Identification of a cleaved aberrant RNA associated with the initiation of transgene silencing

Abstract The success of many engineered crop traits depends on the stable expression of transgenes, but their effectiveness is frequently at risk due to transgene silencing. The reason why certain transgenes are targeted by silencing pathways while others remain highly expressed and durable has remained a major question for decades due to the lack of technologies to study the initiation of transgene silencing. We developed two technologies to identify the trigger of transgene silencing in Arabidopsis (Arabidopsis thaliana) and in lettuce (Latuca sativa): one using the RUBY transgene to visualize the precise developmental time point of transgene silencing and the second to identify all transcripts produced from a transgene. By combining these two methods with Machine Learning, we identified an aberrant transgene-derived RNA that accumulates to high levels and closely correlates with the onset of transgene silencing. Our data suggest that a ribosome stalled at an unusual three-consecutive-histidine peptide sequence on the RUBY transcript triggers No-Go RNA Decay and cleavage of the RUBY mRNA. The production of this cleaved aberrant RNA precedes RNA interference during the triggering of transgene silencing; it is innate to the transgene coding sequence, independent of the promoter used or whether it is transformed into a model plant or crop.