Comprehensive Scanning Mutagenesis of Human Retrotransposon LINE-1 Identifies Motifs Essential for Function
Comprehensive Scanning Mutagenesis of Human Retrotransposon LINE-1 Identifies Motifs Essential for Function
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DOI：
10.1534/genetics.119.302601
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发表时间：
2019-12-01
期刊：
影响因子：
3.3
通讯作者：
Boeke, Jef D.
中科院分区：
文献类型：
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作者：
Adney, Emily M.;Ochmann, Matthias T.;Boeke, Jef D.
长散布核元件-1（LINE-1，L1）是人类基因组中唯一的自主活性转座元件。L1编码的蛋白质ORF 1 p和ORF 2 p使元件能够通过“复制粘贴”机制从一个位点跳到另一个位点。ORF 1 p是一种RNA结合蛋白，ORF 2 p具有核酸内切酶和逆转录酶活性。人类基因组中大量截短的L1残基表明，宿主可能已经进化出阻止L1完全复制的机制，从而减少活性元件的增殖并降低L1的致突变潜力。反过来，L1似乎具有最小化的长度以增加成功全长复制的概率。预计这种精简将导致高信息密度。在这里，我们描述了538个连续的三丙氨酸取代，扫描沿着ORF 1 p和ORF 2 p，以确定功能上重要的区域的库的建设和初步表征。根据简化假说，反转录转座总体上对ORF 1 p和ORF 2 p中的突变非常敏感;只有16%的三丙氨酸突变体保留了近野生型（WT）活性。所有ORF 1 p突变体形成近WT水平的mRNA转录物，75%形成近WT水平的蛋白质。两个ORF 1 p突变体呈现独特的核仁重定位表型。还鉴定了对诱变敏感但缺乏系统发育保守性的ORF 2 p区域。我们提供关于对逆转录最关键的区域的全面信息。这一资源将指导未来的研究与RNA，蛋白质和目标DNA形成的分子间相互作用，在整个L1的生命周期。Adney等。描述了完整和全面的密码子置换诱变人类逆转录转座子LINE-1使用合成DNA的方法。这个实验是第一次对任何转座子进行这种实验。
Long Interspersed Nuclear Element-1 (LINE-1, L1) is the only autonomous active transposable element in the human genome. The L1-encoded proteins ORF1p and ORF2p enable the element to jump from one locus to another via a "copy-and-paste" mechanism. ORF1p is an RNA-binding protein, and ORF2p has endonuclease and reverse transcriptase activities. The huge number of truncated L1 remnants in the human genome suggests that the host has likely evolved mechanisms to prevent full L1 replication, and thereby decrease the proliferation of active elements and reduce the mutagenic potential of L1. In turn, L1 appears to have a minimized length to increase the probability of successful full-length replication. This streamlining would be expected to lead to high information density. Here, we describe the construction and initial characterization of a library of 538 consecutive trialanine substitutions that scan along ORF1p and ORF2p to identify functionally important regions. In accordance with the streamlining hypothesis, retrotransposition was overall very sensitive to mutations in ORF1p and ORF2p; only 16% of trialanine mutants retained near-wild-type (WT) activity. All ORF1p mutants formed near-WT levels of mRNA transcripts and 75% formed near-WT levels of protein. Two ORF1p mutants presented a unique nucleolar-relocalization phenotype. Regions of ORF2p that are sensitive to mutagenesis but lack phylogenetic conservation were also identified. We provide comprehensive information on the regions most critical to retrotransposition. This resource will guide future studies of intermolecular interactions that form with RNA, proteins, and target DNA throughout the L1 life cycle.Adney et al. describe the complete and comprehensive codon substitution mutagenesis of human retrotransposon LINE-1 using a synthetic DNA approach. This experiment is the first of its kind for any transposon...