<i>P</i> values for ssRNA injections.

Genetic robustness can be achieved via several mechanisms including transcriptional adaptation (TA), a sequence similarity-driven process whereby mutant mRNA degradation products modulate, directly or indirectly, the expression of so-called adapting genes. To identify the sequences required for this process, we utilized a transgenic approach in Caenorhabditis elegans, combining an overexpression construct for a mutant gene (act-5) and a fluorescent reporter for the corresponding adapting gene (act-3). Analyzing a series of modifications for each construct, we identified, in the 5’ regulatory region of the act-3 locus, a 25-base pair (bp) element which exhibits 60% identity with a sequence in the act-5 mRNA and which, in the context of a minimal promoter, is sufficient to induce ectopic expression of the fluorescent reporter. The 25 nucleotide (nt) element in the act-5 mRNA lies between the premature termination codon (PTC) and the next exon/exon junction, suggesting the importance of this region of the mutant mRNA for TA. Additionally, we found that single-stranded RNA injections of this 25 nt element from act-5 into the intestine of wild-type larvae led to higher levels of adapting gene (act-3) mRNA. Different models have been proposed to underlie the modulation of gene expression during TA including chromatin remodeling, the inhibition of antisense RNAs, the release of transcriptional pausing, and the suppression of premature transcription termination, and our data clearly show the importance of the regulatory region of the adapting gene in this particular act-5/act-3 TA model. Our findings also suggest that RNA fragments can modulate the expression of loci exhibiting limited sequence similarity, possibly a critical observation when designing RNA based therapies.遗传稳健性可通过多种机制实现，其中包括转录适应（transcriptional adaptation, TA）：这是一种由序列相似性驱动的过程，突变mRNA的降解产物可直接或间接调控所谓的适配基因的表达。为鉴定该过程所需的序列，我们在秀丽隐杆线虫（Caenorhabditis elegans）中采用转基因策略，将突变基因act-5的过表达载体与对应适配基因act-3的荧光报告基因相结合。通过分析每个载体的一系列修饰，我们在act-3基因座的5'调控区中鉴定出一段25碱基对（base pair, bp）的元件，该元件与act-5 mRNA中的一段序列具有60%的同源性；且在最小启动子的背景下，该元件足以诱导荧光报告基因的异位表达。act-5 mRNA中的这段25核苷酸（nucleotide, nt）元件位于提前终止密码子（premature termination codon, PTC）与下一个外显子-外显子连接区之间，这提示突变mRNA的该区域在TA过程中具有重要作用。此外，我们发现将act-5来源的该25 nt元件的单链RNA注射到野生型幼虫的肠道中，可使适配基因act-3的mRNA水平升高。目前已提出多种模型来解释TA过程中基因表达的调控机制，包括染色质重塑、反义RNA抑制、转录暂停释放以及提前转录终止抑制；我们的数据明确证实了适配基因的调控区在该特定act-5/act-3 TA模型中的重要性。我们的研究结果还提示，RNA片段可调控序列相似性有限的基因座的表达，这一发现或许在设计基于RNA的治疗方案时具有关键参考价值。