T cell receptor (TCR) mini-gene mRNA expression regulated by nonsense codons: a nuclear-associated translation-like mechanism.
Premature termination codons (PTCs) are known to decrease mRNA levels. Here, we report our investigation of the mechanism for this downregulation using the TCR-beta gene, which acquires PTCs as a result of programmed rearrangements that occur during normal thymic development. We found that a mini-gene version of this gene, which contains only three TCR-beta exons, exhibited efficient downregulation in response to PTCs. This demonstrates that the full coding sequence is not necessary for appropriate regulation. Mutation of the translation start AUG and a downstream in-frame AUG that displayed similarity to the Kozak consensus sequence reversed the downregulatory response to PTCs. Thus, an AUG start codon is required to define the reading frame of a PTC. Specific suppressor tRNAs also reversed the downregulatory response, strongly implicating the involvement of a translation-like process. Remarkably, the addition of suppressor tRNAs or the inactivation of the start AUGs caused a dramatic rise in the levels of PTC-bearing transcripts in the nuclear fraction prepared by two independent methods. Collectively, our results provide evidence for a codon-based surveillance mechanism associated with the nucleus that downregulates aberrant transcripts encoding potentially toxic polypeptides from nonproductively rearranged genes.
Li
S
S
Department of Immunology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Leonard
D
D
Wilkinson
M F
MF
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D003062:Codon; D015536:Down-Regulation; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D008322:Mammals; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D014162:Transfection
D003062:Codon; D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins