The key role of terminators on the expression and post-transcriptional gene silencing of transgenes
The key role of terminators on the expression and post-transcriptional gene silencing of transgenes
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DOI：
10.1111/tpj.14907
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发表时间：
2020-07-26
期刊：
影响因子：
7.2
通讯作者：
Waterhouse, Peter M.
中科院分区：
文献类型：
--
作者：
F. de Felippes, Felipe;McHale, Marcus;Waterhouse, Peter M.
转基因已成为现代生物学的重要组成部分，是功能基因组研究和生物技术产品开发的重要工具。转基因系的主要挑战之一是转基因基因的表达，与内源基因相比，转基因基因特别容易受到小rna （sRNAs）介导的沉默的影响。人们提出了几个原因来解释为什么转基因经常触发sRNAs的产生，例如由常用的强组成启动子诱导的高水平表达，缺乏内含子，以及类似病毒和其他外源序列的特征。然而，不同基因组元件在保护基因免受沉默机制影响方面的相对贡献及其分子机制尚不清楚。在这里，我们展示了诱变筛选的结果，旨在识别参与保护内源性基因免受成为sRNAs生产模板的特征。有趣的是，所有恢复的突变体都有转录终止功能基因的改变，这表明终止子在这一过程中起着核心作用。事实上，使用GFP报告系统，我们发现，在测试的不同遗传元件中，终止序列对转基因衍生的sRNA积累的影响最大，并且定义明确的poly(a)位点可能特别重要。最后，我们描述了一种意想不到的机制，其中含有某些内含子/终止子组合的转基因导致sRNAs的产生增加，这似乎干扰了剪接。
Transgenes have become essential to modern biology, being an important tool in functional genomic studies and also in the development of biotechnological products. One of the major challenges in the generation of transgenic lines concerns the expression of transgenes, which, compared to endogenes, are particularly susceptible to silencing mediated by small RNAs (sRNAs). Several reasons have been put forward to explain why transgenes often trigger the production of sRNAs, such as the high level of expression induced by commonly used strong constitutive promoters, the lack of introns, and features resembling viral and other exogenous sequences. However, the relative contributions of the different genomic elements with respect to protecting genes from the silencing machinery and their molecular mechanisms remain unclear. Here, we present the results of a mutagenesis screen conceived to identify features involved in the protection of endogenes against becoming a template for the production of sRNAs. Interestingly, all of the recovered mutants had alterations in genes with proposed function in transcription termination, suggesting a central role of terminators in this process. Indeed, using a GFP reporter system, we show that, among different genetic elements tested, the terminator sequence had the greatest effect on transgene-derived sRNA accumulation and that a well-defined poly(A) site might be especially important. Finally, we describe an unexpected mechanism, where transgenes containing certain intron/terminator combinations lead to an increase in the production of sRNAs, which appears to interfere with splicing.