The efficiency of nonsense-mediated mRNA decay is an inherent character and varies among different cells

Department of Genetics, Life Sciences Institute, The Hebrew University, Jerusalem, Israel.
European Journal of HumanGenetics (Impact Factor: 4.23). 12/2007; 15(11):1156-62. DOI: 10.1038/sj.ejhg.5201889
Source: PubMed

ABSTRACT Nonsense-mediated mRNA decay (NMD) is a mechanism, which selectively degrades transcripts carrying premature termination codons (PTCs) and a variety of physiologic transcripts containing NMD-inducing features. In a recent study, we have found variable NMD efficiency among nasal epithelial cells obtained from cystic fibrosis (CF) patients. This variability was found for CF transmembrane conductance regulator (CFTR) transcripts carrying the W1282X PTC, as well as for several NMD physiologic substrates. Here, we aimed to investigate the possibility that variability in NMD efficiency is a more generalized phenomenon and is not restricted to nasal epithelial cells. To investigate this possibility, we analyzed the NMD efficiency of both a CFTR constructs carrying the W1282X PTC and beta-globin constructs carrying the NS39 PTC, in HeLa and MCF7 cells. Variability in NMD efficiency was found for both constructs between the cells, such that in HeLa cells the NMD was highly efficient and in MCF7 the efficiency was significantly lower. Moreover, similar differences in the efficiency of NMD were found for five endogenous NMD physiologic transcripts. Altogether, our results demonstrate existence of cells in which NMD of all transcripts is efficient, whereas others in which the NMD is less efficient, suggesting that the efficiency of NMD is an inherent character of cells. Our results also suggest that variability in the efficiency of NMD is a general phenomenon and is not restricted to nasal epithelial cells. As NMD affects the level of many transcripts, variability in the NMD efficiency might play a role as a genetic modifier of different cellular functions.

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    • "Beside the common F508del variant, our CF patients carry missense and nonsense alterations in the CFTR gene. Indeed, different studies have shown that the effect of nonsense mutations on CFTR mRNA levels are very variable [16] and nonsense-mediated decay (NMD) is addressed as the major mechanism for rapid degradation of aberrant transcripts harboring premature termination codons. Interestingly, microRNAs have recently been defined as important regulators of the NMD efficiency [17]. "
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    ABSTRACT: MicroRNAs (miRNAs) have recently emerged as important gene regulators in Cystic Fibrosis (CF), a common monogenic disease characterized by severe infection and inflammation, especially in the airway compartments. In the current study, we show that both miR-145 and miR-494 are significantly up-regulated in nasal epithelial tissues from CF patients compared with healthy controls (p<0.001 and p<0.01, respectively) by Quantitative Real-Time PCR. Only miR-494 levels showed a trend of correlation with reduced CFTR mRNA expression and positive sweat test values, supporting the negative regulatory role of this miRNA on CFTR synthesis. Using computational prediction algorithms and luciferase reporter assays, SMAD family member 3 (SMAD3), a key element of the TGF-β1 inflammatory pathway, was identified as a target of miR-145. Indeed, miR-145 synthetic mimics suppressed by approximately 40% the expression of a reporter construct containing the SMAD3 3'-UTR. Moreover, we observed an inverse correlation between SMAD3 mRNA expression and miR-145 in CF nasal tissues (r=-0.68, p=0.0018, Pearson's correlation). Taken together, these results confirm the pivotal role of miRNAs in the CF physio-pathogenesis and suggest that miRNA deregulation play a role in the airway disease severity by modulating CFTR levels as well as the expression of important molecules involved in the inflammatory response. miR-494 and miR-145 may, therefore, be potential biomarker and therapeutic target to specific CF clinical manifestations.
    Journal of cystic fibrosis: official journal of the European Cystic Fibrosis Society 04/2013; 12(6). DOI:10.1016/j.jcf.2013.03.007 · 3.82 Impact Factor
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    • "Gehring et al. reported that NMD can be triggered in a UPF2-inde- pendent manner [36], while Chan et al. describe an UPF3b-indepen- dent NMD pathway [37]. These studies highlight variants of the NMD mechanism and may explain why NMD efficiency varies significantly across different cell types [38]. Given the expression profile of ROD1 across different tissues, it is thus plausible that it could be another factor adding to the heterogeneity of NMD. "
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    ABSTRACT: RNA-binding proteins play a crucial role in the post-transcriptional regulation of gene expression. Polypyrimidine tract binding protein (PTB in humans) has been extensively characterized as an important splicing factor, and has additional functions in 3' end processing and translation. ROD1 is a PTB paralog containing four RRM (RNA recognition motif) domains. Here, we discover a function of ROD1 in nonsense-mediated mRNA decay (NMD). We show that ROD1 and the core NMD factor UPF1 interact and co-regulate an extensive number of target genes. Using a reporter system, we demonstrate that ROD1, similarly to UPF1 and UPF2, is required for the destabilization of a known NMD substrate. Finally, we show through RIP-seq that ROD1 and UPF1 associate with a significant number of common transcripts.
    FEBS letters 04/2012; 586(8):1101-10. DOI:10.1016/j.febslet.2012.03.015 · 3.34 Impact Factor
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    • "If so, reducing the protein expression level should reduce or eliminate the complexes. HeLa cells are frequently used for studying nonsense mediated mRNA decay due to their low transfection efficiency and low levels of protein expression (Kang et al., 2009; Linde et al., 2007). Therefore we first determined the relative expression levels of wild-type γ2S subunits when coexpressed with α1 and β2 subunits in HeLa cells with the same amounts of cDNA used for HEK 293T cell transfections and measured subunit expression levels using the quantitative method of flow cytometry to compare subunit expression levels in each cell type (Figure 2A). "
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    ABSTRACT: The GABA(A) receptor γ2 subunit nonsense mutation Q351X has been associated with the genetic epilepsy syndrome generalized epilepsy with febrile seizures plus, which includes a spectrum of seizures types from febrile seizures to Dravet syndrome. Although most genetic epilepsy syndromes are mild and remit with age, Dravet syndrome has a more severe clinical course with refractory seizures associated with developmental delay and cognitive impairment. The basis for the broad spectrum of seizure phenotypes is uncertain. We demonstrated previously that the GABA(A) receptor γ2 subunit gene Q351X mutation suppressed biogenesis of wild-type partnering α1 and β2 subunits in addition to its loss of function. Here we show that γ2S(Q351X) subunits have an additional impairment of biogenesis. Mutant γ2(Q351X) subunits were degraded more slowly than wild-type γ2 subunits and formed SDS-resistant, high-molecular-mass complexes or aggregates in multiple cell types, including neurons. The half-life of γ2S(Q351X) subunits was ∼4 h, whereas that of γ2S subunits was ∼2 h. Mutant subunits formed complexes rapidly after synthesis onset. Using multiple truncated subunits, we demonstrated that aggregate formation was a general phenomenon for truncated γ2S subunits and that their Cys-loop cysteines were involved in aggregate formation. Protein aggregation is a hallmark of neurodegenerative diseases, but the effects of the mutant γ2S(Q351X) subunit aggregates on neuronal function and survival are unclear. Additional validation of the mutant subunit aggregation in vivo and determination of the involved signaling pathways will help reveal the pathological effects of these mutant subunit aggregates in the pathogenesis of genetic epilepsy syndromes.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 10/2010; 30(41):13895-905. DOI:10.1523/JNEUROSCI.2320-10.2010 · 6.75 Impact Factor
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