Posttranscriptional gene silencing of gn1 in tobacco triggers accumulation of truncated gn1-derived RNA species.

Posttranscriptional silencing of basic beta-1,3-glucanase genes in the tobacco line T17 is manifested by reduced transcript levels of the gn1 transgene and homologous, endogenous basic beta-1,3-glucanase genes. An RNA ligation-mediated rapid amplification of cDNA ends (RLM-RACE) technique was used to compare the 3' termini of gn1 RNAs present in expressing (hemizygous and young homozygous) and silenced (mature homozygous) T17 plants. Full-length, polyadenylated gn1 transcripts primarily accumulated in expressing plants, whereas in silenced T17 plants, mainly 3'-truncated, nonpolyadenylated gn1 RNAs were detected. The relative abundance of these 3'-truncated gn1 RNA species gradually increased during the establishment of silencing in homozygous T17 plants. Similar 3'-truncated, nonpolyadenylated gn1 RNA products were observed in an independent case of beta-1,3-glucanase posttranscriptional gene silencing. This suggests that these 3'-truncated gn1 RNAs are a general feature of tobacco plants showing posttranscriptional silencing of the gn1 transgene.

Sequences throughout the basic beta-1,3-glucanase mRNA coding region are targets for homology dependent post-transcriptional gene silencing.

In the transgenic tobacco line T17, plants homozygous for the gn1 transgene display developmentally regulated post-transcriptional silencing of basic beta-1,3-glucanase genes. Previously, it has been shown that silencing involves a markedly increased turnover of silencing-target glucanase mRNAs. Using a two-component viral reporter system facilitated a comparison, in a quantitat- ive manner, of the relative silencing efficiencies of various sequences derived from the gn1 transgene. The results show that target sites for the silencing mechanism are present throughout the coding region of the gn1 mRNA. Similar-sized coding region sequences along the entire gn1 mRNA display a similar susceptibility to the silencing mechanism. The susceptibility to silencing increases as the coding region elements increase in size. Relative to internal sequences, the 5' and 3' terminal regions of the gn1 mRNA are inefficient targets for the silencing machinery. Importantly, sequences of the gn1 transgene that are not part of the mature gn1 mRNA are not recognized by the silencing machinery when expressed in chimeric viral RNAs. These results show that the glucanase silencing mechanism in T17 plants is primarily directed against gn1 mRNA-internal sequences and that terminal sequences of the gn1 mRNA are relatively unaffected by the silencing mechanism.

Silencing of beta-1,3-glucanase genes in tobacco correlates with an increased abundance of RNA degradation intermediates.

Post-transcriptional gene silencing of beta-1,3 glucanase genes in the transgenic tobacco line T17 is characterised by an increased turnover and, as a consequence, reduced levels of gn1 transgene and endogenous beta-1,3 glucanase mRNAs. Here, additional gn1 RNAs, both larger and smaller than the full-length messenger, are shown to accumulate in silenced plants of the transgenic tobacco line T17. The longer-than-full-length gn1 RNAs are the result of cryptic processing of the gn1 messenger. The small gn1 RNAs in silenced plants correspond to distal and proximal parts of the mature gn1 messenger. The proximal RNA products are intact at their 5' extremity, but terminate at different positions at the 3'-end. The distal RNA products contain a poly(A) tail and are truncated to various positions at the 5'-end. These observations indicate that degradation of the mature gn1 transcript does not start at the 5'- or 3'-end, but rather are consistent with degradation of the gn1 transcript starting with an endonucleolytic cleavage followed by internal exonuclease digestion. Importantly, the truncated products are more abundant in silenced plants than in expressing plants. This suggests, together with the previously reported silencing-related increased gn1 mRNA turnover and the similar rates of gn1 transcription in silenced and expressing T17 plants, that the predominant decay route for the gn1 transcripts differs between silenced and expressing conditions.