An elongation factor-associating domain is inserted into human cysteinyl-tRNA synthetase by alternative splicing

The amino acid sequence of human cytoplasmic cysteinyl-tRNA synthetase (CRS) was examined by analyzing sequences of genomic and expressed sequence tag fragments. From theses analyses, a few interesting possibilities were suggested for the structure of human CRS. First, different isoforms of CRS may result from alternative splicing. Second, the largest one would comprise 831 amino acids. Third, a new exon was identified encoding an 83 amino acid domain that is homologous to parts of elongation factor-1 subunits as well as other proteins involved in protein synthesis. Northern blot analysis showed three different mRNAs for CRS (of ∼3.0, 2.7 and 2.0 kb) from human testis while only the 2.7 kb mRNA was commonly detected in other tissues. Expression of the exon 2-containing transcript in testis was confirmed by RT–PCR and northern blotting. CRS containing the exon 2-encoded peptide retained catalytic activity comparable to that lacking this peptide. This peptide was responsible for the specific interaction of CRS with elongation factor-1γ.

通过分析基因组序列与表达序列标签（expressed sequence tag, EST）片段，对人类细胞质半胱氨酰-tRNA合成酶（cytoplasmic cysteinyl-tRNA synthetase, CRS）的氨基酸序列进行了系统研究。经上述分析，针对人类CRS的结构提出了三项值得关注的推测：其一，CRS的不同同工型可通过可变剪接（alternative splicing）产生；其二，其中最大的同工型包含831个氨基酸残基；其三，本研究鉴定出一段编码83个氨基酸结构域的新外显子，该结构域与延伸因子-1（elongation factor-1）亚基的部分序列以及其他参与蛋白质合成的蛋白具有同源性。Northern印迹（Northern blot）分析结果显示，人类睾丸组织中存在3种不同的CRS信使RNA（mRNA），其长度分别约为3.0、2.7和2.0 kb；而在其他组织中通常仅能检测到2.7 kb的mRNA。通过逆转录聚合酶链反应（RT-PCR）与Northern印迹实验，证实了包含外显子2的转录本在睾丸组织中的表达。携带外显子2编码肽段的CRS，其催化活性与不含该肽段的CRS相当；该肽段正是CRS与延伸因子-1γ（elongation factor-1γ）发生特异性相互作用的关键结构。