RESUMEN
RIG-I-like receptors (RLRs: RIG-I, MDA5 and LGP2) play a major role in the innate immune response against viral infections and detect patterns on viral RNA molecules that are typically absent from host RNA. Upon RNA binding, RLRs trigger a complex downstream signaling cascade resulting in the expression of type I interferons and proinflammatory cytokines. In the past decade extensive efforts were made to elucidate the nature of putative RLR ligands. In vitro and transfection studies identified 5'-triphosphate containing blunt-ended double-strand RNAs as potent RIG-I inducers and these findings were confirmed by next-generation sequencing of RIG-I associated RNAs from virus-infected cells. The nature of RNA ligands of MDA5 is less clear. Several studies suggest that double-stranded RNAs are the preferred agonists for the protein. However, the exact nature of physiological MDA5 ligands from virus-infected cells needs to be elucidated. In this work, we combine a crosslinking technique with next-generation sequencing in order to shed light on MDA5-associated RNAs from human cells infected with measles virus. Our findings suggest that RIG-I and MDA5 associate with AU-rich RNA species originating from the mRNA of the measles virus L gene. Corresponding sequences are poorer activators of ATP-hydrolysis by MDA5 in vitro, suggesting that they result in more stable MDA5 filaments. These data provide a possible model of how AU-rich sequences could activate type I interferon signaling.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Virus del Sarampión/metabolismo , Sarampión/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Proteínas Virales/biosíntesis , Línea Celular Tumoral , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , Células HEK293 , Humanos , Helicasa Inducida por Interferón IFIH1 , Sarampión/genética , Virus del Sarampión/genética , ARN Mensajero/genética , ARN Viral/genética , Receptores Inmunológicos , Proteínas Virales/genéticaRESUMEN
Translocations or mutations of FUS, EWSR1, and TAF15 (FET) result in distinct genetic diseases. N-terminal translocations of any FET protein to a series of transcription factors yields chimeric proteins that contribute to sarcomagenesis, whereas mutations in the conserved COOH-terminal domain of wild-type FUS were recently shown to cause familial amyotrophic lateral sclerosis. We thus investigated whether the loss of one FUS allele by translocation in liposarcoma may be followed by mutations in either the remaining FUS allele or the paralogous EWSR1. Furthermore, we investigated the strength of the FET promoters and their contributions to sarcomagenesis given the proteins' frequent involvement in oncogenic translocations. We sequenced the respective genomic regions of both FUS and EWSR1 in 96 liposarcoma samples. Additionally, we determined FET transcript and protein levels in several liposarcoma cell lines. We did not observe sequence variations in either FUS or EWSR1. However, protein copy numbers reached an impressive 0.9 and 5.5 Mio of FUS and EWSR1 per tumor cell, respectively. Compared with adipose-derived stem cells, FUS and EWSR1 protein expression levels were elevated on average 28.6-fold and 7.3-fold, respectively. TAF15 mRNA levels were elevated on average 3.9-fold, although with a larger variation between samples. Interestingly, elevated TAF15 mRNA levels did not translate to strongly elevated protein levels, consistent with its infrequent occurrence as translocation partner in tumors. These results suggest that the powerful promoters of FET genes are predominantly responsible for the oncogenic effect of transcription factor translocations in sarcomas.
Asunto(s)
Proteínas de Unión a Calmodulina/genética , Liposarcoma/genética , ARN Mensajero/genética , Proteína FUS de Unión a ARN/genética , Proteínas de Unión al ARN/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Alelos , Animales , Secuencia de Bases , Proteínas de Unión a Calmodulina/biosíntesis , Línea Celular Transformada , Línea Celular Tumoral , Exones , Células HEK293 , Humanos , Liposarcoma/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Proteína EWS de Unión a ARN , Proteína FUS de Unión a ARN/biosíntesis , Proteínas de Unión al ARN/biosíntesis , Spodoptera/genética , Spodoptera/metabolismo , Factores Asociados con la Proteína de Unión a TATA/biosíntesis , Transfección , Translocación Genética , Regulación hacia ArribaRESUMEN
Unicellular organisms that live in marine environments must cope with considerable fluctuations in the availability of inorganic phosphate (Pi). Here, we investigated the extracellular Pi concentration-dependent expression, as well as the intracellular or extracellular localization, of phosphatases and phosphate transporters of the diatom Phaeodactylum tricornutum. We identified Pi-regulated plasma membrane-localized, ER-localized, and secreted phosphatases, in addition to plasma membrane-localized, vacuolar membrane-localized, and plastid-surrounding membrane-localized phosphate transporters that were also regulated in a Pi concentration-dependent manner. These studies not only add further knowledge to already existing transcriptomic data, but also highlight the capacity of the diatom to distribute Pi intracellularly and to mobilize Pi from extracellular and intracellular resources.
RESUMEN
FUS, EWSR1 and TAF15, constituting the FET protein family, are abundant, highly conserved RNA-binding proteins with important roles in oncogenesis and neuronal disease, yet their RNA targets and recognition elements are unknown. Using PAR-CLIP, we defined global RNA targets for all human FET proteins and two ALS-causing human FUS mutants. FET members showed similar binding profiles, whereas FUS mutants showed a drastically altered binding pattern, consistent with changes in subcellular localization.