RESUMEN
In mammalian cells, spurious transcription results in a vast repertoire of unproductive non-coding RNAs, whose deleterious accumulation is prevented by rapid decay. The nuclear exosome targeting (NEXT) complex plays a central role in directing non-functional transcripts to exosome-mediated degradation, but the structural and molecular mechanisms remain enigmatic. Here, we elucidated the architecture of the human NEXT complex, showing that it exists as a dimer of MTR4-ZCCHC8-RBM7 heterotrimers. Dimerization preconfigures the major MTR4-binding region of ZCCHC8 and arranges the two MTR4 helicases opposite to each other, with each protomer able to function on many types of RNAs. In the inactive state of the complex, the 3' end of an RNA substrate is enclosed in the MTR4 helicase channel by a ZCCHC8 C-terminal gatekeeping domain. The architecture of a NEXT-exosome assembly points to the molecular and regulatory mechanisms with which the NEXT complex guides RNA substrates to the exosome.
Asunto(s)
Exosomas , ARN , Núcleo Celular/genética , Núcleo Celular/metabolismo , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Exosomas/genética , Exosomas/metabolismo , Humanos , Unión Proteica , ARN/genética , ARN/metabolismo , ARN Helicasas/metabolismo , Estabilidad del ARN/genéticaRESUMEN
Termination of RNA polymerase II (RNAPII) transcription in metazoans relies largely on the cleavage and polyadenylation (CPA) and integrator (INT) complexes originally found to act at the ends of protein-coding and small nuclear RNA (snRNA) genes, respectively. Here, we monitor CPA- and INT-dependent termination activities genome-wide, including at thousands of previously unannotated transcription units (TUs), producing unstable RNA. We verify the global activity of CPA occurring at pA sites indiscriminately of their positioning relative to the TU promoter. We also identify a global activity of INT, which is largely sequence-independent and restricted to a ~3-kb promoter-proximal region. Our analyses suggest two functions of genome-wide INT activity: it dampens transcriptional output from weak promoters, and it provides quality control of RNAPII complexes that are unfavorably configured for transcriptional elongation. We suggest that the function of INT in stable snRNA production is an exception from its general cellular role, the attenuation of non-productive transcription.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Proteínas de Unión al ADN/metabolismo , ARN Polimerasa II/metabolismo , ARN Nuclear Pequeño/biosíntesis , Terminación de la Transcripción Genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Poliadenilación , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , ARN Nuclear Pequeño/genéticaRESUMEN
The fidelity of transcription initiation is essential for accurate gene expression, but the determinants of start site selection are not fully understood. Rap1 and other general regulatory factors (GRFs) control the expression of many genes in yeast. We show that depletion of these factors induces widespread ectopic transcription initiation within promoters. This generates many novel non-coding RNAs and transcript isoforms with diverse stability, drastically altering the coding potential of the transcriptome. Ectopic transcription initiation strongly correlates with altered nucleosome positioning. We provide evidence that Rap1 can suppress ectopic initiation by a "place-holder" mechanism whereby it physically occludes inappropriate sites for pre-initiation complex formation. These results reveal an essential role for GRFs in the fidelity of transcription initiation and in the suppression of pervasive transcription, profoundly redefining current models for their function. They have important implications for the mechanism of transcription initiation and the control of gene expression.
