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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation
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Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation

机译：线性II组内含子RNA可以在真核生物中逆行，并且可以使用非同源末端连接进行cDNA连接

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摘要

Mobile group II introns retrohome by an RNP-based mechanism in which the excised intron lariat RNA fully reverse splices into a DNA site via 2 sequential transesterification reactions and is reverse transcribed by the associated intron-encoded protein. However, linear group II intron RNAs, which can arise by either hydrolytic splicing or debranching of lariat RNA, cannot carry out both reverse-splicing steps and were thus expected to be immobile. Here, we used facile microinjection assays in 2 eukaryotic systems, Xenopus laevis oocyte nuclei and Drosophila melanogaster embryos, to show that group II intron RNPs containing linear intron RNA can retrohome by carrying out the first step of reverse splicing into a DNA site, thereby ligating the 3' end of the intron RNA to the 5' end of the downstream exon DNA. The attached linear intron RNA is then reverse transcribed, yielding an intron cDNA whose free end is linked to the upstream exon DNA. Some of these retrohoming events result in the precise insertion of full-length intron. Most, however, yield aberrant 5' junctions with 5' exon resections, 5' intron truncations, and/or extra nucleotide residues, hallmarks of nonhomologous end-joining. Our findings reveal a mobility mechanism for linear group II intron RNAs, show how group II introns can co-opt different DNA repair pathways for retrohoming, and suggest that linear group II intron RNAs might be used for site-specific DNA integration in gene targeting.
机译：可移动的II组内含子通过基于RNP的机制逆行，其中被切除的内含子套索状RNA通过2个连续的酯交换反应完全反向剪接成DNA位点，并被相关的内含子编码蛋白反向转录。但是，通过套索状RNA的水解剪接或去分支而产生的线性II族内含子RNA不能同时进行两个反向剪接步骤，因此不能固定。在这里，我们在两个真核生物系统（非洲爪蟾卵母细胞核和果蝇果蝇）胚胎中使用了简便的显微注射测定法，以显示包含线性内含子RNA的II组内含子RNPs可以通过进行反向剪接至DNA位点的第一步从而连接而进行归巢。内含子RNA的3'末端到下游外显子DNA的5'末端。然后将附着的线性内含子RNA反转录，得到内含子cDNA，其自由端与上游外显子DNA连接。这些回溯事件中的一些会导致全长内含子的精确插入。然而，大多数产生具有5'外显子切除，5'内含子截短和/或额外的核苷酸残基的异常5'连接，这是非同源末端连接的标志。我们的发现揭示了线性II组内含子RNA的迁移机制，表明II组内含子如何可以选择不同的DNA修复途径进行归巢，并暗示线性II组内含子RNA可用于基因靶向中的位点特异性DNA整合。

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    Fanglei Zhuang; Marta Mastroianni; Travis B. White; Alan M. Lambowitz;

  • 作者单位

    Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology,School of Biological Sciences, University of Texas, Austin, TX 78712;

    Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology,School of Biological Sciences, University of Texas, Austin, TX 78712;

    Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology,School of Biological Sciences, University of Texas, Austin, TX 78712;

    Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology,School of Biological Sciences, University of Texas, Austin, TX 78712;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    DNA repair; gene targeting; retrotransposition; reverse transcriptase; ribozyme;

    机译：DNA修复;基因靶向逆转座逆转录酶核酶;

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