The human lysyl-tRNA synthetase gene encodes both the cytoplasmic and mitochondrial enzymes by means of an unusual alternative splicing of the primary transcript
The human lysyl-tRNA synthetase gene encodes both the cytoplasmic and mitochondrial enzymes by means of an unusual alternative splicing of the primary transcript
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DOI：
10.1074/jbc.m006265200
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发表时间：
2000-11-10
影响因子：
4.8
通讯作者：
Davidson, E
中科院分区：
文献类型：
--
作者：
Tolkunova, E;Park, H;Davidson, E
已经鉴定了两个编码人赖氨酰-tRNA合成酶的cDNA。一个编码先前鉴定的酶的细胞质形式。第二个cDNA包含相同的序列，但在mRNA的5 '端插入了180个碱基对。这导致预测的蛋白质，其羧基576个氨基酸与细胞质酶的那些相同，但具有不同的49个氨基酸的氨基末端，其含有推定的线粒体靶向序列。两种赖氨酰-tRNA合成酶-绿色荧光蛋白基因融合体在人细胞系中的表达证实了细胞质形式靶向细胞质，线粒体形式靶向线粒体。基因组赖氨酰-tRNA合成酶基因由15个外显子组成。这两种异构体是通过基因前三个外显子的选择性剪接产生的。通过将外显子1剪接到外显子3来产生细胞质形式。在外显子1和3之间包含外显子2产生编码线粒体同种型的mRNA，其具有额外的上游小开放阅读框，主要由细胞质同种型的5'编码区的一部分组成。这是在基因的第二外显子上编码的线粒体靶向序列的第一个实例。核糖核酸酶保护分析表明，编码细胞质同种型的mRNA约占赖氨酰-tRNA合成酶基因成熟转录物的70%，线粒体同种型约占30%。在大肠杆菌中表达后纯化的酶的线粒体形式，氨酰化的体外转录物对应于细胞质和线粒体tRNA（Lys），尽管这些tRNA的受体茎中的碱基序列存在差异。
Two cDNAs encoding human lysyl-tRNA synthetase have been identified. One encodes the cytoplasmic form of the enzyme identified previously. The second cDNA contains the same sequence but with a 180-bp insertion at the 5'-end of the mRNA. This results in a predicted protein whose carboxyl 576 amino acids are identical to those of the cytoplasmic enzyme but with a different amino terminus of 49 amino acids that contains a putative mitochondrial targeting sequence. Expression of the two lysyl-tRNA synthetase-green fluorescent protein gene fusions in a human cell line confirmed that the cytoplasmic form was targeted to the cytoplasm and the mitochondrial form to mitochondria, The genomic lysyl-tRNA synthetase gene consisted of 15 exons. The two isoforms were created by alternative splicing of the first three exons of the gene. The cytoplasmic form was created by splicing exon 1 to exon 3, The inclusion of exon 2 between exons 1 and 3 produced an mRNA encoding the mitochondrial isoform with an additional upstream small open reading frame, consisting mainly of a portion of the 5' coding region of the cytoplasmic isoform, This is the first example of mitochondrial targeting sequence being encoded on the second exon of a gene. Ribonuclease protection analysis showed that the mRNA encoding the cytoplasmic isoform makes up approximately 70%, and the mitochondrial isoform approximately 30%, of the mature transcripts from the lysyl-tRNA synthetase gene. The mitochondrial form of the enzyme, purified after expression in Escherichia coli, aminoacylated in vitro transcripts corresponding to both the cytoplasmic and mitochondrial tRNA(Lys), despite the difference in the discriminator base sequence in the acceptor stems of these tRNAs.