Asunto(s)
Regulación Fúngica de la Expresión Génica , ARN de Hongos/biosíntesis , ARN Mensajero/biosíntesis , ARN no Traducido/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Sitios de Unión , Ensamble y Desensamble de Cromatina , Nucleosomas/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN de Hongos/genética , ARN Mensajero/genética , ARN no Traducido/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Complejo Shelterina , Proteínas de Unión a Telómeros/genética , Factores de Transcripción/genética , Sitio de Iniciación de la Transcripción , Iniciación de la Transcripción GenéticaRESUMEN
Pervasive transcription initiates from cryptic promoters and is observed in eukaryotes ranging from yeast to mammals. The Set2-Rpd3 regulatory system prevents cryptic promoter function within expressed genes. However, conserved systems that control pervasive transcription within intergenic regions have not been well established. Here we show that Mot1, Ino80 chromatin remodeling complex (Ino80C), and NC2 co-localize on chromatin and coordinately suppress pervasive transcription in S. cerevisiae and murine embryonic stem cells (mESCs). In yeast, all three proteins bind subtelomeric heterochromatin through a Sir3-stimulated mechanism and to euchromatin via a TBP-stimulated mechanism. In mESCs, the proteins bind to active and poised TBP-bound promoters along with promoters of polycomb-silenced genes apparently lacking TBP. Depletion of Mot1, Ino80C, or NC2 by anchor away in yeast or RNAi in mESCs leads to near-identical transcriptome phenotypes, with new subtelomeric transcription in yeast, and greatly increased pervasive transcription in both yeast and mESCs.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Células Madre Embrionarias/enzimología , Fosfoproteínas/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/genética , Sitios de Unión , Línea Celular , Proteínas de Unión al ADN , Eucromatina/genética , Eucromatina/metabolismo , Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Genotipo , Heterocromatina/genética , Heterocromatina/metabolismo , Fenotipo , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , Proteínas Represoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIID , Factores de Transcripción/genética , TransfecciónRESUMEN
In Escherichia coli and Salmonella, many genes silenced by the nucleoid structuring protein H-NS are activated upon inhibiting Rho-dependent transcription termination. This response is poorly understood and difficult to reconcile with the view that H-NS acts mainly by blocking transcription initiation. Here we have analyzed the basis for the up-regulation of H-NS-silenced Salmonella pathogenicity island 1 (SPI-1) in cells depleted of Rho-cofactor NusG. Evidence from genetic experiments, semiquantitative 5' rapid amplification of complementary DNA ends sequencing (5' RACE-Seq), and chromatin immunoprecipitation sequencing (ChIP-Seq) shows that transcription originating from spurious antisense promoters, when not stopped by Rho, elongates into a H-NS-bound regulatory region of SPI-1, displacing H-NS and rendering the DNA accessible to the master regulator HilD. In turn, HilD's ability to activate its own transcription triggers a positive feedback loop that results in transcriptional activation of the entire SPI-1. Significantly, single-cell analyses revealed that this mechanism is largely responsible for the coexistence of two subpopulations of cells that either express or do not express SPI-1 genes. We propose that cell-to-cell differences produced by stochastic spurious transcription, combined with feedback loops that perpetuate the activated state, can generate bimodal gene expression patterns in bacterial populations.
Asunto(s)
Proteínas Bacterianas , Proteínas de Unión al ADN , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Salmonella , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Silenciador del Gen , Salmonella/genética , Salmonella/patogenicidad , Análisis de la Célula Individual , Transcripción Genética , Virulencia/genéticaRESUMEN
Nonadaptive hypotheses on the evolution of eukaryotic genome size predict an expansion when the process of purifying selection becomes weak. Accordingly, species with huge genomes, such as lungfish, are expected to show a genome-wide relaxation signature of selection compared with other organisms. However, few studies have empirically tested this prediction using genomic data in a comparative framework. Here, we show that 1) the newly assembled transcriptome of the Australian lungfish, Neoceratodus forsteri, is characterized by an excess of pervasive transcription, or transcriptional leakage, possibly due to suboptimal transcriptional control, and 2) a significant relaxation signature in coding genes in lungfish species compared with other vertebrates. Based on these observations, we propose that the largest known animal genomes evolved in a nearly neutral scenario where genome expansion is less efficiently constrained.
Asunto(s)
Peces , Genómica , Animales , Australia , Peces/genética , Tamaño del Genoma , Selección GenéticaRESUMEN
Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a threat to proper gene expression that needs to be controlled. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here, we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II, and structurally characterize its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires CTD recognition by the N-terminal domain of Sen1. We provide evidence that the Sen1-CTD interaction does not promote initial Sen1 recruitment, but rather enhances Sen1 capacity to induce the release of paused RNAPII from the DNA. Our results shed light on the network of protein-protein interactions that control termination of non-coding transcription by Sen1.
