O'Sullivan D M, Noonan D, Quaranta V
J Exp Med. 1987 Aug 1;166(2):444-60. doi: 10.1084/jem.166.2.444.
We determined the structural basis for the presence of electrophoretically-distinct, antigenically-related forms of invariant chains in Ia oligomers, and established the mechanisms by which they can be expressed from a single gene. S1 nuclease protection assays indicated that, in B cells, transcription of this gene initiates at a minimum of three sites. Thus, unlike previously thought, invariant chain mRNAs have heterogeneous 5' untranslated segments that may differentially affect initiation of translation. Further, restriction mapping and nucleotide sequencing of cDNAs revealed two kinds of invariant chain mRNAs differing by an internal coding segment of 192 bp. This segment represents an alternatively spliced exon, as demonstrated by nucleotide sequencing of corresponding genomic regions. The exon (exon X) encodes a cysteine-rich stretch of 64 amino acids near the COOH terminus that displays a striking and surprising homology to an internal amino acid repeat of thyroglobulin, suggesting an evolutionary mechanism of exon shuffling. Transient expression of cDNAs indicated that both types of alternatively spliced mRNAs contain two in-frame AUGs functioning as alternate start sites for translation. Thus, transfections with exon X-lacking cDNAs resulted in the expression of Mr 33,000 and 31,000 proteins, detected by immunoprecipitation with anti-invariant chain antisera, and identical by two-dimensional gel (2-D) analyses to the B cell invariant-chain forms gamma 1 (Mr 31,000), gamma 2, and gamma 3 (Mr 33,000). Similarly, exon X-containing cDNAs expressed Mr 43,000 and 41,000 proteins, also identical by 2-D migration to Ia-associated proteins. Thus, human Ia molecules contain four forms of invariant chain of closely related but nonidentical primary structure that are generated from a single gene by a complex pattern of alternate transcriptional start, exon splicing, and translational start.
我们确定了Ia寡聚体中电泳性质不同但抗原相关的恒定链形式存在的结构基础，并建立了它们可从单个基因表达的机制。S1核酸酶保护试验表明，在B细胞中，该基因的转录至少起始于三个位点。因此，与之前的想法不同，恒定链mRNA具有异质性的5'非翻译区，这可能会对翻译起始产生不同影响。此外，cDNA的限制性图谱分析和核苷酸测序揭示了两种恒定链mRNA，它们相差一个192 bp的内部编码片段。该片段代表一个可变剪接外显子，相应基因组区域的核苷酸测序证明了这一点。该外显子（外显子X）在COOH末端附近编码一段富含64个氨基酸的富含半胱氨酸序列，与甲状腺球蛋白的内部氨基酸重复序列具有显著且惊人的同源性，提示了外显子重排的进化机制。cDNA的瞬时表达表明，两种可变剪接的mRNA均含有两个读框内的AUG，作为翻译的交替起始位点。因此，用缺乏外显子X的cDNA转染导致表达出33,000和31,000 Mr的蛋白质，用抗恒定链抗血清进行免疫沉淀检测到，经二维凝胶（2-D）分析与B细胞恒定链形式γ1（31,000 Mr）、γ2和γ3（33,000 Mr）相同。同样，含有外显子X的cDNA表达出43,000和41,000 Mr的蛋白质，经2-D迁移分析也与Ia相关蛋白相同。因此，人Ia分子含有四种一级结构密切相关但不完全相同的恒定链形式，它们通过交替转录起始、外显子剪接和翻译起始的复杂模式从单个基因产生。
