T-CELL RECEPTOR-BETA MESSENGER-RNA SPLICING DURING THYMIC MATURATION IN-VIVO AND IN AN INDUCIBLE T-CELL CLONE IN-VITRO

The expression of TCR-beta mRNAs competent to encode functional V(D)JCbeta proteins requires the activation of programmed DNA rearrangement events. It is not known whether other regulatory mechanisms control the steady-state levels of mature TCR-beta transcripts during thymic ontogeny. In this report, we demonstrate that TCR-beta pre-mRNAs accumulate in T cells, thus implicating RNA splicing as another potential level of regulation. Three methods were used to characterize the intron content of these pre-mRNA: Northern blot analysis, ribonuclease H mapping, and reverse transcription polymerase chain reaction analysis. Using these methods, we demonstrate that intron-containing TCR-beta transcripts derived from both the JC(beta1) and JC(beta2) loci accumulate in murine fetal and adult thymus. (VD)JC(beta1) pre-mRNAs that accumulate in the thymus possess unusually long poly(A) tails (greater-than-or-equal-to 300 nucleotides) and contain different combinations of four introns: the large intron between the J(beta1) and C(beta1) elements and the three introns within the C(beta1) element. The presence of an unusual transcript possessing IVS2(Cbeta1) at the 5' terminus suggests that cleavage of its splice acceptor is inefficient or negatively regulated. The profile of incompletely spliced TCR-beta transcripts present in the thymus ig vivo is identical in intron content to those that we previously showed accumulate in the nucleus of the immature SL12.4 T lymphoma cell clone. An unstable negative regulatory protein may control TCR-beta expression in this cell clone because fully spliced TCR-beta transcripts are dramatically induced in the cytoplasm after treatment with any of five different protein synthesis inhibitors (cycloheximide, anisomyosin, emetine, puromycin, and pactamycin), all of which act by distinct mechanisms to inhibit protein synthesis.