Asunto(s)
ADN Helicasas/química , ADN Helicasas/metabolismo , ARN Helicasas/química , ARN Helicasas/metabolismo , ARN Polimerasa II/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sitios de Unión , Regulación Fúngica de la Expresión Génica , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , ARN de Hongos/metabolismo , ARN no Traducido/metabolismo , Saccharomyces cerevisiae/genética , Terminación de la Transcripción GenéticaRESUMEN
A substantial part of living cells activity involves transcription regulation. The RNA polymerases responsible for this job need to know 'where/when' to start and stop in the genome, answers that may change throughout life and upon external stimuli. In Saccharomyces cerevisiae, RNA Pol II transcription termination can follow two different routes: the poly(A)-dependent one used for most of the mRNAs and the Nrd1/Nab3/Sen1 (NNS) pathway for non-coding RNAs (ncRNA). The NNS targets include snoRNAs and cryptic unstable transcripts (CUTs) generated by pervasive transcription. This review recapitulates the state of the art in structural biology and biophysics of the Nrd1, Nab3 and Sen1 components of the NNS complex, with special attention to their domain structures and interactions with peptide and RNA motifs, and their heterodimerization. This structural information is put into the context of the NNS termination mechanism together with possible prospects for evolution in the field.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , ARN Helicasas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN Polimerasa II/metabolismo , Regulación Fúngica de la Expresión GénicaRESUMEN
Analysis of genome wide transcription start sites (TSSs) revealed an unexpected complexity since not only canonical TSS of annotated genes are recognized by RNA polymerase. Non-canonical TSS were detected antisense to, or within, annotated genes as well new intergenic (orphan) TSS, not associated with known genes. Previously, it was hypothesized that many such signals represent noise or pervasive transcription, not associated with a biological function. Here, a modified Cappable-seq protocol allows determining the primary transcriptome of the enterohemorrhagic E. coli O157:H7 EDL933 (EHEC). We used four different growth media, both in exponential and stationary growth phase, replicated each thrice. This yielded 19,975 EHEC canonical and non-canonical TSS, which reproducibly occurring in three biological replicates. This questions the hypothesis of experimental noise or pervasive transcription. Accordingly, conserved promoter motifs were found upstream indicating proper TSSs. More than 50% of 5,567 canonical and between 32% and 47% of 10,355 non-canonical TSS were differentially expressed in different media and growth phases, providing evidence for a potential biological function also of non-canonical TSS. Thus, reproducible and environmentally regulated expression suggests that a substantial number of the non-canonical TSSs may be of unknown function rather than being the result of noise or pervasive transcription.
Asunto(s)
Escherichia coli Enterohemorrágica , Escherichia coli O157 , Escherichia coli O157/genética , Sitio de Iniciación de la Transcripción , Ciclo Celular , Medios de CultivoRESUMEN
Transcription termination delimits transcription units but also plays important roles in limiting pervasive transcription. We have previously shown that transcription termination occurs when elongating RNA polymerase II (RNAPII) collides with the DNA-bound general transcription factor Reb1. We demonstrate here that many different DNA-binding proteins can induce termination by a similar roadblock (RB) mechanism. We generated high-resolution transcription maps by the direct detection of RNAPII upon nuclear depletion of two essential RB factors or when the canonical termination pathways for coding and non-coding RNAs are defective. We show that RB termination occurs genomewide and functions independently of (and redundantly with) the main transcription termination pathways. We provide evidence that transcriptional readthrough at canonical terminators is a significant source of pervasive transcription, which is controlled to a large extent by RB termination. Finally, we demonstrate the occurrence of RB termination around centromeres and tRNA genes, which we suggest shields these regions from RNAPII to preserve their functional integrity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Terminación de la