RESUMEN
Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterized, as most disease models have been based on overexpressing mutant FUS, which will alter RNA processing due to FUS autoregulation. We and others have recently created knockin models that overcome the overexpression problem, and have generated high depth RNA-sequencing on FUS mutants in parallel to FUS knockout, allowing us to compare mutation-induced changes to genuine loss of function. We find that FUS-ALS mutations induce a widespread loss of function on expression and splicing. Specifically, we find that mutant FUS directly alters intron retention levels in RNA-binding proteins. Moreover, we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS levels have been linked to disease, and we show here that this novel autoregulation mechanism is altered by FUS mutations. Crucially, we also observe this phenomenon in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Homeostasis/genética , Proteína FUS de Unión a ARN/genética , Animales , Citoplasma/genética , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Humanos , Intrones/genética , Mutación con Pérdida de Función , Ratones , Ratones Noqueados , Mutación/genética , Empalme del ARN/genética , Superóxido Dismutasa-1/genética , Proteína que Contiene Valosina/genéticaRESUMEN
Nonsense-mediated mRNA decay (NMD), which is best known for degrading mRNAs with premature termination codons (PTCs), is thought to be triggered by aberrant translation termination at stop codons located in an environment of the mRNP that is devoid of signals necessary for proper termination. In mammals, the cytoplasmic poly(A)-binding protein 1 (PABPC1) has been reported to promote correct termination and therewith antagonize NMD by interacting with the eukaryotic release factors 1 (eRF1) and 3 (eRF3). Using tethering assays in which proteins of interest are recruited as MS2 fusions to a NMD reporter transcript, we show that the three N-terminal RNA recognition motifs (RRMs) of PABPC1 are sufficient to antagonize NMD, while the eRF3-interacting C-terminal domain is dispensable. The RRM1-3 portion of PABPC1 interacts with eukaryotic initiation factor 4G (eIF4G) and tethering of eIF4G to the NMD reporter also suppresses NMD. We identified the interactions of the eIF4G N-terminus with PABPC1 and the eIF4G core domain with eIF3 as two genetically separable features that independently enable tethered eIF4G to inhibit NMD. Collectively, our results reveal a function of PABPC1, eIF4G and eIF3 in translation termination and NMD suppression, and they provide additional evidence for a tight coupling between translation termination and initiation.
Asunto(s)
Factor 4G Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Degradación de ARNm Mediada por Codón sin Sentido , Codón sin Sentido/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factor 4G Eucariótico de Iniciación/química , Humanos , Proteína I de Unión a Poli(A)/química , Proteína I de Unión a Poli(A)/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína , Proteínas Proto-Oncogénicas c-ets/química , Proteínas Proto-Oncogénicas c-ets/metabolismo , Ribonucleósido Difosfato Reductasa/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
During B cell maturation, immunoglobulin (Ig) genes frequently acquire premature translation-termination codons (PTCs) as a result of the somatic rearrangement of V, D, and J gene segments. However, it is essential for a B lymphocyte to produce only one kind of antibody and therefore to ensure that the heavy and light chain polypeptides are expressed exclusively from the corresponding functional alleles, whereas no protein is made from the nonproductively rearranged alleles. At the post-transcriptional level, a well-studied mRNA quality control mechanism, termed nonsense-mediated mRNA decay (NMD), recognizes and degrades PTC-containing mRNAs in a translation-dependent manner. In addition, transcriptional silencing of PTC-containing Ig-mu and Ig-gamma heavy chain reporter genes was observed in HeLa cells. To investigate the silencing of nonproductively rearranged Ig genes in a more physiological context, we analyzed a monoclonal line of immortalized murine pro-B cells harboring one productively (PTC-) and one nonproductively (PTC+) rearranged Ig-mu heavy chain allele. We show that the steady-state abundance of PTC+ mRNA was approximately 40-fold lower when compared to that of the PTC- mRNA. However, both the PTC+ and PTC- allele seemed to be equally well transcribed since the abundances of PTC+ and PTC- pre-mRNA were very similar and chromatin immunoprecipitations revealed comparable occupancy of RNA polymerase II and acetylated histone H3 on both alleles. Altogether, we found no evidence for transcriptional silencing of the PTC+ allele in this pro-B cell line; hence, the efficient down-regulation of the PTC+ Ig-mu mRNA results entirely from NMD.