RNA Target Sequences Promote Spreading of RNA Silencing

Abstract

It is generally recognized that a silencing-inducing locus can efficiently reduce the expression of genes that give rise to transcripts partially homologous to those produced by the silencing-inducing locus (primary targets). Interestingly, the expression of genes that produce transcripts without homology to the silencing-inducing locus (secondary targets) can also be decreased dramatically via transitive RNA silencing. This phenomenon requires primary target RNAs that contain sequences homologous to secondary target RNAs. Sequences upstream from the region homologous to the silencing inducer in the primary target transcripts give rise to approximately 22-nucleotide small RNAs, coinciding with the region homologous to the secondary target. The presence of these small RNAs corresponds with reduced expression of the secondary target whose transcripts are not homologous to the silencing inducer. The data suggest that in transgenic plants, targets of RNA silencing are involved in the expansion of the pool of functional small interfering RNAs. Furthermore, methylation of target genes in sequences without homology to the initial silencing inducer indicates not only that RNA silencing can expand across target RNAs but also that methylation can spread along target genes.

A single transgene locus triggers both transcriptional and post-transcriptional silencing through double-stranded RNA production

“…Indeed, previous run-on analyses on methylated versus non-methylated transgenes have revealed that methylation of transcribed sequences does not aVect the rate of transcription (Elmayan et al 1998). However, in one case, methylation has also been shown to spread from coding to promoter sequences, resulting in TGS (Van Houdt et al 2003). It is conceivable that such a spread of methylation from the 271 transgene sequences into the adjacent plant promoter has happened in the epigenetic variant (271*) that we recovered by plating 50,000 hemizygous 271 seeds on a medium supplemented with kanamycin.…”

The Frequency and Efficiency of Endogene Suppression by Transitive Silencing Signals Is Influenced by the Length of Sequence Homology

“…Here, we study in more detail the 3# to 5# spreading previously observed with the XYZ-silencing system (Van Houdt et al, 2003;Bleys et al, 2006a) by using primary targets Y 98 , Y 250 , and Y 500 in which 98 nt, 250 nt, and 500 nt of the CAT2 sequence, respectively, were inserted between the nptII-3#chs end targeted by the silencing inducer X 21 and the upstream region homologous to a transgenic gus target Z c . We demonstrate that the length of the CAT2 inserts influences the timing and efficiency of silencing induced by secondary CAT2-and gus-specific siRNAs originating from the primary target transcripts.…”

“…In an XYZ system in tobacco (Nicotiana tabacum), we have shown that transitive amplification products that arise from silenced targets containing transgenic b-glucuronidase (gus) sequences are able to trigger silencing of a gus transgene (Van Houdt et al, 2003). We have now implemented this system in Arabidopsis (Arabidopsis thaliana) and showed that both transgenes and endogenes can be silenced by the secondary transitive signals (Bleys et al, 2006a).…”

“…We assessed the occurrence of transitive silencing of both secondary targets CAT and gus, looked at the relationship between silencing efficiency and secondary siRNA production, and checked the accumulation of tertiary endogenous siRNAs. In tobacco, a posttranscriptionally silenced IR of a T-DNA with a highly transcribed transgene, designated as locus X, can trigger transitive silencing of a secondary target Z when a stepwise homology is created through the introduction of a chimeric primary target Y with homology to both the silencing inducer X and the secondary target Z (Van Houdt et al, 2003). Similarly, in Arabidopsis, locus X 21 ( Fig.…”

“…In this organism, spreading of silencing is not limited to transgenic targets, but endogenous transcripts can also be used as template for amplification (Sijen et al, 2001;Alder et al, 2003). In plants, transitive silencing can occur in both the 3# to 5# and the 5# to 3# direction along transgenic target RNAs in a primer-dependent or primer-independent manner (Braunstein et al, 2002;Vaistij et al, 2002;Himber et al, 2003;Van Houdt et al, 2003;Kościań ska et al, 2005;Miki et al, 2005;Petersen and Albrechtsen, 2005). Virus-induced gene silencing has been shown to spread over a distance of at least 1,000 nt from the 5# end to the 3# end of the target mRNA, while 3# to 5# spreading can extend at least through 332 nt, with a possible limit of 600 nt (Vaistij et al, 2002;Petersen and Albrechtsen, 2005).…”