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

Abstract

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-␤ pre-mRNAs (intron-bearing) are frequently derived from ptc-bearing genes but such transcripts rarely accumulate as mature (fully spliced) TCR-␤ transcripts. Transfection studies in the SL12.4 T-cell line showed that the presence of a ptc in any of several TCR-␤ exons triggered a decrease in mRNA levels. Ptc-bearing TCR-␤ 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 downregulatory 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-␤ and ␤-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. T-cell receptor (TCR)1 and immunoglobulin (Ig) genes undergo programmed rearrangement events during lymphocyte ontogeny. During this process, variable (V) elements are juxtaposed to joining (J) elements to create functional genes (1, 2).In some TCR and Ig genes, diversity (D) elements are also included in this rearrangement process. The tremendous combinatorial possibilities afforded by this rearrangement mechanism permit the generation of a wide variety of antigen receptors. Additional variability is provided by the enzyme terminal transferase which introduces random nucleotides at the junctions between V, D, and, J elements (1, 2). Variability is also engendered by the low fidelity of the rearrangement event itself; the borders of each element are not fixed, sometimes leading to small deletions at the junctions between the V, D, and J elements. The collective result of these insertional and deletional events is that a large fraction of rearrangement events will generate out-of-frame (nonproductive) genes that contain premature termination codons (ptcs).Since out-of-frame TCR and Ig genes are commonly generated during normal lymphocyte development, there may exist a mechanism that diminishes the expression of these nonfunctional ptc-containing genes. Consistent with...

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