RESUMEN
The introduction of insertion-deletions (INDELs) by non-homologous end-joining (NHEJ) pathway underlies the mechanistic basis of CRISPR-Cas9-directed genome editing. Selective gene ablation using CRISPR-Cas9 is achieved by installation of a premature termination codon (PTC) from a frameshift-inducing INDEL that elicits nonsense-mediated decay (NMD) of the mutant mRNA. Here, by examining the mRNA and protein products of CRISPR targeted genes in a cell line panel with presumed gene knockouts, we detect the production of foreign mRNAs or proteins in ~50% of the cell lines. We demonstrate that these aberrant protein products stem from the introduction of INDELs that promote internal ribosomal entry, convert pseudo-mRNAs (alternatively spliced mRNAs with a PTC) into protein encoding molecules, or induce exon skipping by disruption of exon splicing enhancers (ESEs). Our results reveal challenges to manipulating gene expression outcomes using INDEL-based mutagenesis and strategies useful in mitigating their impact on intended genome-editing outcomes.
Asunto(s)
Edición Génica/métodos , Mutagénesis , ARN Mensajero/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sistemas CRISPR-Cas , Línea Celular , Línea Celular Tumoral , Codón sin Sentido/genética , Mutación del Sistema de Lectura , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Mutación INDEL , Estabilidad del ARN , ARN Mensajero/químicaRESUMEN
The maintenance of adult animal tissues depends upon highly conserved intercellular signaling molecules that include the secreted WNT proteins. Although it is generally accepted that lipidation of WNTs by the acyltransferase Porcupine (PORCN) and their subsequent recognition by the Wntless (WLS) protein is essential for their cellular secretion, the molecular understanding of this process remains limited. Using structurally diverse fatty acyl donor analogs and mouse embryonic fibroblasts expressing PORCN protein from different metazoan phyla, we demonstrate here that PORCN active-site features, which are conserved across the animal kingdom, enforce cis-Δ9 fatty acylation of WNTs. Aberrant acylation of a WNT with an exogenously supplied trans-Δ9 fatty acid induced the accumulation of WNT-PORCN complexes, suggesting that the fatty acyl species is critical for the extrication of lipidated WNTs from PORCN. Our findings reveal a previously unrecognized fatty acyl-selective checkpoint in the manufacturing of a lipoprotein that forms a basis for WNT signaling sensitivity to trans fats and to PORCN inhibitors in clinical development.
Asunto(s)
Aciltransferasas/metabolismo , Ácidos Grasos/metabolismo , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Vía de Señalización Wnt , Acilación , Aciltransferasas/genética , Animales , Células COS , Caenorhabditis elegans , Pollos , Chlorocebus aethiops , Ácidos Grasos/genética , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Schistosoma mansoni , XenopusRESUMEN
Tankyrases (TNKS and TNKS2) are enzymes that catalyze poly-ADP-ribosylation (PARsylation) of their target proteins. Tankyrase-mediated PARsylation plays critical regulatory roles in cell signaling, particularly in the Wnt/ß-catenin pathway. The sterile alpha motif (SAM) domain in tankyrases mediates their oligomerization, which is essential for tankyrase function. The oligomerization regulates the catalytic activity of tankyrases, but the underlying mechanism is unclear. Our analyses of crystal structures of the tankyrase catalytic domain suggest that formation of a head-to-head dimer regulates the catalytic activity. Our activity assays show that residues in the catalytic domain dimer interface are important for the PARsylation activity of tankyrases both in solution and cells. The dimer is weak and may only form in the context of the SAM domain-mediated oligomers of tankyrases, consistent with the dependence of the tankyrase activity on the SAM domain.