RESUMEN
Polyadenylation controls mRNA biogenesis, nucleo-cytoplasmic export, translation and decay. These processes are interdependent and coordinately regulated by poly(A)-binding proteins (PABPs), yet how PABPs are themselves regulated is not fully understood. Here, we report the discovery that human nuclear PABPN1 is phosphorylated by mitotic kinases at four specific sites during mitosis, a time when nucleoplasm and cytoplasm mix. To understand the functional consequences of phosphorylation, we generated a panel of stable cell lines inducibly over-expressing PABPN1 with point mutations at these sites. Phospho-inhibitory mutations decreased cell proliferation, highlighting the importance of PABPN1 phosphorylation in cycling cells. Dynamic regulation of poly(A) tail length and RNA stability have emerged as important modes of gene regulation. We therefore employed long-read sequencing to determine how PABPN1 phospho-site mutants affected poly(A) tails lengths and TimeLapse-seq to monitor mRNA synthesis and decay. Widespread poly(A) tail lengthening was observed for phospho-inhibitory PABPN1 mutants. In contrast, expression of phospho-mimetic PABPN1 resulted in shorter poly(A) tails with increased non-A nucleotides, in addition to increased transcription and reduced stability of a distinct cohort of mRNAs. Taken together, PABPN1 phosphorylation remodels poly(A) tails and increases mRNA turnover, supporting the model that enhanced transcriptome dynamics reset gene expression programs across the cell cycle.
Asunto(s)
Mitosis , Poli A , Proteína I de Unión a Poli(A) , Poliadenilación , Estabilidad del ARN , ARN Mensajero , Transcriptoma , Humanos , Mitosis/genética , Proteína I de Unión a Poli(A)/metabolismo , Proteína I de Unión a Poli(A)/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Fosforilación , Poli A/metabolismo , Estabilidad del ARN/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , Células HeLaRESUMEN
Thousands of genes produce polyadenylated mRNAs that still contain one or more introns. These transcripts are known as retained intron RNAs (RI-RNAs). In the past 10 years, RI-RNAs have been linked to post-transcriptional alternative splicing in a variety of developmental contexts, but they can also be dead-end products fated for RNA decay. Here we discuss the role of intron retention in shaping gene expression programs, as well as recent evidence suggesting that the biogenesis and fate of RI-RNAs is regulated by nuclear organization. We discuss the possibility that proximity of RNA to nuclear speckles - biomolecular condensates that are highly enriched in splicing factors and other RNA binding proteins - is associated with choices ranging from efficient co-transcriptional splicing, export and stability to regulated post-transcriptional splicing and possible vulnerability to decay.
Asunto(s)
Intrones/genética , Precursores del ARN/genética , Empalme del ARN/genética , ARN Mensajero/genética , Empalme Alternativo/genética , Núcleo Celular/genética , Regulación de la Expresión Génica/genética , Humanos , Procesamiento Postranscripcional del ARN/genética , Estabilidad del ARN/genéticaRESUMEN
Execution of the meiotic and mitotic cell division programs requires distinct gene expression patterns. Unlike mitotic cells, meiotic cells reduce ploidy by following one round of DNA replication with two rounds of chromosome segregation (meiosis I and meiosis II). However, the mechanisms by which cells prevent DNA replication between meiosis I and meiosis II are not fully understood. Here, we show that transcriptional repression of two essential DNA replication genes, CDC6 and SLD2, is associated with production of shorter meiosis-specific RNAs containing the 3' end of both genes. Despite the short CDC6 RNA coding for a short protein (Cdc6short), this protein is not essential for meiosis and it does not have either a positive or negative impact on DNA replication. Production of CDC6short mRNA does not require the upstream CDC6 promoter (PCDC6) and is not a processed form of the full-length RNA. Instead, CDC6short depends on transcription initiation from within the ORF upon repression of PCDC6. Finally, using CDC6 genes from related yeast, we show that repression of full-length CDC6 mRNA is evolutionarily conserved and that this repression is consistently associated with production of unique short CDC6 RNAs. Together, these data demonstrate that meiotic cells transcriptionally repress full-length CDC6 and SLD2, and that inactivation of PCDC6 results in heterogeneous transcription initiation from within the CDC6 ORF.
Asunto(s)
Proteínas de Ciclo Celular/genética , Meiosis , Isoformas de ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces/genética , Factores de Elongación Transcripcional/genética , Proteínas de Ciclo Celular/metabolismo , Codón Iniciador , Evolución Molecular , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Regiones Promotoras Genéticas , Isoformas de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Elongación Transcripcional/metabolismoRESUMEN
Meiotic cells undergo a single round of DNA replication followed by two rounds of chromosome segregation (the meiotic divisions) to produce haploid gametes. Both DNA replication and chromosome segregation are similarly regulated by CDK oscillations in mitotic cells. Yet how these two events are uncoupled between the meiotic divisions is unclear. Using Saccharomyces cerevisiae, we show that meiotic cells inhibit both helicase loading and helicase activation to prevent DNA replication between the meiotic divisions. CDK and the meiosis-specific kinase Ime2 cooperatively inhibit helicase loading, and their simultaneous inhibition allows inappropriate helicase reloading. Further analysis uncovered two previously unknown mechanisms by which Ime2 inhibits helicase loading. Finally, we show that CDK and the polo-like kinase Cdc5 trigger degradation of Sld2, an essential helicase-activation protein. Together, our data demonstrate that multiple kinases inhibit both helicase loading and activation between the meiotic divisions, thereby ensuring reductive cell division.
Asunto(s)
Segregación Cromosómica , Quinasas Ciclina-Dependientes/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , Meiosis , Saccharomyces cerevisiae/crecimiento & desarrollo , Redes Reguladoras de Genes , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mapas de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Spliced messages constitute one-fourth of expressed mRNAs in the yeast Saccharomyces cerevisiae, and most mRNAs in metazoans. Splicing requires 5' splice site (5'SS), branch point (BP), and 3' splice site (3'SS) elements, but the role of the BP in splicing control is poorly understood because BP identification remains difficult. We developed a high-throughput method, Branch-seq, to map BPs and 5'SSs of isolated RNA lariats. Applied to S. cerevisiae, Branch-seq detected 76% of expressed, annotated BPs and identified a comparable number of novel BPs. We performed RNA-seq to confirm associated 3'SS locations, identifying some 200 novel splice junctions, including an AT-AC intron. We show that several yeast introns use two or even three different BPs, with effects on 3'SS choice, protein coding potential, or RNA stability, and identify novel introns whose splicing changes during meiosis or in response to stress. Together, these findings show unanticipated complexity of splicing in yeast.
