Molina-Sánchez Maria D, García-Rodríguez Fernando M, Toro Nicolás
Structure, Dynamics and Function of Rhizobacterial Genomes, Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas Granada, Spain.
Front Mol Biosci. 2016 Sep 27;3:58. doi: 10.3389/fmolb.2016.00058. eCollection 2016.
The functional unit of mobile group II introns is a ribonucleoprotein particle (RNP) consisting of the intron-encoded protein (IEP) and the excised intron RNA. The IEP has reverse transcriptase activity but also promotes RNA splicing, and the RNA-protein complex triggers site-specific DNA insertion by reverse splicing, in a process called retrohoming. reconstituted ribonucleoprotein complexes from the group II intron Ll.LtrB, which produce a double strand break, have recently been studied as a means of developing group II intron-based gene targeting methods for higher organisms. The group II intron RmInt1 is an efficient mobile retroelement, the dispersal of which appears to be linked to transient single-stranded DNA during replication. The RmInt1IEP lacks the endonuclease domain (En) and cannot cut the bottom strand to generate the 3' end to initiate reverse transcription. We used an expression system to produce soluble and active RmInt1 IEP and reconstituted RNPs with purified components . The RNPs generated were functional and reverse-spliced into a single-stranded DNA target. This work constitutes the starting point for the use of group II introns lacking DNA endonuclease domain-derived RNPs for highly specific gene targeting methods.
移动II组内含子的功能单位是一种核糖核蛋白颗粒（RNP），它由内含子编码蛋白（IEP）和切除的内含子RNA组成。IEP具有逆转录酶活性，但也能促进RNA剪接，并且这种RNA-蛋白质复合物通过反向剪接触发位点特异性DNA插入，这一过程称为归巢。最近，对来自II组内含子Ll.LtrB的重组核糖核蛋白复合物进行了研究，该复合物会产生双链断裂，以此作为开发用于高等生物的基于II组内含子的基因靶向方法的一种手段。II组内含子RmInt1是一种高效的移动反转录元件，其扩散似乎与复制过程中的瞬时单链DNA有关。RmInt1 IEP缺乏核酸内切酶结构域（En），无法切割底部链以产生3'端来启动逆转录。我们使用一种表达系统来产生可溶性且有活性的RmInt1 IEP，并使用纯化的组分重构核糖核蛋白。产生的核糖核蛋白具有功能，并反向剪接到单链DNA靶标中。这项工作构成了将缺乏DNA核酸内切酶结构域的II组内含子来源的核糖核蛋白用于高度特异性基因靶向方法的起点。