Transcripción Genética , Transcripción Genética , Proteínas de Unión al ADN/genética , Genoma Fúngico , ARN Polimerasa II/genética , ARN de Hongos , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Widespread intragenic transcription initiation has been observed in many species. Here we show that the Escherichia coli ehxCABD operon contains numerous intragenic promoters in both sense and antisense orientations. Transcription from these promoters is silenced by the histone-like nucleoid structuring (H-NS) protein. On a genome-wide scale, we show that 46% of H-NS-suppressed transcripts in E. coli are intragenic in origin. Furthermore, many intergenic promoters repressed by H-NS are for noncoding RNAs (ncRNAs). Thus, a major overlooked function of H-NS is to prevent transcription of spurious RNA. Our data provide a molecular description for the toxicity of horizontally acquired DNA and explain how this is counteracted by H-NS.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Regulación Bacteriana de la Expresión Génica , Intrones/genética , Silenciador del Gen , Operón/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
The mitochondrial genome of the pathogenic yeast Candida albicans displays a typical organization of several (eight) primary transcription units separated by noncoding regions. Presence of genes encoding Complex I subunits and the stability of its mtDNA sequence make it an attractive model to study organellar genome expression using transcriptomic approaches. The main activity responsible for RNA degradation in mitochondria is a two-component complex (mtEXO) consisting of a 3'-5' exoribonuclease, in yeasts encoded by the DSS1 gene, and a conserved Suv3p helicase. In C. albicans, deletion of either DSS1 or SUV3 gene results in multiple defects in mitochondrial genome expression leading to the loss of respiratory competence. Transcriptomic analysis reveals pervasive transcription in mutants lacking the mtEXO activity, with evidence of the entire genome being transcribed, whereas in wild-type strains no RNAs corresponding to a significant fraction of the noncoding genome can be detected. Antisense ('mirror') transcripts, absent from normal mitochondria are also prominent in the mutants. The expression of multiple mature transcripts, particularly those translated from bicistronic mRNAs, as well as those that contain introns is affected in the mutants, resulting in a decreased level of proteins and reduced respiratory complex activity. The phenotype is most severe in the case of Complex IV, where a decrease of mature COX1 mRNA level to ~5% results in a complete loss of activity. These results show that RNA degradation by mtEXO is essential for shaping the mitochondrial transcriptome and is required to maintain the functional demarcation between transcription units and non-coding genome segments.
Asunto(s)
Candida albicans/genética , ADN Mitocondrial/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Genoma Mitocondrial , Mitocondrias/genética , Proteínas Mitocondriales/metabolismo , Mutación , Candida albicans/enzimología , ADN Mitocondrial/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Mitocondrias/enzimología , Proteínas Mitocondriales/genética , Estabilidad del ARN , Transcripción GenéticaRESUMEN
The genome is pervasively transcribed across various species, yielding numerous non-coding RNAs. As a counterbalance for pervasive transcription, various organisms have a nuclear RNA exosome complex, whose structure is well conserved between yeast and mammalian cells. The RNA exosome not only regulates the processing of stable RNA species, such as rRNAs, tRNAs, small nucleolar RNAs, and small nuclear RNAs, but also plays a central role in RNA surveillance by degrading many unstable RNAs and misprocessed pre-mRNAs. In addition, associated cofactors of RNA exosome direct the exosome to distinct classes of RNA substrates, suggesting divergent and/or multi-layer control of RNA quality in the cell. While the RNA exosome is essential for cell viability and influences various cellular processes, mutations and alterations in the RNA exosome components are linked to the collection of rare diseases and various diseases including cancer, respectively. The present review summarizes the relationships between pervasive transcription and RNA exosome, including evolutionary crosstalk, mechanisms of RNA exosome-mediated RNA surveillance, and physiopathological effects of perturbation of RNA exosome.
Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma/fisiología , Estabilidad del ARN/fisiología , Transcripción Genética/genética , Animales , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Genoma/genética , Humanos , ARN/genética , ARN/metabolismo , Estabilidad del ARN/genética , ARN Nuclear/genética , ARN Nuclear/metabolismoRESUMEN
The combination of pervasive transcription and prolific alternative splicing produces a mammalian transcriptome of great breadth and diversity. The majority of transcribed genomic bases are intronic, antisense, or intergenic to protein-coding genes, yielding a plethora of short and long non-protein-coding regulatory RNAs. Long noncoding RNAs (lncRNAs) share most aspects of their biogenesis, processing, and regulation with mRNAs. However, lncRNAs are typically expressed in more restricted patterns, frequently from enhancers, and exhibit almost universal alternative splicing. These features are consistent with their role as modular epigenetic regulators. We describe here the key studies and technological advances that have shaped our understanding of the dimensions, dynamics, and biological relevance of the mammalian noncoding transcriptome.
Asunto(s)
ARN no Traducido/genética , Transcriptoma , Empalme Alternativo , Animales , Exones , HumanosRESUMEN
Long noncoding RNAs (lncRNAs), which are longer than 200 nucleotides but often unstable, contribute a substantial and diverse portion to pervasive noncoding transcriptomes. Most lncRNAs are poorly annotated and understood, although several play important roles in gene regulation and diseases. Here we systematically uncover and analyze lncRNAs in Schizosaccharomyces pombe. Based on RNA-seq data from twelve RNA-processing mutants and nine physiological conditions, we identify 5775 novel lncRNAs, nearly 4× the previously annotated lncRNAs. The expression of most lncRNAs becomes strongly induced under the genetic and physiological perturbations, most notably during late meiosis. Most lncRNAs are cryptic and suppressed by three RNA-processing pathways: the nuclear exosome, cytoplasmic exonuclease, and RNAi. Double-mutant analyses reveal substantial coordination and redundancy among these pathways. We classify lncRNAs by their dominant pathway into cryptic unstable transcripts (CUTs), Xrn1-sensitive unstable transcripts (XUTs), and Dicer-sensitive unstable transcripts (DUTs). XUTs and DUTs are enriched for antisense lncRNAs, while CUTs are often bidirectional and actively translated. The cytoplasmic exonuclease, along with RNAi, dampens the expression of thousands of lncRNAs and mRNAs that become induced during meiosis. Antisense lncRNA expression mostly negatively correlates with sense mRNA expression in the physiological, but not the genetic conditions. Intergenic and bidirectional lncRNAs emerge from nucleosome-depleted regions, upstream of positioned nucleosomes. Our results highlight both similarities and differences to lncRNA regulation in budding yeast. This broad survey of the lncRNA repertoire and characteristics in S. pombe, and the interwoven regulatory pathways that target lncRNAs, provides a rich framework for their further functional analyses.
Asunto(s)
Exonucleasas/metabolismo , Exosomas/metabolismo , ARN Largo no Codificante/genética , Schizosaccharomyces/genética , Análisis de Secuencia de ARN/métodos , Núcleo Celular/metabolismo , Citoplasma/enzimología , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Fúngica de la Expresión Génica , Meiosis , Anotación de Secuencia Molecular , Mutación , Interferencia de ARN , Estabilidad del ARN , ARN de Hongos/genética , ARN Largo no Codificante/química , Schizosaccharomyces/química , Schizosaccharomyces/enzimologíaRESUMEN
Recent experimental and computational work revealed that transcriptional terminators in Saccharomyces cerevisiae can terminate transcription coming from both directions. This mechanism helps budding yeast cope with the pervasive nature of transcription by limiting aberrant transcription from invading neighboring genes.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/genética , Terminación de la Transcripción Genética , Transcripción Genética , Regiones Promotoras Genéticas , Estabilidad del ARN/genética , ARN de Hongos/genética , ARN Mensajero/genéticaRESUMEN
Populations of genetically identical microorganisms exhibit high degree of cell-to-cell phenotypic diversity even when grown in uniform environmental conditions. Heterogeneity is a genetically determined trait, which ensures bacterial adaptation and survival in the ever changing environmental conditions. Fluctuations in gene expression (noise) at the level of transcription initiation largely contribute to cell-to-cell variability within population. Not surprisingly, the analyses of the mechanisms driving phenotypic heterogeneity are mainly focused on the activity of promoters and transcriptional factors. Less attention is currently given to a role of intrinsic and factor-dependent transcription terminators. Here, we discuss recent advances in understanding the regulatory role of the multi-functional transcription termination factor Rho, the major inhibitor of pervasive transcription in bacteria and the emerging global regulator of gene expression. We propose that termination activity of Rho might be among the mechanisms by which cells manage the intensity of transcriptional noise, thus affecting population heterogeneity.
