Madrenas J, Vincent D H, Kriangkum J, Elliott J F, Halloran P F
Department of Immunology, University of Alberta, Edmonton, Canada.
Mol Immunol. 1994 Sep;31(13):993-1004. doi: 10.1016/0161-5890(94)90094-9.
We recently reported the expression of a truncated T cell receptor (TCR) alpha mRNA in kidney and brain of normal mice. In the kidney, the truncated TCR alpha transcript was expressed by bone marrow-dependent, non-T large interstitial cells located predominantly in the medulla. Here, we report the molecular characterization of the truncated TCR alpha transcript from kidney. Using a modified anchored-PCR (A-PCR) technique and directional cloning, 37 cDNA clones extending 5' of the C alpha region were generated. cDNA sequencing showed that 29 of the clones (78%) originated in the J alpha 11-2 region. Of these clones, 17 started upstream or in the J alpha 11-2 exon and contained the entire J alpha 11-2 sequence correctly spliced to the first C alpha exon. Analysis of the sequence revealed the presence of multiple stop codons in all three reading frames. The other 12 clones originated further upstream of the J alpha 11-2 exon and did not include the J alpha 11-2 exon, but rather arose from the joining of a cryptic splice donor signal to the usual TCR alpha C splice acceptor. This alternatively spliced transcript contained an open reading frame extending from the upstream J alpha 11-2 region to 82 nucleotides downstream of the beginning of the TCR C alpha region, and potentially encoded a 36 amino acid polypeptide. The remaining eight clones all contained the J alpha TA61 region correctly spliced to C alpha with two of these extending upstream of the J alpha TA61 exon. The predominance of J alpha 11-2-C alpha containing clones was confirmed by RNase protection assay using total RNA from kidney and spleen of scid mice. The 3' region of the transcript contained a fully conserved, correctly spliced TCR alpha C region which was polyadenylated at the 3' end. The truncated TCR alpha mRNA could be detected in preparations of cytoplasmic RNA, indicating that this transcript follows a normal RNA processing pathway. Our results demonstrate that the truncated TCR alpha mRNA expressed in normal mouse kidney is a germline J-C transcript resulting from transcription initiated predominantly upstream of the J alpha 11-2 region. This germline transcript in the kidney is undergoing alternative splicing leading to the appearance of an open reading frame coding for a short polypeptide. These results suggest that the product of this transcript may be functionally relevant.
我们最近报道了正常小鼠肾脏和大脑中截短的T细胞受体（TCR）α mRNA的表达情况。在肾脏中，截短的TCR α转录本由主要位于髓质的骨髓依赖性非T大间质细胞表达。在此，我们报告来自肾脏的截短TCR α转录本的分子特征。使用改良的锚定PCR（A-PCR）技术和定向克隆，生成了37个延伸至C α区域5'端的cDNA克隆。cDNA测序显示，其中29个克隆（78%）起源于J α 11 - 2区域。在这些克隆中，17个起始于J α 11 - 2外显子上游或位于该外显子内，并包含正确剪接至第一个C α外显子的完整J α 11 - 2序列。序列分析显示在所有三个阅读框中均存在多个终止密码子。另外12个克隆起源于J α 11 - 2外显子更上游，不包含J α 11 - 2外显子，而是由一个隐蔽的剪接供体信号与通常的TCR α C剪接受体连接产生。这种选择性剪接的转录本包含一个开放阅读框，从上游J α 11 - 2区域延伸至TCR C α区域起始点下游82个核苷酸处，并可能编码一个36个氨基酸的多肽。其余8个克隆均包含正确剪接至C α的J α TA61区域，其中两个延伸至J α TA61外显子上游。使用重度联合免疫缺陷（scid）小鼠肾脏和脾脏的总RNA进行的核糖核酸酶保护试验证实了含J α 11 - 2 - C α克隆的优势。转录本的3'区域包含一个完全保守、正确剪接的TCR α C区域，其3'端进行了多聚腺苷酸化。在细胞质RNA制剂中可检测到截短的TCR α mRNA，表明该转录本遵循正常的RNA加工途径。我们的结果表明，正常小鼠肾脏中表达的截短TCR α mRNA是一种种系J - C转录本，主要由起始于J α 11 - 2区域上游的转录产生。肾脏中的这种种系转录本正在进行选择性剪接，导致出现一个编码短多肽的开放阅读框。这些结果表明该转录本的产物可能具有功能相关性。
