RESUMEN
Translation initiation at alternative start sites can dynamically control the synthesis of two or more functionally distinct protein isoforms from a single mRNA. Alternate isoforms of the developmental transcription factor CCAAT/enhancer-binding protein α (C/EBPα) produced from different start sites exert opposing effects during myeloid cell development. This choice between alternative start sites depends on sequence features of the CEBPA transcript, including a regulatory uORF, but the molecular basis is not fully understood. Here, we identify the factors that affect C/EBPα isoform choice using a sensitive and quantitative two-color fluorescent reporter coupled with CRISPRi screening. Our screen uncovered a role of the ribosome rescue factor PELOTA (PELO) in promoting the expression of the longer C/EBPα isoform by directly removing inhibitory unrecycled ribosomes and through indirect effects mediated by the mechanistic target of rapamycin kinase. Our work uncovers further links between ribosome recycling and translation reinitiation that regulate a key transcription factor, with implications for normal hematopoiesis and leukemogenesis.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT , Biosíntesis de Proteínas , Isoformas de Proteínas , Ribosomas , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Humanos , Ribosomas/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Iniciación de la Cadena Peptídica Traduccional , Ratones , Serina-Treonina Quinasas TOR/metabolismo , Células HEK293RESUMEN
Translation initiation at alternative start sites can dynamically control the synthesis of two or more functionally distinct protein isoforms from a single mRNA. Alternate isoforms of the hematopoietic transcription factor CCAAT-enhancer binding protein α (C/EBPα) produced from different start sites exert opposing effects during myeloid cell development. This alternative initiation depends on sequence features of the CEBPA transcript, including a regulatory upstream open reading frame (uORF), but the molecular basis is not fully understood. Here we identify trans-acting factors that affect C/EBPα isoform choice using a sensitive and quantitative two-color fluorescence reporter coupled with CRISPRi screening. Our screen uncovered a role for the ribosome rescue factor PELOTA (PELO) in promoting expression of the longer C/EBPα isoform, by directly removing inhibitory unrecycled ribosomes and through indirect effects mediated by the mechanistic target of rapamycin (mTOR) kinase. Our work provides further mechanistic insights into coupling between ribosome recycling and translation reinitiation in regulation of a key transcription factor, with implications for normal hematopoiesis and leukemiagenesis.
RESUMEN
DNA damage activates a robust transcriptional stress response, but much less is known about how DNA damage impacts translation. The advent of genome editing with Cas9 has intensified interest in understanding cellular responses to DNA damage. Here, we find that DNA double-strand breaks (DSBs), including those induced by Cas9, trigger the loss of ribosomal protein RPS27A from ribosomes via p53-independent proteasomal degradation. Comparisons of Cas9 and dCas9 ribosome profiling and mRNA-seq experiments reveal a global translational response to DSBs that precedes changes in transcript abundance. Our results demonstrate that even a single DSB can lead to altered translational output and ribosome remodeling, suggesting caution in interpreting cellular phenotypes measured immediately after genome editing.
Asunto(s)
Roturas del ADN de Doble Cadena , Edición Génica , Sistemas CRISPR-Cas , Daño del ADN/genética , Reparación del ADN , Edición Génica/métodos , Proteínas Ribosómicas/genéticaRESUMEN
The human genome is structurally organized in three-dimensional space to facilitate functional partitioning of transcription. We learned that the latent episome of the human Epstein-Barr virus (EBV) preferentially associates with gene-poor chromosomes and avoids gene-rich chromosomes. Kaposi's sarcoma-associated herpesvirus behaves similarly, but human papillomavirus does not. Contacts on the EBV side localize to OriP, the latent origin of replication. This genetic element and the EBNA1 protein that binds there are sufficient to reconstitute chromosome association preferences of the entire episome. Contacts on the human side localize to gene-poor and AT-rich regions of chromatin distant from transcription start sites. Upon reactivation from latency, however, the episome moves away from repressive heterochromatin and toward active euchromatin. Our work adds three-dimensional relocalization to the molecular events that occur during reactivation. Involvement of myriad interchromosomal associations also suggests a role for this type of long-range association in gene regulation.IMPORTANCE The human genome is structurally organized in three-dimensional space, and this structure functionally affects transcriptional activity. We set out to investigate whether a double-stranded DNA virus, Epstein-Barr virus (EBV), uses mechanisms similar to those of the human genome to regulate transcription. We found that the EBV genome associates with repressive compartments of the nucleus during latency and with active compartments during reactivation. This study advances our knowledge of the EBV life cycle, adding three-dimensional relocalization as a novel component to the molecular events that occur during reactivation. Furthermore, the data add to our understanding of nuclear compartments, showing that disperse interchromosomal interactions may be important for regulating transcription.
Asunto(s)
Cromatina/genética , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiología , Plásmidos/genética , Línea Celular , Núcleo Celular/genética , Núcleo Celular/virología , Cromatina/virología , Cromosomas Humanos/genética , Cromosomas Humanos/virología , Humanos , Células K562 , Origen de RéplicaRESUMEN
Lytic infection by the Epstein-Barr virus (EBV) poses numerous health risks, such as infectious mononucleosis and lymphoproliferative disorder. Proteins in the bromodomain and extraterminal (BET) family regulate multiple stages of viral life cycles and provide promising intervention targets. Synthetic small molecules can bind to the bromodomains and disrupt function by preventing recognition of acetylated lysine substrates. We demonstrate that JQ1 and other BET inhibitors block two different steps in the sequential cascade of the EBV lytic cycle. BET inhibitors prevent expression of the viral immediate-early protein BZLF1. JQ1 alters transcription of genes controlled by the host protein BACH1, and BACH1 knockdown reduces BZLF1 expression. BET proteins also localize to the lytic origin of replication (OriLyt) genetic elements, and BET inhibitors prevent viral late gene expression. There JQ1 reduces BRD4 recruitment during reactivation to preclude replication initiation. This represents a rarely observed dual mode of action for drugs.
Asunto(s)
Antivirales/farmacología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/antagonistas & inhibidores , Proteínas del Grupo de Complementación de la Anemia de Fanconi/antagonistas & inhibidores , Regulación Viral de la Expresión Génica/efectos de los fármacos , Herpesvirus Humano 4/efectos de los fármacos , Proteínas Nucleares/antagonistas & inhibidores , Transactivadores/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Acetilación , Azepinas/farmacología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Lisina/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , Origen de Réplica/efectos de los fármacos , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Triazoles/farmacología , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismo , Activación Viral/efectos de los fármacos , Fenómenos Fisiológicos de los Virus/efectos de los fármacosRESUMEN
The human Epstein-Barr virus (EBV) evades the immune system by entering a transcriptionally latent phase in B cells. EBV in tumor cells expresses distinct patterns of genes referred to as latency types. Viruses in tumor cells also display varying levels of lytic transcription resulting from spontaneous reactivation out of latency. We measured this dynamic range of lytic transcription with RNA deep sequencing and observed no correlation with EBV latency types among genetically different viruses, but type I cell lines reveal more spontaneous reactivation than isogenic type III cultures. We further determined that latency type and spontaneous reactivation levels predict the relative amount of induced reactivation generated by cytotoxic chemotherapy drugs. Our work has potential implications for personalizing medicine against EBV-transformed malignancies. Identifying latency type or measuring spontaneous reactivation may provide predictive power in treatment contexts where viral production should be either avoided or coerced.