Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712-0159, USA.
RNA. 2010 Apr;16(4):732-47. doi: 10.1261/rna.2007310. Epub 2010 Feb 23.
Mobile group II introns encode a reverse transcriptase that binds the intron RNA to promote RNA splicing and intron mobility, the latter via reverse splicing of the excised intron into DNA sites, followed by reverse transcription. Previous work showed that the Lactococcus lactis Ll.LtrB intron reverse transcriptase, denoted LtrA protein, binds with high affinity to DIVa, a stem-loop structure at the beginning of the LtrA open reading frame and makes additional contacts with intron core regions that stabilize the active RNA structure for forward and reverse splicing. LtrA's binding to DIVa down-regulates its translation and is critical for initiation of reverse transcription. Here, by using high-throughput unigenic evolution analysis with a genetic assay in which LtrA binding to DIVa down-regulates translation of GFP, we identified regions at LtrA's N terminus that are required for DIVa binding. Then, by similar analysis with a reciprocal genetic assay, we confirmed that residual splicing of a mutant intron lacking DIVa does not require these N-terminal regions, but does require other reverse transcriptase (RT) and X/thumb domain regions that bind the intron core. We also show that N-terminal fragments of LtrA by themselves bind specifically to DIVa in vivo and in vitro. Our results suggest a model in which the N terminus of nascent LtrA binds DIVa of the intron RNA that encoded it and nucleates further interactions with core regions that promote RNP assembly for RNA splicing and intron mobility. Features of this model may be relevant to evolutionarily related non-long-terminal-repeat (non-LTR)-retrotransposon RTs.
移动群 II 内含子编码一种逆转录酶，该酶结合内含子 RNA 以促进 RNA 剪接和内含子移动，后者通过切除内含子的反向剪接进入 DNA 位点，然后进行逆转录。先前的工作表明，乳球菌 Ll.LtrB 内含子逆转录酶，称为 LtrA 蛋白，与 DIVa 具有高亲和力，DIVa 是 LtrA 开放阅读框起始处的茎环结构，并与稳定正向和反向剪接的内含子核心区域进行额外接触。LtrA 与 DIVa 的结合下调其翻译，这对于逆转录的起始至关重要。在这里，我们通过使用高通量单基因进化分析和一种遗传测定，其中 LtrA 与 DIVa 的结合下调 GFP 的翻译，鉴定了 LtrA 中需要结合 DIVa 的 N 端区域。然后，通过类似的遗传测定的分析，我们证实缺乏 DIVa 的突变内含子的残余剪接不需要这些 N 端区域，但确实需要其他结合内含子核心的逆转录酶 (RT) 和 X/拇指结构域。我们还表明，LtrA 的 N 端片段本身在体内和体外都可以特异性地结合 DIVa。我们的结果表明，新生 LtrA 的 N 端与编码它的内含子 RNA 的 DIVa 结合，并引发与核心区域的进一步相互作用，促进 RNP 组装以进行 RNA 剪接和内含子移动。该模型的特征可能与进化上相关的非长末端重复（非-LTR）-逆转录转座子 RT 相关。
