Yoshihisa Tohru, Ohshima Chié, Yunoki-Esaki Kaori, Endo Toshiya
Research Center for Materials Science, Nagoya University, Japan.
Genes Cells. 2007 Mar;12(3):285-97. doi: 10.1111/j.1365-2443.2007.01056.x.
The splicing of nuclear encoded RNAs, including tRNAs, has been widely believed to occur in the nucleus. However, we recently found that one of the tRNA splicing enzymes, splicing endonuclease, is localized to the outer surface of mitochondria in Saccharomyces cerevisiae. These results suggested the unexpected possibility of tRNA splicing in the cytoplasm. To investigate this possibility, we examined whether cytoplasmic pre-tRNAs are bona fide intermediates for tRNA maturation in vivo. We isolated a new reversible allele of temperature-sensitive (ts) sen2 (HA-sen2-42), which encodes a mutant form of one of the catalytic subunits of yeast splicing endonuclease. The HA-sen2-42 cells accumulated large amounts of pre-tRNAs in the cytoplasm at a restrictive temperature, but the pre-tRNAs were diminished when the cells were transferred to a permissive temperature. Using pulse-chase/hybrid-precipitation techniques, we showed that the pre-tRNAs were not degraded but rather converted into mature tRNAs during incubation at the permissive temperature. These and other results indicate that, in S. cerevisiae, pre-tRNAs in the cytoplasm are genuine substrates for splicing, and that the splicing is indeed carried out in the cytoplasm.
包括转运RNA（tRNA）在内的核编码RNA的剪接，一直被广泛认为发生在细胞核中。然而，我们最近发现，其中一种tRNA剪接酶，即剪接内切核酸酶，定位于酿酒酵母线粒体的外表面。这些结果提示了tRNA在细胞质中进行剪接这种意想不到的可能性。为了研究这种可能性，我们检测了细胞质中的前体tRNA在体内是否是tRNA成熟的真正中间体。我们分离出了一个新的温度敏感型（ts）sen2的可逆等位基因（HA-sen2-42），它编码酵母剪接内切核酸酶的一个催化亚基的突变形式。HA-sen2-42细胞在限制温度下在细胞质中积累了大量前体tRNA，但当细胞转移到允许温度时，前体tRNA减少。使用脉冲追踪/杂交沉淀技术，我们表明前体tRNA在允许温度下孵育期间没有被降解，而是转化为成熟的tRNA。这些以及其他结果表明，在酿酒酵母中，细胞质中的前体tRNA是剪接的真正底物，并且剪接确实在细胞质中进行。
