Postdoctoral Fellow Donald Danforth Plant Science Center Chesterfield, Missouri
Body of Abstract: While transgene silencing is a common phenomenon, it is generally not well understood why certain transgenes are targeted for silencing and others are stably expressed for generations. It is generally agreed that the key trigger molecule required to transgene silencing is an ‘aberrant RNA,’ but the field does not understand what these aberrant RNAs look like or how they function to trigger RNAi. Identification and characterization of these aberrant RNAs has remained elusive because the technologies to address this question have been lacking. To this end, we developed a qualitative and quantitative expression assay specifically designed to examine all RNAs derived from a subset of loci across any transcriptome in extreme depth using long-read sequencing.
We examined all RNAs, both polyadenylated (polyA) and non-polyA, in functional transgenic lines as well as a system developed using the RUBY transgene wherein the silencing state can be derived from the phenotype of the plant in Arabidopsis. Using this technique we can examine features such as percent antisense RNAs, percent polyA RNAs, and can examine all RNAs produced from protein-coding genes and the transgenes. Overall, we found that transgenes have a different RNA fingerprint than endogenous genes. Thus, even when a transgene is functional, it is not as ‘transcriptionally-clean’ as endogenous genes, and rather subject to different layers of regulation that are intermediate between protein-coding genes and TE transcripts. We now have a tool powerful enough to examine and identify the aberrant RNAs that cause transgene silencing that has evaded researchers for decades.