Asunto(s)
Heterogeneidad Genética , Factor Rho/genética , Transcripción Genética , Bacterias/genética , Regiones Promotoras Genéticas , ARN Bacteriano/genéticaRESUMEN
40 years ago, organelle genomes were assumed to be streamlined and, perhaps, unexciting remnants of their prokaryotic past. However, the field of organelle genomics has exposed an unparallel diversity in genome architecture (i.e. genome size, structure, and content). The transcription of these eccentric genomes can be just as elaborate - organelle genomes are pervasively transcribed into a plethora of RNA types. However, while organelle protein-coding genes are known to produce polycistronic transcripts that undergo heavy posttranscriptional processing, the nature of organelle noncoding transcriptomes is still poorly resolved. Here, we review how wet-lab experiments and second-generation sequencing data (i.e. short reads) have been useful to determine certain types of organelle RNAs, particularly noncoding RNAs. We then explain how third-generation (long-read) RNA-Seq data represent the new frontier in organelle transcriptomics. We show that public repositories (e.g. NCBI SRA) already contain enough data for inter-phyla comparative studies and argue that organelle biologists can benefit from such data. We discuss the prospects of using publicly available sequencing data for organelle-focused studies and examine the challenges of such an approach. We highlight that the lack of a comprehensive database dedicated to organelle genomics/transcriptomics is a major impediment to the development of a field with implications in basic and applied science.
RESUMEN
RNA-Seq is a recently developed sequencing technology, that through the analysis of cDNA allows for unique insights into the transcriptome of a cell. The data generated by RNA-Seq provides information on gene expression, alternative splicing events and the presence of non-coding RNAs. It has been realised non-coding RNAs are more then just artefacts of erroneous transcription and play vital regulatory roles at the genomic, transcriptional and translational level. Transcription of the DNA sense strand produces antisense transcripts. This is known as antisense transcription and often results in the production of non-coding RNAs that are complementary to their associated sense transcripts. Antisense tran-scription has been identified in bacteria, fungi, protozoa, plants, invertebrates and mammals. It seems that antisense tran-scriptional 'hot spots' are located around nucleosome-free regions such as those associated with promoters, indicating that it is likely that antisense transcripts carry out important regulatory functions. This underlines the importance of identifying the presence and understanding the function of these antisense non-coding RNAs. The information concerning strand ori-gin is often lost during conventional RNA-Seq; capturing this information would substantially increase the worth of any RNA-Seq experiment. By manipulating the input cDNA during the template preparation stage it is possible to retain this vital information. This forms the basis of strand-specific RNA-Seq. With an ability to unlock immense portions of new in-formation surrounding the transcriptome, this cutting edge technology may hold the key to developing a greater under-standing of the transcriptome.
RESUMEN
Transcriptome-wide interrogation of eukaryotic genomes has unveiled the pervasive nature of RNA polymerase II transcription. Virtually, any DNA region with an accessible chromatin structure can be transcribed, resulting in a mass production of noncoding RNAs (ncRNAs) with the potential of interfering with gene expression programs. Budding yeast has proved to be a powerful model organism to understand the mechanisms at play to control pervasive transcription and overcome the risks of hazardous disruption of cellular functions. In this review, we focus on the actors and strategies yeasts employ to govern ncRNA production, and we discuss recent findings highlighting the dangers of losing control over pervasive transcription.