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

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 b-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 39 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 39 terminators in the GUS transgene 39 reduced mRNA 39 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.

The gypsy Insulator of Drosophila melanogaster, Together With Its Binding Protein Suppressor of Hairy-Wing, Facilitate High and Precise Expression of Transgenes in Arabidopsis thaliana

“…Position-effect variegation is a well-documented concept in Drosophila, but very little is known about it in Arabidopsis, where the 35S promoter of virus origin has been extensively employed in transgenesis. The variation of the expression level of a transgene driven by this strong and constitutive promoter has been attributed largely to a gene-specific RNA-silencing mechanism (Schubert et al 2004;Luo and Chen 2007). The impact of position effects on the variation of the expression pattern of a transgene driven by a tissue-or stage-specific plant promoter was less documented.…”

“…Increasing evidence suggests that insulators have evolved from promoters (Raab and Kamakaka 2010). The gypsy insulator downstream from the terminator region of transgenes might ensure correct termination of reporter transcripts, because it was documented that RNA silencing triggered by mRNA 39 readthrough was a major cause of reduced expression of transgenes in plants (Luo and Chen 2007). However, terminator activity for the gypsy insulator was recently tested in Drosophila, where it was found not to function (Silicheva et al 2010).…”

“…RNA silencing was triggered if the transcript level of a transgene surpassed a gene-specific threshold (Schubert et al 2004). Improperly terminated, unpolyadenylated mRNA from transgene transcriptions generated by 39 readthrough was subjected to RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-mediated RNA silencing (Luo and Chen 2007).…”

The longer version of Arabidopsis thaliana heat shock protein 18.2 gene terminator contributes to higher expression of stably integrated transgenes in cultured tobacco cells

“…In a previous study, when a transgene without a terminator or with a poor terminator was introduced into a plant genome, aberrant transcripts without polyA tails accumulated, leading to induction of RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent RNA silencing (Luo and Chen 2007). Those authors also reported that silencing was relieved, and the expression level of the gene concomitantly increased, by a double terminator that promoted accurate polyadenylation of mRNA.…”

Reevaluation of the Reliability and Usefulness of the Somatic Homologous Recombination Reporter Lines

“…Boyko et al (2010b) showed that acute exposure to various types of stresses results in the transient change of the 35S promoter-driven transgene expression, whereas chronic exposure to stress does not lead to any significant changes. Additionally, it is known that most terminators used in plant science, such as NOS, 35S, and G7, are somewhat leaky and can lead to read-through transcription (Rose and Last, 1997;Luo and Chen, 2007;Ü lker et al, 2008) or even supply cryptic intron splice sites (Ü lker et al, 2008). Since the sequence details are missing from publications that make use of the SHR lines, it has not been possible to analyze whether these sequences could have stochastic-or pathogen-induced promoter, terminator, or intron activities.…”