Tomita K, Ueda T, Watanabe K
Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Bunkyo-ku, Japan.
Nucleic Acids Res. 1996 Dec 15;24(24):4987-91. doi: 10.1093/nar/24.24.4987.
In squid (Loligo bleekeri) mitochondria, the two 3'-terminal nucleotides (G72-G73) of the tRNA(Tyr) gene overlap with the two 5'-terminal nucleotides (G1-G2) of the downstream tRNA(Cys) gene. To elucidate the processing mechanism(s) of the tRNA molecules derived from this region, tRNAs were analyzed by sequencing cDNAs synthesized from circularized tRNAs. Nucleotides G1-G2 in tRNA(Cys) appeared to be without post-transcriptional conversion, whereas CCA was post-transcriptionally added to the 3'-terminus. In contrast, in the majority of tRNAs(Tyr), G72-G73 were found to be converted to A72-A73, accompanied by the CCA addition. These results indicate that a precursor of tRNA(Tyr) is processed at U71 and two adenosines are attached prior to the CCA addition. Thus, we suggest that 5' processing of the precursor tRNA dominates 3' processing and maturation of the tRNA is mediated by a polyadenylylation enzyme in the mitochondria, a scenario which is consistent with the editing process proposed in land snail mitochondria. We also obtained intermediates, such as a premature tRNA lacking CCA that terminated at U71 and one with a single adenosine attached at position 72, which support the suggested maturation process. However, although we failed to detect a tRNA(Cys) lacking G1-G2 at the 5'-terminus, we obtained cDNAs for tRNA(Tyr) with G72-G73 and the CCA terminus. This inconsistent result suggests the co-existence of another process(es) in the maturation of these tRNA molecules in squid mitochondria.
在鱿鱼（剑尖枪乌贼）线粒体中，tRNA（Tyr）基因的两个3'末端核苷酸（G72 - G73）与下游tRNA（Cys）基因的两个5'末端核苷酸（G1 - G2）重叠。为了阐明源自该区域的tRNA分子的加工机制，通过对由环化tRNA合成的cDNA进行测序来分析tRNA。tRNA（Cys）中的核苷酸G1 - G2似乎没有转录后转化，而CCA是转录后添加到3'末端的。相反，在大多数tRNA（Tyr）中，发现G72 - G73被转化为A72 - A73，并伴随着CCA的添加。这些结果表明，tRNA（Tyr）的前体在U71处进行加工，并且在添加CCA之前连接了两个腺苷。因此，我们认为前体tRNA的5'加工主导3'加工，并且tRNA的成熟由线粒体中的聚腺苷酸化酶介导，这一情况与陆地蜗牛线粒体中提出的编辑过程一致。我们还获得了中间体，例如在U71处终止且缺乏CCA的早熟tRNA以及在位置72处连接有单个腺苷的tRNA，这些都支持了所提出的成熟过程。然而，尽管我们未能检测到5'末端缺乏G1 - G2的tRNA（Cys），但我们获得了具有G72 - G73和CCA末端的tRNA（Tyr）的cDNA。这一不一致的结果表明，在鱿鱼线粒体中这些tRNA分子的成熟过程中存在另一种过程。
