Evidence that translation reinitiation abrogates nonsense-mediated mRNA decay in mammalian cells

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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 ∼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.