Improperly Terminated, Unpolyadenylated mRNA of Sense Transgenes Is Targeted by RDR6-Mediated RNA Silencing in Arabidopsis

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Abstract
RNA silencing can be induced by highly transcribed transgenes through a pathway dependent on RNA-DEPENDENT RNA POLYMERASE6 (RDR6) and may function as a genome protection mechanism against excessively expressed genes. Whether all transcripts or just aberrant transcripts activate this protection mechanism is unclear. Consistent RNA silencing induced by a transgene with three direct repeats of the beta-glucuronidase (GUS) open reading frame (ORF) is associated with high levels of truncated, unpolyadenylated transcripts, probably from abortive transcription elongation. Truncated, unpolyadenylated transcripts from triple GUS ORF repeats were degraded in the wild type but accumulated in an rdr6 mutant, suggesting targeting for degradation by RDR6-mediated RNA silencing. A GUS transgene without a 3' transcription terminator produced unpolyadenylated readthrough mRNA and consistent RDR6-dependent RNA silencing. Both GUS triple repeats and terminator-less GUS transgenes silenced an expressed GUS transgene in trans in the wild type but not in the rdr6 mutant. Placing two 3' terminators in the GUS transgene 3' reduced mRNA 3' readthrough, decreased GUS-specific small interfering RNA accumulation, and enhanced GUS gene expression. Moreover, RDR6 was localized in the nucleus. We propose that improperly terminated, unpolyadenylated mRNA from transgene transcription is subject to RDR6-mediated RNA silencing, probably by acting as templates for the RNA polymerase, in Arabidopsis thaliana.