Abstract
Sense transgene-induced post-transcriptional gene silencing (S-PTGS) is thought to be a type of RNA silencing in which ARGONAUTE1 directs the small interfering RNA (siRNA)-mediated cleavage of a target mRNA in the cytoplasm. Here, we report that the altered splicing of endogenous counterpart genes is a main cause for the reduction of their mature mRNA levels. After the S-PTGS of a tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) gene, 3′-truncated, polyadenylated endo-NtFAD3 transcripts and 5′-truncated, intron-containing endo-NtFAD3 transcripts were detected in the total RNA fraction. Although transcription proceeded until the last exon of the endogenous NtFAD3 gene, intron-containing NtFAD3 transcripts accumulated in the nucleus of the S-PTGS plants. Several intron-containing NtFAD3 transcripts harboring most of the exon sequences were generated when an endogenous silencing suppressor gene, rgs-CaM, was overexpressed in the S-PTGS plants. These intron-containing NtFAD3 splice variants were generated in the presence of NtFAD3 siRNAs that are homologous to the nucleotide sequences of these splice variants. The results of this study indicate that the inhibition of endo-NtFAD3 gene expression is primarily directed via the alteration of splicing and not by cytoplasmic slicer activity. Our results suggest that the transgene and intron-containing endogenous counterpart genes are differentially suppressed in S-PTGS plants.
Highlights
In higher plants, a transgene often induces the inactivation of itself and homologous endogenous counterpart genes [1,2], a phenomenon called sense transgene-induced post-transcriptional gene silencing (S-PTGS)
We found that a high transcription rate of the transgene was necessary for the generation of NtFAD3 small interfering RNA (siRNA); when the promoter of the trans-NtFAD3 gene was inactivated by RNA-directed DNA methylation (RdDM), the transcription rate of the transgene was reduced, followed by the disappearance of NtFAD3 siRNA
RNA viruses replicate exclusively in the cytoplasm [29,30], and the suppression of viral replication by RNA silencing should be associated with the cytoplasmic RNA cleavage activity of the siRNA-AGO1 protein complex [6]
Summary
A transgene often induces the inactivation of itself and homologous endogenous counterpart genes [1,2], a phenomenon called sense transgene-induced post-transcriptional gene silencing (S-PTGS). In S-PTGS, aberrant transcripts from transgene loci recruit RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) for the synthesis of complementary RNA strands [3,4]. Singlestranded siRNAs are incorporated into ARGONAUTE proteins (AGOs); in particular, siRNAs incorporated into AGO1 ( called slicer) act as a guide for mRNA degradation and/or translational inhibition [6,7]. This silencing step is likely to occur at unique cytoplasmic foci, termed processing bodies (PBs), where AGO1, DCP2, and XRN4 are localized [8]. The above-mentioned 24-nt-long siRNAs guide the methylation of homologous genomic DNA in a pathway referred to as RNA-directed DNA methylation (RdDM) [11,12]
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