A REGULATORY MECHANISM THAT DETECTS PREMATURE NONSENSE CODONS IN T-CELL RECEPTOR TRANSCRIPTS IN-VIVO IS REVERSED BY PROTEIN-SYNTHESIS INHIBITORS IN-VITRO
A REGULATORY MECHANISM THAT DETECTS PREMATURE NONSENSE CODONS IN T-CELL RECEPTOR TRANSCRIPTS IN-VIVO IS REVERSED BY PROTEIN-SYNTHESIS INHIBITORS IN-VITRO
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DOI：
10.1074/jbc.270.48.28995
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发表时间：
1995-12-01
影响因子：
4.8
通讯作者：
WILKINSON, MF
中科院分区：
文献类型：
--
作者：
CARTER, MS;DOSKOW, J;WILKINSON, MF
在T细胞和B细胞的个体发育期间的基因重排产生大量的T细胞受体（TCR）和免疫球蛋白（IG）基因库。由于这种重排过程的易错性质，预期三分之二的重排的TCR和IG基因是框外的，因此含有提前终止密码子（ptc），我们对来自胎儿和成人胸腺的逆转录-聚合酶链反应产物进行了序列分析，发现新转录的TCR-β前体mRNA（携带内含子的）通常来源于携带ptc的基因，但此类转录物很少积累为成熟（完全剪接的）TCR-β转录物。在SL 12.4 T细胞系中的转染研究表明，在几个TCR-β外显子中的任何一个中存在ptc都触发mRNA水平的降低，在含有框内和框外基因的细胞克隆中，携带Ptc的TCR-β转录物的水平被选择性地抑制，从而证明了这种下调反应的等位基因特异性，具有不同作用机制的蛋白质合成抑制剂（茴香霉素、放线菌酮、恩替卡韦、帕他霉素、嘌呤霉素和脊髓灰质炎病毒）都逆转了下调反应，在放线菌酮处理后0.5小时内诱导了携带Pt c的转录物，蛋白质合成抑制剂的逆转不限于淋巴样细胞，如在HeLa细胞中转染的TCR-β和β-珠蛋白构建体所示。总的来说，数据表明PTC介导的mRNA衰变途径需要不稳定的蛋白质、核糖体或核糖体样实体，蛋白质合成抑制剂可能是阐明ptc介导的mRNA衰变的分子机制的有用工具，ptc介导的mRNA衰变是一种可能发生在哺乳动物细胞核部分中的神秘反应。
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.