Data_Sheet_2_Identification of a Transferrable Terminator Element That Inhibits Small RNA Production and Improves Transgene Expression Levels.docx

The role of terminators is more commonly associated with the polyadenylation and 3′ end formation of new transcripts. Recent evidence, however, suggests that this regulatory region can have a dramatic impact on gene expression. Nonetheless, little is known about the molecular mechanisms leading to the improvements associated with terminator usage in plants and the different elements in a plant terminator. Here, we identified an element in the Arabidopsis HSP18.2 terminator (tHSP) to be essential for the high level of expression seen for transgenes under the regulation of this terminator. Our molecular analyses suggest that this newly identified sequence acts to improve transcription termination, leading to fewer read-through events and decreased amounts of small RNAs originating from the transgene. Besides protecting against silencing, the tHSP-derived sequence positively impacts splicing efficiency, helping to promote gene expression. Moreover, we show that this sequence can be used to generate chimeric terminators with enhanced efficiency, resulting in stronger transgene expression and significantly expanding the availability of efficient terminators that can be part of good expression systems. Thus, our data make an important contribution toward a better understanding of plant terminators, with the identification of a new element that has a direct impact on gene expression, and at the same time, creates new possibilities to modulate gene expression via the manipulation of 3′ regulatory regions.

终止子（terminator）的经典功能多与新生转录本的多聚腺苷酸化及3'端形成过程相关。但新近研究证据表明，这类调控区域可对基因表达产生显著调控作用。尽管如此，学界对于植物中使用终止子带来表达提升的分子机制，以及植物终止子内部的各类组成元件，仍缺乏深入认知。本研究中，我们在拟南芥HSP18.2终止子（tHSP）中鉴定出一段核心序列，其对于该终止子调控下的转基因实现高水平表达不可或缺。分子分析结果显示，这段新鉴定的序列可增强转录终止效率，减少通读转录事件的发生，并降低源自转基因的小RNA（small RNAs）的积累量。该tHSP来源序列除了能够抵御基因沉默之外，还可正向调控剪接效率，进而助力基因表达的提升。此外，我们证实该序列可用于构建具有更高终止效率的嵌合终止子（chimeric terminators），从而实现更强的转基因表达，并大幅扩充可用于优质表达系统的高效终止子资源库。综上，本研究通过鉴定出一段直接影响基因表达的新型元件，为深入解析植物终止子的功能机制做出了重要贡献，同时也为通过操控3'调控区域实现基因表达调控开辟了全新路径。