Abstract
Nonsense-mediated mRNA decay (NMD) is a molecular pathway of mRNA surveillance that ensures the rapid degradation of mRNAs containing premature translation termination codons in all studied eukaryotes. Originally, NMD was thought of as a quality control pathway that targets non-functional mRNAs arising from mutations and splicing errors. More recently, NMD has been shown to also regulate normal gene expression and NMD thus emerged as one of the key post-transcriptional mechanisms of gene regulation. Despite the progress in the understanding of the role and mechanism of this pathway, the physiological impact of NMD on humans is not yet fully uncovered. To explore the functions of NMD in humans, I combined RNAi against the essential NMD factor UPF1 with genome-wide microarray analysis. My research indicate that NMD affects the expression of a large number of genes implicated in a wide diversity of functions although a majority seems to be affected indirectly and – consequently – do not represent legitimate NMD targets. The validation of five bona fide NMD transcripts allowed me to develop an assay to quantitate differences in NMD efficiency. Using three different strains of HeLa cells as a simple model, I have systematically analysed the molecular mechanism underlying quantitative differences in NMD efficiency. The results of this analysis show that the quantitative differences in NMD efficiency represent a stable characteristic of the investigated strains. Low NMD efficiency is shown to be functionally related to the reduced abundance of the exon junction component RNPS1 in one of the analysed HeLa strains. Furthermore, restoration of functional RNPS1 expression, but not of NMD-inactive mutant proteins, also restores efficient NMD in the RNPS1 deficient cell line. I conclude that cellular concentrations of RNPS1 can modify NMD efficiency and propose that the cell type specific co-factor availability represents a novel principle that quantitatively controls NMD. I also tested the hypothesis of NMD as a genetic modifier in the phenotypic expression of disease. A specific -thalassemia – common in Mediterranean Asia – was assayed as a model. My results do not support a role of NMD for the variable severity of this specific mutation leading to anemia.
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