ABCMdb

PMID: 17625509

Linde L, Boelz S, Neu-Yilik G, Kulozik AE, Kerem B
The efficiency of nonsense-mediated mRNA decay is an inherent character and varies among different cells.
Eur J Hum Genet. 2007 Nov;15(11):1156-62. Epub 2007 Jul 11., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Trp1282* This variability was found for CF transmembrane conductance regulator (CFTR) transcripts carrying the W1282X PTC, as well as for several NMD physiologic substrates. Login to comment 4 ABCC7 p.Trp1282* To investigate this possibility, we analyzed the NMD efficiency of both a CFTR constructs carrying the W1282X PTC and b-globin constructs carrying the NS39 PTC, in HeLa and MCF7 cells. Login to comment 13 ABCC7 p.Trp1282* Tel: þ 49 972 2 6585689; Fax: þ 49 972 2 6584810; E-mail: kerem@cc.huji.ac.il European Journal of Human Genetics (2007) 15, 1156-1162 & 2007 Nature Publishing Group All rights reserved 1018-4813/07 $30.00 www.nature.com/ejhg In a recent study, we have shown that NMD efficiency considerably varies among three cell lines derived from nasal epithelium of cystic fibrosis (CF) patients.7 We found variability in NMD efficiency for CF transmembrane conductance regulator (CFTR) transcripts carrying a disease-causing PTC, the W1282X mutation. Login to comment 21 ABCC7 p.Trp1282* Variability in NMD efficiency was found for two different transcripts carrying disease-causing PTCs: CFTR constructs carrying the W1282X nonsense mutation and b-globin transcripts carrying the NS39 nonsense mutation. Login to comment 30 ABCC7 p.Trp1282* The CFTR mutant construct was generated using the same cloning approach on DNA extracted from an individual homozygous for the W1282X mutation. Login to comment 31 ABCC7 p.Trp1282* Both constructs were sequenced and asides from the W1282X mutation no variation was identified in all exons and 199 bp of each intron. Login to comment 41 ABCC7 p.Trp1282* For quantification of CFTR W1282X transcript levels in HeLa and MCF7 cells, which had been transfected with the CFTR constructs, we performed real-time PCR in the LightCycler (software version 3.5), using a FastStart DNA Master SYBR Green I kit (Roche Diagnostics). Login to comment 43 ABCC7 p.Trp1282* The level of mRNA transcribed from the WT or W1282X CFTR construct was normalized to the mRNA level of GFP. Login to comment 59 ABCC7 p.Trp1282* We analyzed the efficiency of NMD for two different transcripts carrying disease-causing PTCs: CFTR constructs carrying the CF-causing PTC W1282X and the b-globin constructs carrying the b-thalassemia-causing PTC NS39. Login to comment 61 ABCC7 p.Trp1282* In our previous study, we analyzed the efficiency of NMD for endogenous CFTR transcripts carrying the W1282X nonsense mutation, in nasal epithelial cells derived from CF patients. Login to comment 62 ABCC7 p.Trp1282* As HeLa and MCF7 cells do not express CFTR gene, we transfected them with CFTR constructs carrying the W1282X PTC, which is located 455 nucleotides upstream to the final exon-exon junction, or the normal sequence (WT) (Figure 1a). Login to comment 63 ABCC7 p.Trp1282* We examined the levels of transfected W1282X transcripts following treatment with CHX, a general inhibitor of translation elongation and as NMD is a translation-dependent mechanism, CHX treatment leads indirectly to inhibition of NMD.13 A GFP plasmid was used as a reference for transfection efficiency in each cell line. Login to comment 64 ABCC7 p.Trp1282* The levels of mRNA transcribed from the WT or W1282X CFTR construct was normalized to the mRNA level of GFP. Login to comment 67 ABCC7 p.Trp1282* These results suggest that in MCF7 cells, the NMD of CFTR transcripts carrying the W1282X PTC is inefficient, as the transcript level showed only a marginal increase following the NMD indirect inhibition. Login to comment 70 ABCC7 p.Trp1282* The levels of the a b Figure 1 Effect of CHX treatment on the level of CFTR mRNA transcribed from constructs carrying the W1282X PTC. Login to comment 71 ABCC7 p.Trp1282* (a) A scheme of the WT (upper panel) and W1282X (lower panel) constructs, which contained all CFTR exons (marked in the boxes by numbers) and part of the intronic sequences between exons 20-22. Login to comment 74 ABCC7 p.Trp1282* The level of mRNA transcribed from CFTR constructs carrying either the normal sequence or the W1282X PTC was normalized to the mRNA level of GFP. Login to comment 76 ABCC7 p.Trp1282* The fold increase in the level of CFTR W1282X transcripts is shown as mean7SEM. Login to comment 81 ABCC7 p.Trp1282* Following CHX treatment, no increase in the level of NS39 transcripts was observed in MCF7 cells, whereas in HeLa cells a significant increase (4.470.20-fold) was observed (Figure 2b), similar to the variable effect of CHX on the level of CFTR W1282X transcripts in these cell lines. Login to comment