RESUMEN
Dissecting the regulatory mechanisms controlling mammalian transcripts from production to degradation requires quantitative measurements of mRNA flow across the cell. We developed subcellular TimeLapse-seq to measure the rates at which RNAs are released from chromatin, exported from the nucleus, loaded onto polysomes, and degraded within the nucleus and cytoplasm in human and mouse cells. These rates varied substantially, yet transcripts from genes with related functions or targeted by the same transcription factors and RNA-binding proteins flowed across subcellular compartments with similar kinetics. Verifying these associations uncovered a link between DDX3X and nuclear export. For hundreds of RNA metabolism genes, most transcripts with retained introns were degraded by the nuclear exosome, while the remaining molecules were exported with stable cytoplasmic lifespans. Transcripts residing on chromatin for longer had extended poly(A) tails, whereas the reverse was observed for cytoplasmic mRNAs. Finally, machine learning identified molecular features that predicted the diverse life cycles of mRNAs.
Asunto(s)
Núcleo Celular , Cromatina , ARN Helicasas DEAD-box , ARN Mensajero , Animales , Humanos , Ratones , ARN Mensajero/metabolismo , ARN Mensajero/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Cromatina/metabolismo , Cromatina/genética , Citoplasma/metabolismo , Citoplasma/genética , Estabilidad del ARN , Transporte Activo de Núcleo Celular , Polirribosomas/metabolismo , Polirribosomas/genética , Aprendizaje Automático , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Exosomas/metabolismo , Exosomas/genéticaRESUMEN
Oxidative phosphorylation (OXPHOS) complexes, encoded by both mitochondrial and nuclear DNA, are essential producers of cellular ATP, but how nuclear and mitochondrial gene expression steps are coordinated to achieve balanced OXPHOS subunit biogenesis remains unresolved. Here, we present a parallel quantitative analysis of the human nuclear and mitochondrial messenger RNA (mt-mRNA) life cycles, including transcript production, processing, ribosome association, and degradation. The kinetic rates of nearly every stage of gene expression differed starkly across compartments. Compared with nuclear mRNAs, mt-mRNAs were produced 1,100-fold more, degraded 7-fold faster, and accumulated to 160-fold higher levels. Quantitative modeling and depletion of mitochondrial factors LRPPRC and FASTKD5 identified critical points of mitochondrial regulatory control, revealing that the mitonuclear expression disparities intrinsically arise from the highly polycistronic nature of human mitochondrial pre-mRNA. We propose that resolving these differences requires a 100-fold slower mitochondrial translation rate, illuminating the mitoribosome as a nexus of mitonuclear co-regulation.
Asunto(s)
Mitocondrias , Ribosomas Mitocondriales , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Ribosomas Mitocondriales/metabolismo , Biosíntesis de Proteínas , Fosforilación Oxidativa , Proteínas Mitocondriales/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismoRESUMEN
Combinatorially, intron excision within a given nascent transcript could proceed down any of thousands of paths, each of which would expose different dynamic landscapes of cis-elements and contribute to alternative splicing. In this study, we found that post-transcriptional multi-intron splicing order in human cells is largely predetermined, with most genes spliced in one or a few predominant orders. Strikingly, these orders were conserved across cell types and stages of motor neuron differentiation. Introns flanking alternatively spliced exons were frequently excised last, after their neighboring introns. Perturbations to the spliceosomal U2 snRNA altered the preferred splicing order of many genes, and these alterations were associated with the retention of other introns in the same transcript. In one gene, early removal of specific introns was sufficient to induce delayed excision of three proximal introns, and this delay was caused by two distinct cis-regulatory mechanisms. Together, our results demonstrate that multi-intron splicing order in human cells is predetermined, is influenced by a component of the spliceosome and ensures splicing fidelity across long pre-mRNAs.
Asunto(s)
Precursores del ARN , Empalme del ARN , Humanos , Intrones/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN/genética , Empalme Alternativo/genética , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
Oxidative phosphorylation (OXPHOS) complexes, encoded by both mitochondrial and nuclear DNA, are essential producers of cellular ATP, but how nuclear and mitochondrial gene expression steps are coordinated to achieve balanced OXPHOS biogenesis remains unresolved. Here, we present a parallel quantitative analysis of the human nuclear and mitochondrial messenger RNA (mt-mRNA) life cycles, including transcript production, processing, ribosome association, and degradation. The kinetic rates of nearly every stage of gene expression differed starkly across compartments. Compared to nuclear mRNAs, mt-mRNAs were produced 700-fold higher, degraded 5-fold faster, and accumulated to 170-fold higher levels. Quantitative modeling and depletion of mitochondrial factors, LRPPRC and FASTKD5, identified critical points of mitochondrial regulatory control, revealing that the mitonuclear expression disparities intrinsically arise from the highly polycistronic nature of human mitochondrial pre-mRNA. We propose that resolving these differences requires a 100-fold slower mitochondrial translation rate, illuminating the mitoribosome as a nexus of mitonuclear co-regulation.
