Selective Translational Repression Of Truncated Proteins ... - PLOS

Identification of NMD-Escape PTC-Containing mRNAs

We analyzed mRNA expression of the 12 genes by reverse transcriptase PCR (RT-PCR) in seven MMR-deficient (LS174T, HCT-8, SNU C2A, SNU C4, DLD-1, HCT116, and LOVO) and three MMR-proficient (NCI-H508, SW480, and HT29) colorectal cancer cell lines. Primers were designed to contain at least one exon–exon junction region and to amplify the coding repeat sequences (Table S2). Of the 20 frameshift mutations in the genomic DNA, mutation-derived transcripts were detected from ten alleles representing six genes (hMSH3, TAF1B, TGFBR2, ACVR2, MARCKS, and TCF-4), whereas ten alleles representing six genes (ABCF1, hMSH6, hRad50, PRKWNK1, RFC3, and SEC63) did not generate frameshift mutation-derived transcripts. No differences in expression of frameshift mutation-derived mRNA were observed between cell lines. Of the ten transcripts with frameshift mutations, five transcripts (representing three genes: hMSH3, TAF1B, and TGFBR2) had PTCs more than 50–55 nt upstream of the last exon–exon junction and were therefore expected to be degraded by NMD but instead escaped from NMD (NMD-escape). On the other hand, the five remaining transcripts (representing three genes: ACVR2, MARCKS, and TCF-4) had PTCs within 50–55 nt upstream of the last exon–exon junction and were therefore expected to be irrelevant to NMD (NMD-irrelevant). Accordingly, the 20 transcripts from 12 genes were classified as NMD-sensitive, NMD-escape, and NMD-irrelevant (Table 1).

In order to confirm the effect of NMD on the NMD-sensitive and NMD-escape PTC-containing mRNAs, we used RT-PCR and a ribonuclease protection assay (RPA) to analyze the expression of the target gene mRNAs after treatment with puromycin, a translation inhibitor. In the five NMD-escape alleles that generated detectable frameshift mutation-derived mRNAs, no expression differences were found after puromycin treatment. In the ten NMD-sensitive alleles that produced no detectable frameshift mutation-derived mRNAs, mutant transcripts were detected after puromycin treatment (Figure S2). We analyzed the amount of two degraded NMD-sensitive transcripts, hRad50 and hMSH6, by RPA and found a total loss of mutant transcripts, as evidenced by a 2-fold increase in hRad50 and hMSH6 products after puromycin treatment. In contrast, there was no loss of TGFBR2 mutant mRNA, an NMD-escape transcript, because the amount of product was unchanged after puromycin treatment (Figure 1).

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Figure 1. Measurement of Degraded Premature Termination Codon-Containing mRNAs by RPA Analysis

No loss of TGFBR2 mutant transcripts was noted after puromycin treatment. In contrast, the amount of hRad50 and hMSH6 mRNA doubled after puromycin treatment. GAPDH was used for standardization. The asterisk indicates mutation status of each gene: (−1) denotes a 1-bp deletion in the cMNR, (w) denotes no mutation in the cMNR, (+1) denotes a 1-bp insertion in the cMNR, and (−2) denotes a 2-bp deletion in the cMNR.

https://doi.org/10.1371/journal.pbio.0050109.g001

Next, we evaluated the effect of down-regulating UPF1 or UPF2, which are key NMD factors, on the stability of the frameshift mutation-derived mRNAs, hRad50 and hMSH6, using specific small interfering RNA (siRNA). Upon the treatment of luciferase siRNA, expression of the mutation-derived hRad50 and hMSH6 mRNAs were not detected in the cell lines with hRad50 and hMSH6 mutations. In contrast, down-regulating UPF1 or UPF2 abundantly increased the frameshift mutation-derived mRNAs, as confirmed by RT-PCR, and sequence analysis. These findings indicate that frameshift mutation-derived mRNAs of hRad50 and hMSH6 are recognized and degraded by the NMD system (Figure S3).