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 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-beta pre-mRNAs (intron-bearing) are frequently derived from ptc-bearing genes but such transcripts rarely accumulate as mature (fully spliced) TCR-beta transcripts. Transfection studies in the SL12.4 T-cell line showed that the presence of a ptc in any of several TCR-beta exons triggered a decrease in mRNA levels, Ptc-bearing TCR-beta 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-beta and beta-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.