A Regulatory Mechanism That Detects Premature Nonsense Codons in T-cell Receptor Transcripts in Vivo Is Reversed by Protein Synthesis Inhibitors in Vitro

Gene rearrangement during the ontogeny of T- and B-cells generates an enormous repertoire of T-cell receptor (TCR) and immunoglobulin (Ig) genes. Because of the error-prone nature of this rearrangement process, two-thirds of rearranged TCR and Ig genes are expected to be out-of-frame and thus contai...

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Bibliographic Details
Published in:The Journal of biological chemistry 1995-12, Vol.270 (48), p.28995-29003
Main Authors: Carter, Mark S., Doskow, Jessica, Morris, Phillip, Li, Shulin, Nhim, Ronald P., Sandstedt, Sara, Wilkinson, Miles F.
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Codon
Down-Regulation - drug effects
HeLa Cells
Humans
Mice
Molecular Sequence Data
Oligodeoxyribonucleotides - chemistry
Protein Synthesis Inhibitors - pharmacology
Receptors, Antigen, T-Cell - genetics
RNA Precursors - genetics
RNA, Messenger - chemistry
RNA, Messenger - genetics
Terminator Regions, Genetic
Thymus Gland - cytology
Thymus Gland - metabolism
Tumor Cells, Cultured
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Summary:Gene rearrangement during the ontogeny of T- and B-cells generates an enormous repertoire of T-cell receptor (TCR) and immunoglobulin (Ig) genes. Because of the error-prone nature of this rearrangement process, two-thirds of rearranged TCR and Ig genes are expected to be out-of-frame and thus contain premature terminations codons (ptcs). We performed sequence analysis of reverse transcriptase-polymerase chain reaction products from fetal and adult thymus and found that newly transcribed TCR-β pre-mRNAs (intron-bearing) are frequently derived from ptc-bearing genes but such transcripts rarely accumulate as mature (fully spliced) TCR-β transcripts. Transfection studies in the SL12.4 T-cell line showed that the presence of a ptc in any of several TCR-β exons triggered a decrease in mRNA levels. Ptc-bearing TCR-β transcripts were selectively depressed in levels in a cell clone that contained both an in-frame and an out-of-frame gene, thus demonstrating the allelic specificity of this down-regulatory response. Protein synthesis inhibitors with different mechanism of action (anisomysin, cycloheximide, emetine, pactamycin, puromycin, and polio virus) all reversed the down-regulatory response. Ptc-bearing transcripts were induced within 0.5 h after cycloheximide treatment. The reversal by protein synthesis inhibitors was not restricted to lymphoid cells, as shown with TCR-β and β-globin constructs transfected in HeLa cells. Collectively, the data suggest that the ptc-mediated mRNA decay pathway requires an unstable protein, a ribosome, or a ribosome-like entity. Protein synthesis inhibitors may be useful tools toward elucidating the molecular mechanism of ptc-mediated mRNA decay, an enigmatic response that can occur in the nuclear fraction of mammalian cells.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.270.48.28995