Inconsistent with prevailing models for nonsense-mediated mRNA decay (NMD) in mammals, the mRNA levels of immunoglobulin-mu (Ig-mu) genes with premature termination codons (PTCs) in the penultimate exon are still reduced by NMD when the intron furthest downstream is deleted. As in yeast, this exon junction complex-independent NMD of Ig-mu mRNAs depends on the distance between the termination codon and the poly(A) tail and suggests an evolutionarily conserved mode of PTC recognition.
Aberrant mRNAs whose open reading frame (ORF) is truncated by the presence of a premature translation-termination codon (PTC) are recognized and degraded in eukaryotic cells by a process called nonsense-mediated mRNA decay (NMD). Here, we report the development of a reporter system that allows monitoring of NMD in mammalian cells by measuring the fluorescence of green fluorescent protein (GFP). The NMD reporter gene consists of a T-cell receptor-β minigene construct, in which the GFP-ORF was inserted such that the stop codon of GFP is recognized as PTC. The reporter mRNA is therefore subjected to NMD, resulting in a low steady-state mRNA level, an accordingly low protein level and hence a very low green fluorescence in normal, NMD-competent cells that express this reporter gene. We show that the inactivation of NMD by RNAi-mediated knockdown of the essential NMD factor hUpf1 or hSmg6 increases the NMD reporter mRNA level, resulting in a proportional increase of the green fluorescence that can be detected by flow cytometry, spectrofluorometry and fluorescence microscopy. With these properties, our GFP-based NMD reporter system could be used for large-scale screenings to identify NMD-inhibiting drugs or NMD-deficient mutant cells.
Purpose: To investigate the mechanism(s) of resistance to the RAF-inhibitor vemurafenib, we conducted a comprehensive analysis of the genetic alterations occurring in metastatic lesions from a patient with a BRAF V600E -mutant cutaneous melanoma who, after a first response, underwent subsequent rechallenge with this drug. Experimental Design: We obtained blood and tissue samples from a patient diagnosed with a BRAF V600E -mutant cutaneous melanoma that was treated with vemurafenib and achieved a near-complete response. At progression, he received additional lines of chemo/immunotherapy and was successfully rechallenged with vemurafenib. Exome and RNA sequencing were conducted on a pretreatment tumor and two subcutaneous resistant metastases, one that was present at baseline and previously responded to vemurafenib (PV1) and one that occurred de novo after reintroduction of the drug (PV2). A culture established from PV1 was also analyzed.Results: We identified two NRAS-activating somatic mutations, Q61R and Q61K, affecting two main subpopulations in the metastasis PV1 and a BRAF alternative splicing, involving exons 4-10, in the metastasis PV2. These alterations, known to confer resistance to RAF inhibitors, were tumor-specific, mutually exclusive, and were not detected in pretreatment tumor samples. In addition, the oncogenic PIK3CA H1047R mutation was detected in a subpopulation of PV1, but this mutation did not seem to play a major role in vemurafenib resistance in this metastasis.Conclusions: This work describes the coexistence within the same patient of different molecular mechanisms of resistance to vemurafenib affecting different metastatic sites. These findings have direct implications for the clinical management of BRAF-mutant melanoma.
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