Dujon B, Colleaux L, Jacquier A, Michel F, Monteilhet C
Basic Life Sci. 1986;40:5-27. doi: 10.1007/978-1-4684-5251-8_2.
Introns of organelle genes share distinctive RNA secondary structures that allow their classification into two known families. These structures are believed to play an essential role in splicing, and members of both structural classes have recently been shown to perform self-splicing reactions in vitro. In lower eukaryotes, many structured introns also contain long internal open reading frames (ORFs), which are able to code for hydrophilic proteins. Several properties of self-splicing structured introns suggest that they resemble mobile genetic elements, even though no actual transposition event involving these introns has yet been found. We report here on the characterization of two intron-encoded proteins that strongly support this attractive idea. First, we show that the class I intron of the 21S ribosomal RNA (rRNA) gene of Saccharomyces cerevisiae omega+ strains (rl intron) encodes a specific transposase. This protein has been partially purified from Escherichia coli cells that overexpress it from an artificial universal code equivalent to the rl intronic ORF. The omega transposase shows a double-strand endonuclease activity in vitro. This activity creates a 4-bp staggered cut with 3' OH overhangs within a specific sequence of the 21S rRNA gene of omega- strains. It is precisely within this sequence that the rl intron inserts by a duplicative transposition. Second, we report on the synthesis, in E. coli, of a putative reverse transcriptase encoded by the class II intron of the cytochrome b gene of Schizosaccharomyces pombe. This synthesis was obtained from E. coli expression vectors, using the class II intronic ORF linked to an artificial initiator sequence. As further support of the idea that structured introns are mobile, we show, from a systematic screening of introns in various yeast species, that the rl intron has transposed into the ATPase subunit 9 gene of Kluyveromyces fragilis. Structural features observed at the new intron homing site may be relevant to the transposition event.
细胞器基因的内含子具有独特的RNA二级结构，可据此将其分为两个已知家族。这些结构被认为在剪接过程中起关键作用，最近已证明这两个结构类别的成员在体外都能进行自我剪接反应。在低等真核生物中，许多有结构的内含子还包含长的内部开放阅读框（ORF），这些阅读框能够编码亲水性蛋白质。自我剪接的有结构内含子的几个特性表明它们类似于可移动遗传元件，尽管尚未发现涉及这些内含子的实际转座事件。我们在此报告了两种内含子编码蛋白的特性，这些特性有力地支持了这一引人关注的观点。首先，我们表明酿酒酵母ω + 菌株21S核糖体RNA（rRNA）基因的I类内含子（rl内含子）编码一种特定的转座酶。该蛋白已从大肠杆菌细胞中部分纯化，这些细胞通过与rl内含子ORF等效的人工通用密码子过量表达该蛋白。ω转座酶在体外显示出双链内切核酸酶活性。这种活性在ω - 菌株21S rRNA基因的特定序列内产生一个带有3' OH突出端的4碱基对交错切口。rl内含子正是通过重复转座插入到这个序列中。其次，我们报告了粟酒裂殖酵母细胞色素b基因II类内含子编码的一种假定逆转录酶在大肠杆菌中的合成。这种合成是通过使用与人工起始序列相连的II类内含子ORF，从大肠杆菌表达载体中获得的。作为对有结构内含子具有可移动性这一观点的进一步支持，我们通过对各种酵母物种内含子的系统筛选表明，rl内含子已转座到脆壁克鲁维酵母的ATP酶亚基9基因中。在新的内含子归巢位点观察到的结构特征可能与转座事件有关。
