RESUMEN
hnRNP UL1 plays an important role in cell nuclei, where it is recruited to DNA damage sites and is involved in the repair of DNA double-strand breaks. Furthermore, this protein is known as a transcriptional repressor of RNA polymerase II genes. In the present study, we have shown that hnRNP UL1 is also localized in the nucleoli of human cells. Upon investigating its function, we found that hnRNP UL1 stimulates ribosomal DNA (rDNA) gene transcription. Moreover, we observed that cells with hnRNP UL1 silencing exhibited increased sensitivity to DNA damage. We also showed that hnRNP UL1 interacts with γH2A.X, RPA32, XRCC1, and Chk1 in cell nucleoli, suggesting its involvement in the repair of rDNA damage.
Asunto(s)
Nucléolo Celular , Reparación del ADN , Ribonucleoproteínas Nucleares Heterogéneas , Proteínas Nucleares , Factores de Transcripción , Nucléolo Celular/genética , Roturas del ADN de Doble Cadena , ADN Ribosómico/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Proteínas Nucleares/genética , Factores de Transcripción/genética , Transcripción Genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genéticaRESUMEN
Genes encoding replication-dependent histones lack introns, and the mRNAs produced are a unique class of RNA polymerase II transcripts in eukaryotic cells that do not end in a polyadenylated tail. Mature mRNAs are thus formed by a single endonucleolytic cleavage that releases the pre-mRNA from the DNA and is the only processing event necessary. U7 snRNP is one of the key factors that determines the cleavage site within the 3'UTR of replication-dependent histone pre-mRNAs. We have previously showed that the FUS protein interacts with U7 snRNA/snRNP and regulates the expression of histone genes by stimulating transcription and 3' end maturation. Mutations in the FUS gene first identified in patients with amyotrophic lateral sclerosis (ALS) lead to the accumulation of the FUS protein in cytoplasmic inclusions. Here, we report that mutations in FUS lead to disruption of the transcriptional activity of FUS and mislocalization of U7 snRNA/snRNP in cytoplasmic aggregates in cellular models and primary neurons. As a consequence, decreased transcriptional efficiency and aberrant 3' end processing of histone pre-mRNAs were observed. This study highlights for the first time the deregulation of replication-dependent histone gene expression and its involvement in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Regulación de la Expresión Génica , Histonas/metabolismo , Mutación , Proteína FUS de Unión a ARN/genética , Ribonucleoproteína Nuclear Pequeña U7/genética , Regiones no Traducidas 3' , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Perfilación de la Expresión Génica , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Neurociencias , Plásmidos/metabolismo , ARN Nuclear Pequeño/genética , Ribonucleoproteínas Nucleares Pequeñas/genéticaRESUMEN
Most U-rich small nuclear ribonucleoproteins (snRNPs) are complexes that mediate the splicing of pre-mRNAs. U7 snRNP is an exception in that it is not involved in splicing but is a key factor in the unique 3' end processing of replication-dependent histone mRNAs. However, by introducing controlled changes in the U7 snRNA histone binding sequence and in the Sm motif, it can be used as an effective tool for gene therapy. The modified U7 snRNP (U7 Sm OPT) is thus not involved in the processing of replication-dependent histone pre-mRNA but targets splicing by inducing efficient skipping or inclusion of selected exons. U7 Sm OPT is of therapeutic importance in diseases that are an outcome of splicing defects, such as myotonic dystrophy, Duchenne muscular dystrophy, amyotrophic lateral sclerosis, ß-thalassemia, HIV-1 infection and spinal muscular atrophy. The benefits of using U7 Sm OPT for gene therapy are its compact size, ability to accumulate in the nucleus without causing any toxic effects in the cells, and no immunoreactivity. The risk of transgene misregulation by using U7 Sm OPT is also low because it is involved in correcting the expression of an endogenous gene controlled by its own regulatory elements. Altogether, using U7 Sm OPT as a tool in gene therapy can ensure lifelong treatment, whereas an oligonucleotide or other drug/compound would require repeated administration. It would thus be strategic to harness these unique properties of U7 snRNP and deploy it as a tool in gene therapy.