Comparison of nonsense-mediated mRNA decay efficiency in various murine tissues

Abstract

Background: The Nonsense-Mediated mRNA Decay (NMD) pathway detects and degrades mRNAs containing premature termination codons, thereby preventing the accumulation of potentially detrimental truncated proteins. Intertissue variation in the efficiency of this mechanism has been suggested, which could have important implications for the understanding of genotype-phenotype correlations in various genetic disorders. However, compelling evidence in favour of this hypothesis is lacking. Here, we have explored this question by measuring the ratio of mutant versus wild-type Men1 transcripts in thirteen tissues from mice carrying a heterozygous truncating mutation in the ubiquitously expressed Men1 gene.

Results: Significant differences were found between two groups of tissues. The first group, which includes testis, ovary, brain and heart, displays a strong decrease of the nonsense transcript (average ratio of 18% of mutant versus wild-type Men1 transcripts, identical to the value measured in murine embryonic fibroblasts). The second group, comprising lung, intestine and thymus, shows much less pronounced NMD (average ratio of 35%). Importantly, the extent of degradation by NMD does not correlate with the expression level of eleven genes encoding proteins involved in NMD or with the expression level of the Men1 gene.

Conclusion: Mouse models are an attractive option to evaluate the efficiency of NMD in multiple mammalian tissues and organs, given that it is much easier to obtain these from a mouse than from a single individual carrying a germline truncating mutation. In this study, we have uncovered in the thirteen different murine tissues that we examined up to a two-fold difference in NMD efficiency.

Figure 1

Description of the Men1 mouse model used in the analysis. (a) Schematic representation of the mutant and of the wild-type allele of the Men1 gene in the heterozygous Men1 mutant mice. Black boxes and lines represent exons and introns respectively. The grey horizontal arrow depicts a loxP sequence, and the black vertical arrow shows the location of the premature stop codon generated by exon 3 deletion. The strategy used to delete Men1 exon 3 in mice has been described previously [17]. (b) Transcript copy number were measured by quantitative RT-PCR using 50 pg of RNA extracted from Men1^+/Δ mouse embryonic fibroblasts (MEF) untreated or treated with puromycin (Puro). Specific amplification of either the wild-type (WT) or the mutant (Mu) transcript lacking exon 3 was achieved using the primers described in the Methods section.

Figure 2

NMD efficiency in 13 tissues of mice heterozygous for a truncating mutation in the Men1 gene. NMD efficiency is expressed as a ratio of the amount of mutant Men1 transcripts versus wild-type species, as measured by quantitative RT-PCR. Each bar on the graph represents the mean value of at least 3 independent measurements (± standard deviation) in four samples of the same tissue (two samples only were used for testis and ovary).

Figure 3

Men1 transcript levels in 13 murine tissues. Transcript levels (copy number per 50 pg of RNA extracted from each of the mentioned tissue) were measured by quantitative RT-PCR, and normalized to the level of Hprt1 and β-actin transcripts. Each bar on the graph represents the mean value of 3 independent measurements (± standard deviation) in four samples of the same tissue (two samples only were used for testis and ovary).

Figure 4

Transcript levels of the Upf1-3 and Smg1 genes in 13 murine tissues. Transcript levels (copy number per 50 pg of RNA extracted from each of the mentioned tissues) were measured by quantitative RT-PCR using the same samples as previously, and normalized to the level of Hprt1 and β-actin transcripts. Each bar on the graph represents the mean value of at least 3 independent measurements (± standard deviation) in four samples of the same tissue (two samples only were used for testis and ovary). Please note that the scale showing the normalized number of transcripts is different for each gene. Ov: ovary; Te: testis; Br: brain; He; heart; St; stomach; Ad: adrenal gland; Li: liver; Sp: spleen; M.G.: mammary gland; Ki: kidney; Lu: lung; In: intestine; Th: thymus.

Figure 5

Transcript levels of the genes encoding the EJC core proteins in 13 murine tissues. Transcript levels (copy number per 50 pg of RNA extracted from each of the mentioned tissues) were measured by quantitative RT-PCR using the same samples as previously, and normalized to the level of Hprt1 and β-actin transcripts, as in Figure 4.

Figure 6

Transcript levels of the genes encoding two splicing factors involved in the NMD mechanism in 13 murine tissues. Transcript levels (copy number per 50 pg of RNA extracted from each of the mentioned tissues) were measured by quantitative RT-PCR using the same samples as previously, and normalized to the level of Hprt1 and β-actin transcripts, as in Figure 4.