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Updated: Aug 14, 2026
Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling
Published on: October 28, 2014
Evidence that translation reinitiation abrogates nonsense-mediated mRNA decay in mammalian cells
1Department of Human Genetics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Abstract:
Nonsense codons upstream of and including position 192 of the human gene for triosephosphate isomerase (TPI) have been found to reduce the abundance of TPI mRNA to approximately 25% of normal. The reduction is due to the decay of newly synthesized TPI mRNA that co-purifies with nuclei. TPI mRNA that co-purifies with cytoplasm is immune to nonsense-mediated decay. Until now, a nonsense codon at position 23 has been the 5'-most nonsense codon that has been analyzed. Here, we provide evidence that a nonsense codon at position 1, 2 or 10 reduces the abundance of nucleus-associated TPI mRNA to an average of only 84% of normal because translation reinitiates at the methionine codon at position 14. First, converting codon 14 to one for valine increased the effectiveness with which an upstream nonsense codon reduces mRNA abundance. Second, when TPI gene sequences, including codon 14, were fused upstream of and in-frame to the translational reading frame of an Escherichia coli chloramphenicol acetyl transferase (CAT) gene that lacked an initiation codon, a nonsense codon at TPI position 1 or 2 allowed for the production of TPI-CAT that was an estimated 14 amino acids smaller than TPI-CAT produced by a nonsense-free gene, whereas a nonsense codon at TPI position 23 precluded the production of TPI-CAT. These and related findings lend credence to the concept that the nonsense-mediated reduction in the half-life of nucleus-associated TPI mRNA involves cytoplasmic ribosomes.
Insights
Early nonsense codons in the triosephosphate isomerase (TPI) gene reduce mRNA levels less than expected due to reinitiation. This suggests cytoplasmic ribosomes are involved in nonsense-mediated mRNA decay.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
- RNA Metabolism
Background:
- Nonsense codons normally trigger mRNA decay, reducing protein production.
- Previous studies showed nonsense codons at position 192 and 23 of the human triosephosphate isomerase (TPI) gene significantly decrease TPI mRNA abundance.
- This decay is linked to nucleus-associated mRNA, while cytoplasmic mRNA is unaffected.
Purpose of the Study:
- To investigate the impact of upstream nonsense codons (positions 1, 2, or 10) on TPI mRNA abundance.
- To determine the role of translation reinitiation at methionine codon 14 in mitigating nonsense-mediated decay.
- To explore the involvement of cytoplasmic ribosomes in the nonsense-mediated decay pathway.
Main Methods:
- Analysis of TPI mRNA abundance in cells with nonsense codons at positions 1, 2, or 10.
- Site-directed mutagenesis to convert codon 14 to valine, preventing reinitiation.
- Construction of fusion genes combining TPI sequences with an Escherichia coli chloramphenicol acetyl transferase (CAT) reporter gene.
Main Results:
- Nonsense codons at TPI positions 1, 2, or 10 reduced nucleus-associated TPI mRNA abundance by only 16% on average.
- Mutating codon 14 to valine enhanced the mRNA-destabilizing effect of upstream nonsense codons.
- Fusion gene experiments showed nonsense codons at TPI positions 1 or 2 allowed truncated TPI-CAT production, unlike nonsense codons at position 23.
Conclusions:
- Translation reinitiation at TPI codon 14 can bypass the effects of upstream nonsense codons, preserving mRNA levels.
- The findings support the model that cytoplasmic ribosomes play a crucial role in the nonsense-mediated decay of nucleus-associated mRNA.
- This study elucidates a novel mechanism of mRNA surveillance and regulation in gene expression.
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