RESUMEN
The MYCN oncoprotein binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3'-5' exoribonuclease complex, suggesting a function in RNA metabolism. Here, we show that MYCN forms stable high-molecular-weight complexes with the exosome and multiple RNA-binding proteins. MYCN binds RNA in vitro and in cells via a conserved sequence termed MYCBoxI. In cells, MYCN associates with thousands of intronic transcripts together with the ZCCHC8 subunit of the nuclear exosome targeting complex and enhances their processing. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. On chromatin, MYCN is then replaced by the MNT(MXD6) repressor protein, inhibiting MYCN-dependent transcription. RNA-binding-deficient alleles show that RNA-binding limits MYCN's ability to activate cell growth-related genes but is required for MYCN's ability to promote progression through S phase and enhance the stress resilience of neuroblastoma cells.
Asunto(s)
Proteína Proto-Oncogénica N-Myc , Proteínas Nucleares , Proteínas Oncogénicas , Proteínas de Unión al ARN , Proteína Proto-Oncogénica N-Myc/metabolismo , Proteína Proto-Oncogénica N-Myc/genética , Humanos , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Oncogénicas/metabolismo , Proteínas Oncogénicas/genética , Regiones Promotoras Genéticas , Línea Celular Tumoral , Neuroblastoma/metabolismo , Neuroblastoma/genética , Neuroblastoma/patología , Exosomas/metabolismo , Exosomas/genética , Intrones , Unión Proteica , Núcleo Celular/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Regulación Neoplásica de la Expresión Génica , ARN/metabolismo , ARN/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Proliferación CelularRESUMEN
Periodic fever is a characteristic clinical feature of human malaria, but how parasites survive febrile episodes is not known. Although the genomes of Plasmodium species encode a full set of chaperones, they lack the conserved eukaryotic transcription factor HSF1, which activates the expression of chaperones following heat shock. Here, we show that PfAP2-HS, a transcription factor in the ApiAP2 family, regulates the protective heat-shock response in Plasmodium falciparum. PfAP2-HS activates the transcription of hsp70-1 and hsp90 at elevated temperatures. The main binding site of PfAP2-HS in the entire genome coincides with a tandem G-box DNA motif in the hsp70-1 promoter. Engineered parasites lacking PfAP2-HS have reduced heat-shock survival and severe growth defects at 37 °C but not at 35 °C. Parasites lacking PfAP2-HS also have increased sensitivity to imbalances in protein homeostasis (proteostasis) produced by artemisinin, the frontline antimalarial drug, or the proteasome inhibitor epoxomicin. We propose that PfAP2-HS contributes to the maintenance of proteostasis under basal conditions and upregulates specific chaperone-encoding genes at febrile temperatures to protect the parasite against protein damage.
Asunto(s)
Fiebre/parasitología , Malaria Falciparum/parasitología , Plasmodium falciparum/fisiología , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Antimaláricos/farmacología , Artemisininas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Calor , Humanos , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Proteostasis/efectos de los fármacos , Proteínas Protozoarias/genética , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Plasmodium parasites undergo several major developmental transitions during their complex lifecycle, which are enabled by precisely ordered gene expression programs. Transcriptomes from the 48-h blood stages of the major human malaria parasite Plasmodium falciparum have been described using cDNA microarrays and RNA-seq, but these assays have not always performed well within non-coding regions, where the AT-content is often 90-95%. RESULTS: We developed a directional, amplification-free RNA-seq protocol (DAFT-seq) to reduce bias against AT-rich cDNA, which we have applied to three strains of P. falciparum (3D7, HB3 and IT). While strain-specific differences were detected, overall there is strong conservation between the transcriptional profiles. For the 3D7 reference strain, transcription was detected from 89% of the genome, with over 78% of the genome transcribed into mRNAs. We also find that transcription from bidirectional promoters frequently results in non-coding, antisense transcripts. These datasets allowed us to refine the 5' and 3' untranslated regions (UTRs), which can be variable, long (> 1000 nt), and often overlap those of adjacent transcripts. CONCLUSIONS: The approaches applied in this study allow a refined description of the transcriptional landscape of P. falciparum and demonstrate that very little of the densely packed P. falciparum genome is inactive or redundant. By capturing the 5' and 3' ends of mRNAs, we reveal both constant and dynamic use of transcriptional start sites across the intraerythrocytic developmental cycle that will be useful in guiding the definition of regulatory regions for use in future experimental gene expression studies.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Malaria Falciparum/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/genética , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Humanos , Estadios del Ciclo de Vida , Técnicas de Amplificación de Ácido Nucleico/métodos , Plasmodium falciparum/clasificación , Plasmodium falciparum/genética , ARN Mensajero/genética , Especificidad de la EspecieRESUMEN
In the malaria parasite Plasmodium falciparum, the switch from asexual multiplication to sexual differentiation into gametocytes is essential for transmission to mosquitos. The transcription factor PfAP2-G is a key determinant of sexual commitment that orchestrates this crucial cell fate decision. Here we identify the direct targets of PfAP2-G and demonstrate that it dynamically binds hundreds of sites across the genome. We find that PfAP2-G is a transcriptional activator of early gametocyte genes, and identify differences in PfAP2-G occupancy between gametocytes derived via next-cycle and same-cycle conversion. Our data implicate PfAP2-G not only as a transcriptional activator of gametocyte genes, but also as a potential regulator of genes important for red blood cell invasion. We also find that regulation by PfAP2-G requires interaction with a second transcription factor, PfAP2-I. These results clarify the functional role of PfAP2-G during sexual commitment and early gametocytogenesis.