation by restricting lineage-specific gene expression. The mechanisms of heterochromatin establishment and of heterochromatin maintenance are separable and involve the ability of sequence-specific factors bound to nascent transcripts to recruit chromatin-modifying enzymes. Heterochromatin can spread along the chromatin from nucleation sites. The propensity of heterochromatin to promote its own spreading and inheritance is counteracted by inhibitory factors. Because of its importance for chromosome function, heterochromatin has key roles in the pathogenesis of various human diseases. In this Review, we discuss conserved principles of heterochromatin formation and function using selected examples from studies of a range of eukaryotes, from yeast to human, with an emphasis on insights obtained from unicellular model organisms.

Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871–base pair designer eukaryotic chromosome, synlll, which is based on the 316,617–base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATα allele on synIII. The complete design and synthesis of synIII establishes S. cerevisiae as the basis for designer eukaryotic genome biology.

Oxidative phosphorylation (OXPHOS) is a vital process for energy generation, and is carried out by complexes within the mitochondria. OXPHOS complexes pose a unique challenge for cells because their subunits are encoded on both the nuclear and the mitochondrial genomes. Genomic approaches designed to study nuclear/cytosolic and bacterial gene expression have not been broadly applied to mitochondria, so the co-regulation of OXPHOS genes remains largely unexplored. Here we monitor mitochondrial and nuclear gene expression in Saccharomyces cerevisiae during mitochondrial biogenesis, when OXPHOS complexes are synthesized. We show that nuclear- and mitochondrial-encoded OXPHOS transcript levels do not increase concordantly. Instead, mitochondrial and cytosolic translation are rapidly, dynamically and synchronously regulated. Furthermore, cytosolic translation processes control mitochondrial translation unidirectionally. Thus, the nuclear genome coordinates mitochondrial and cytosolic translation to orchestrate the timely synthesis of OXPHOS complexes, representing an unappreciated regulatory layer shaping the mitochondrial proteome. Our whole-cell genomic profiling approach establishes a foundation for studies of global gene regulation in mitochondria. The genes encoding the subunits of oxidative phosphorylation complexes are split between the nuclear and mitochondrial genomes, but their translation is synchronized by signalling from the cytosol to the mitochondria. Aligning mitochondrial and nuclear expression The OXPHOS (oxidative phosphorylation) complexes within the mitochondrial inner membrane generate the large majority of the cell's energy through the synthesis of ATP from ADP and inorganic phosphate during the oxidation of NADH by molecular oxygen. As the OXPOS complex contains subunits encoded by both the nuclear and the mitochondrial genomes, it has been widely assumed that there must be communication between the two compartments to coordinate gene expression. Stirling Churchman and colleagues have now characterized synthesis of the OXPHOS subunits. They find that nuclear and mitochondrial transcription programs are independently regulated under the direction of the nuclear genome. Regulation occurs not at the level of transcription, but rather in terms of translation, with mitochondrial translation regulated through the cytosolic ribosomes.