RESUMEN
Understanding the complex network that regulates transcription elongation requires the quantitative analysis of RNA polymerase II (Pol II) activity in a wide variety of regulatory environments. We performed native elongating transcript sequencing (NET-seq) in 41 strains of Saccharomyces cerevisiae lacking known elongation regulators, including RNA processing factors, transcription elongation factors, chromatin modifiers, and remodelers. We found that the opposing effects of these factors balance transcription elongation and antisense transcription. Different sets of factors tightly regulate Pol II progression across gene bodies so that Pol II density peaks at key points of RNA processing. These regulators control where Pol II pauses with each obscuring large numbers of potential pause sites that are primarily determined by DNA sequence and shape. Antisense transcription varies highly across the regulatory landscapes analyzed, but antisense transcription in itself does not affect sense transcription at the same locus. Our findings collectively show that a diverse array of factors regulate transcription elongation by precisely balancing Pol II activity.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Secuencia de Bases , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Factores de Elongación Transcripcional/genéticaRESUMEN
A common feature of eukaryotic centromeres is the presence of large tracts of tandemly arranged repeats, known as satellite DNA. However, these centromeric repeats appear to experience rapid evolution under forces such as molecular drive and centromere drive, seemingly without consequence to the integrity of the centromere. Moreover, blocks of heterochromatin within the karyotype, including the centromere, are hotspots for chromosome rearrangements that may drive speciation events by contributing to reproductive isolation. However, the relationship between the evolution of heterochromatic sequences and the karyotypic dynamics of these regions remains largely unknown. Here, we show that a single conserved satellite DNA sequence in the order Rodentia of the genus Peromyscus localizes to recurrent sites of chromosome rearrangements and heterochromatic amplifications. Peromyscine species display several unique features of chromosome evolution compared to other Rodentia, including stable maintenance of a strict chromosome number of 48 among all known species in the absence of any detectable interchromosomal rearrangements. Rather, the diverse karyotypes of Peromyscine species are due to intrachromosomal variation in blocks of repeated DNA content. Despite wide variation in the copy number and location of repeat blocks among different species, we find that a single satellite monomer maintains a conserved sequence and homogenized tandem repeat structure, defying predictions of molecular drive. The conservation of this satellite monomer results in common, abundant, and large blocks of chromatin that are homologous among chromosomes within one species and among diverged species. Thus, such a conserved repeat may have facilitated the retention of polymorphic chromosome variants within individuals and intrachromosomal rearrangements between species-both factors that have previously been hypothesized to contribute towards the extremely wide range of ecological adaptations that this genus exhibits.
Asunto(s)
Centrómero , ADN Satélite/genética , Cariotipo , Peromyscus/genética , Animales , Secuencia Conservada , Evolución Molecular , Variación Genética , Heterocromatina , Especificidad de la EspecieRESUMEN
Many non-coding transcripts (ncRNA) generated by RNA polymerase II in S. cerevisiae are terminated by the Nrd1-Nab3-Sen1 complex. However, Sen1 helicase levels are surprisingly low compared with Nrd1 and Nab3, raising questions regarding how ncRNA can be terminated in an efficient and timely manner. We show that Sen1 levels increase during the S and G2 phases of the cell cycle, leading to increased termination activity of NNS. Overexpression of Sen1 or failure to modulate its abundance by ubiquitin-proteasome-mediated degradation greatly decreases cell fitness. Sen1 toxicity is suppressed by mutations in other termination factors, and NET-seq analysis shows that its overexpression leads to a decrease in ncRNA production and altered mRNA termination. We conclude that Sen1 levels are carefully regulated to prevent aberrant termination. We suggest that ncRNA levels and coding gene transcription termination are modulated by Sen1 to fulfill critical cell cycle-specific functions.
Asunto(s)
ADN Helicasas/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular , Regulación Fúngica de la Expresión Génica , ARN Helicasas/metabolismo , ARN de Hongos/biosíntesis , ARN Mensajero/biosíntesis , ARN no Traducido/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Terminación de la Transcripción Genética , ADN Helicasas/genética , Viabilidad Microbiana , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , ARN Helicasas/genética , ARN de Hongos/genética , ARN Mensajero/genética , ARN no Traducido/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , UbiquitinaciónRESUMEN
Transcriptome-wide maps of RNA binding protein (RBP)-RNA interactions by immunoprecipitation (IP)-based methods such as RNA IP (RIP) and crosslinking and IP (CLIP) are key starting points for evaluating the molecular roles of the thousands of human RBPs. A significant bottleneck to the application of these methods in diverse cell lines, tissues, and developmental stages is the availability of validated IP-quality antibodies. Using IP followed by immunoblot assays, we have developed a validated repository of 438 commercially available antibodies that interrogate 365 unique RBPs. In parallel, 362 short-hairpin RNA (shRNA) constructs against 276 unique RBPs were also used to confirm specificity of these antibodies. These antibodies can characterize subcellular RBP localization. With the burgeoning interest in the roles of RBPs in cancer, neurobiology, and development, these resources are invaluable to the broad scientific community. Detailed information about these resources is publicly available at the ENCODE portal (https://www.encodeproject.org/).