GROUP II INTRON RNA SPLICING AND MOBILITY

拟议的研究包括继续对II组内含子的蛋白质依赖剪接和迁移反应进行生化遗传学研究。II组内含子的迁移是由内含子编码的逆转录酶介导的，它也作为促进内含子剪接的成熟酶起作用。在目前的资助期间，我们阐明了蛋白质依赖剪接和迁移反应的一般性质，并开发了进一步研究这些反应的工具。首先，我们发现II组内含子的迁移（归巢）是通过靶DNA引物的逆转录机制发生的，涉及一种新的内含子编码的DNA内切酶，该内切酶是一种包含内含子编码蛋白和切除的内含子RNA的RNP复合物。在一种机制中，RNP复合体中的内含子RNA直接反向剪接到DNA靶位点，然后由内含子编码的蛋白质进行逆转录。其次，我们发现确定内含子插入位置的II组内含子内切酶使用其RNA和蛋白质亚基来识别其DNA靶位中的特定序列。在内含子RNA的情况下，这涉及DNA靶位点的13-nt区域与内含子RNA序列之间的碱基配对，因此，可以通过修改内含子RNA来改变内切酶的特异性。这些发现提出了一种可能性，即II组内含子可能被用作靶向基因破坏和在DNA基因组中所需位点插入遗传信息的工具。最后，我们建立了一个高效的乳酸乳球菌II组内含子L1.LtrB的大肠杆菌表达系统。最近的工作扩展包括研究大肠杆菌中乳球菌内含子的后归巢和剪接的遗传系统的发展，以及使用纯化组分研究剪接和迁移反应的方法。具体目的有：(1)继续对依赖成熟酶的II族内含子剪接反应进行生化分析。(2)确定参与II组内含子剪接的其他蛋白因子。(3)分析内含子编码DNA核酸内切酶活性的反应机理和DNA靶位相互作用。(4)支持合作确定II族内含子编码蛋白及其与内含子RNA和RT底物复合物的x射线晶体结构。(5)继续合作研究酵母线粒体和细菌中II族内含子的迁移机制。(6)探索利用II组内含子作为载体在动物细胞中进行靶向基因破坏和位点特异性DNA插入的可行性。

The proposed research involves continued biochemical-genetics studies of the protein-dependent splicing and mobility reactions of group II introns. Group II intron mobility is mediated by intron-encoded reverse transcriptases that also function as maturases to promote splicing of the intron. During the current grant period, we elucidated the general nature of the protein- dependent splicing and mobility reactions, and developed tools for studying the reactions further. First, we showed that group II intron mobility ( homing ) occurs via target-DNA primed reverse transcription mechanisms involving a novel intron-encoded DNA endonuclease, which is an RNP complex containing the intron- encoded protein and the excised intron RNA. In one mechanism, the intron RNA in the RNP complex reverse splices directly into the DNA target site and is then reverse transcribed by the intron-encoded protein. Second, we showed that group II intron endonucleases, which determine the site of intron insertion, use both their RNA and protein subunits to recognize specific sequences in their DNA target sites. In the case of the intron RNA, this involves base pairing between a 13-nt region of the DNA target site and sequences in the intron RNA and consequently, it is possible to change the specificity of the endonuclease simply by modifying the intron RNA. These findings raise the possibility that group II introns might be used as tools for targeted gene disruption and insertion of genetic information at desired sites in DNA genomes. Finally, we developed an efficient E. coli expression system for the Lactococcus lactis group II intron L1.LtrB. Recent extensions of the work include the development of genetic systems for studying both the retrohoming and splicing of the Lactococcal intron in E. coli, and methods for studying the splicing and mobility reactions using purified components. Specific aims are: (1) To continue biochemical analysis of the maturase-dependent group II intron splicing reaction. (2) To identify additional protein factors involved in group II intron splicing. (3) To analyze the reaction mechanism and DNA target-site interactions of the intron-encoded DNA endonuclease activity. (4) To support collaborations to determine X-ray crystal structures of the group II intron-encoded protein and its complexes with intron RNA and RT substrates. (5) To continue collaborations on group II intron mobility mechanisms in yeast mitochondria and bacteria. (6) To explore the feasibility of using group II introns as vectors for targeted gene disruption and site-specific DNA insertion in animal cells.