Introns reduce transitivity proportionally to their length, suggesting that silencing spreads along the pre-mRNA

Abstract

SUMMARYEndogenes rarely support transitive silencing, whereas most transgenes generally allow the spread of silencing to occur along the primary target. To determine whether the presence of introns might explain the difference, we investigated the influence of introns in the primary target on 3¢-5¢ silencing transitivity. When present in a transgene, an intron-containing endogene fragment does not prohibit the spread of silencing across this fragment, indicating that introns do not preclude silencing transitivity along endogenes. Also, a multiple intron-containing genomic gene fragment that had previously been shown not to support transitivity in an endogenous context could support transitivity when present in a transgene. Nevertheless, genomic intron-containing fragments delayed the onset and diminished the efficiency of transitive silencing of a secondary target compared with the corresponding cDNA fragments. Remarkably, transitivity was impaired proportionally with the length of the pre-mRNA, and not of the mRNA. The latter result suggests that the RNAdependent RNA polymerase-based spreading of silencing progresses along the non-spliced rather than the fully processed mature mRNA.

An endogene-resembling transgene is resistant to DNA methylation and systemic silencing

“…In support of our suggestion, while the presence of UTRs and introns is positively correlated with RNA decay, it is negatively correlated with RNA silencing. 23,60,[62][63][64] Spliceosome association likely excludes RDR6 recruitment. 16 Thus, endogenous siRNAs matching the transcripts of intron-containing genes are underrepresented in sRNA libraries of Arabidopsis, while siRNAs derived from intronless gene transcripts are highly abundant.…”

“…3, probes G and P). Secondary siRNAs production may be slow 23 and may take longer (Fig. 3) and the 182 bp and 134 bp regions analyzed by bisulfite sequencing (Fig.…”

“…Thus, transitivity may spread to 5′ and 3′ direction, but not indefinitely. 21,23 Nevertheless, transitivity results in the amplification of siRNAs constituting a self-reinforcing feedback loop of silencing. Silencing may spread through plasmodesmata cell-to-cell and through the vasculare system to systemic parts of the plant.…”

Gene Duplication and RNA Silencing in Soybean

“…The FAD2-A1 intron-induced RNA silencing led to the understanding that RNA degradation can take place in the nucleus [44]. Although whether RNA degradation in the nucleus is inducible for other genes or in other plants has not been known, this phenomenon is intriguing because the involvement of nuclear events has been assumed for amplification of RNA silencing via transitivity [45] or intron-mediated suppression of RNA silencing [46].…”

The spliceosome as a transposon sensor

“…39 These findings echo the earlier observation that introns retard the RdRP-dependent phenomenon of secondary siRNA, in which a single-stranded target of siRNA becomes gradually itself a template for siRNA synthesis. 40 Although the mechanisms by which efficient splicing opposes RNA silencing are unclear, the requirement for RdRP in plant siRNA pathways raises the possibility that efficient splicing acts to prevent transcripts from exhibiting triggers for RdRP activity, which are thought to include absence of a 5′ cap or poly-A tail. 41,42 To summarize, the data to date suggest that efficient splicing impedes RNA silencing in plants.…”