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.

中文翻译：

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终止子在转基因表达和转录后基因沉默中的关键作用。

转基因已成为现代生物学必不可少的工具，是功能基因组研究和生物技术产品开发中的重要工具。转基因品系的产生中的主要挑战之一涉及转基因的表达，与内基因相比，转基因的表达特别容易受到小RNA（sRNA）介导的沉默的影响。提出了几个理由来解释为什么转基因通常会触发sRNA的产生，例如由常用的强组成型启动子诱导的高水平表达，内含子的缺乏以及类似于病毒和其他外源序列的特征。然而，关于保护基因免受沉默机制及其分子机制的影响，不同基因组元件的相对贡献尚不清楚。这里，我们提出了诱变筛选的结果，旨在鉴定参与保护内生基因的功能，以防止其成为生产sRNA的模板。有趣的是，所有回收的突变体在具有拟议的转录终止功能的基因中都发生了改变，这表明终止子在这一过程中起着核心作用。确实，使用GFP报告系统，我们显示出，在测试的不同遗传元件中，终止子序列对转基因衍生的sRNA积累的影响最大，而且明确定义的poly（A）位点可能尤其重要。最后，我们描述了一个出乎意料的机制，其中包含某些内含子/终止子组合的转基因导致sRNA产量增加，这似乎在干扰剪接。