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T cell receptor (TCR) mini-gene mRNA expression regulated by nonsense codons: a nuclear-associated translation-like
S Li1, D Leonard, M F Wilkinson
1Department of Immunology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Abstract:
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.
Insights
Premature termination codons (PTCs) trigger mRNA reduction via a nuclear surveillance mechanism. This process requires a translation start codon (AUG) and involves translation-like activity to degrade aberrant transcripts.
Area of Science:
- Molecular Biology
- Genetics
- Immunology
Background:
- Premature termination codons (PTCs) are known to reduce messenger RNA (mRNA) levels.
- The TCR-beta gene undergoes programmed rearrangements during thymic development, sometimes resulting in PTCs.
Purpose of the Study:
- To investigate the mechanism by which PTCs downregulate mRNA levels.
- To determine the role of the translation start codon and translation-associated processes in PTC-mediated mRNA decay.
Main Methods:
- Utilized a mini-gene version of the TCR-beta gene containing three exons.
- Introduced mutations in the translation start AUG and a downstream AUG.
- Employed specific suppressor transfer RNAs (tRNAs).
- Fractionated nuclear components to analyze transcript levels.
Main Results:
- A mini-gene TCR-beta construct showed efficient PTC-induced downregulation, indicating the full coding sequence is not required.
- Mutating the start AUG or a downstream AUG abolished the downregulatory response.
- Suppressor tRNAs reversed the downregulation, suggesting a translation-dependent mechanism.
- Inactivation of start AUGs or addition of suppressor tRNAs led to increased nuclear levels of PTC-bearing transcripts.
Conclusions:
- Evidence supports a codon-based nuclear surveillance mechanism that downregulates aberrant transcripts.
- This mechanism targets transcripts with PTCs, likely preventing the production of potentially toxic polypeptides from nonproductively rearranged genes.
- The translation start codon is crucial for defining the reading frame and initiating this surveillance pathway.