Transcriptional silencing and promoter methylation triggered by double-stranded RNA

Abstract

Double-stranded RNA induces a post-transcriptional gene silencing process, termed RNAi, in diverse organisms. It is shown here that transcriptional gene silencing accompanied by de novo methylation of a target promoter in plants can be triggered by a double-stranded RNA containing promoter sequences. Similar to the double-stranded RNA involved in RNAi, this promoter double-stranded RNA, which is synthesized in the nucleus, is partially cleaved into small RNAs approximately 23 nucleotides in length. Both transcriptional and post-transcriptional gene silencing can thus be initiated by double-stranded RNAs that enter the same degradation pathway. The results also implicate double-stranded RNA in directing DNA methylation. Different constructs designed to produce double-stranded promoter RNA in various ways were evaluated for their ability to induce gene silencing in tobacco and Arabidopsis. RNA hairpins transcribed from inverted DNA repeats were the most effective trans-acting silencing signals. This strategy could be useful for transcriptionally downregulating genes in a variety of plants.

Fig. 1. DNA constructs designed to produce NOSpro dsRNA and their effectiveness in different plants. NOSpro IR without (A) and with (B) a transcribing 35Spro, which should produce a NOSpro hairpin RNA. (C) Conversion of a transcribed NOSpro DR into an IR via Cre recombinase activity (Odell and Russell, 1994) results in a switch from synthesis of a single-stranded NOSpro RNA to an RNA hairpin. (D) Synthesis of separate sense and antisense NOSpro RNAs from different loci. (E) Production of overlapping sense and antisense NOSpro transcripts at one locus. Three constructs based on this strategy were tested. The 35S and MAS promoters are approximately the same strength; the 19Spro is somewhat weaker. The Arabidopsis column shows the number of silenced plants over the total number of independent transformants tested. The percentage silencing is shown in parentheses. The target gene tested for construct A was a NOSpro-NPTII gene and for constructs B and E an intact nopaline synthase gene. S, sense orientation; AS, antisense orientation; MAS, mannopine synthase promoter. n.d., not determined.

Fig. 2. Detection of NOSpro dsRNA in silenced plant lines. RNA preparations (50 µg/lane) were treated with RNase One (‘+’ lanes), which digests ssRNA and leaves dsRNA intact. (A) Silenced plants contained a resistant NOSpro dsRNA of the expected size (∼280 bp; Mette et al., 1999) (lanes 3 and 4). This NOSpro dsRNA was not detected after introduction of the 271 locus (lanes 5 and 6), which represses the 35S promoter transcribing NOSpro sequences, or in the original target line (lanes 1 and 2). Lanes 7 and 8 show positive controls [0.1 pg (left) and 1 pg (right)] consisting of NOSpro dsRNA (∼300 bp) formed by annealing antisense (AS) and sense (S) transcripts synthesized in vitro. (B) In a non-silenced line, an ∼0.6 kb poly adenylated NOSpro RNA is present (‘–’ lane) but readily degraded by RNase One. (C) Actin mRNA controls for the same RNA preparations used in (A). Size markers are shown to the left.

Fig. 3. A transcribed NOSpro direct repeat (DR) has a negligible effect on methylation of the target NOSpro. (A) Southern analysis demonstrating intact copies of the 35Spro-NOSpro DR construct integrated in three independent tobacco transformants. The first lane of each panel shows a HindIII (H)–PvuII (Pv) double digest probed with a 35Spro probe; in all three plant lines, a band of the expected size was obtained (arrowhead). The second lane of each panel shows triple digests with HPv plus SacII (S), which is methylation sensitive (open circle). Methylation at this S site in the NOSpro has been tightly correlated with silencing of this promoter. The appearance of the unmethylated fragment demonstrates that at least the S₁ site in the first NOSpro copy of the DR is unmethylated in all three lines. (B) RNase protection demonstrating that the entire NOSpro DR is transcribed in all three lines. Because the two NOSpro copies in the DR differ somewhat in size (263 versus 197 bp; see T-DNA constructs, Materials and methods), two sizes of protected fragment were obtained. (C) Methylation analysis of the target NOSpro, which is the same in all lines, in the presence of the transcribed NOSpro DR. EcoRI (E)–PstI (P) digests of either minus (first lanes) or plus (second lanes) methylation-sensitive S were performed. In all cases, little or no methylation is present, as indicated by the complete or nearly complete shift to the smaller fragment after addition of S. The partial methylation seen in the middle panel could result from spontaneous partial methylation of the NOSpro that occurs occasionally in the target plant line (Matzke et al., 1993) or from additional complexity or binary vector sequences in the NOSpro DR locus. Probes are indicated by black bars beneath the maps. Size markers are shown to the right.

Fig. 4. Kanamycin resistance before and after Cre-mediated conversion of a transcribed NOSpro DR into an IR. Silencing of the homozygous target NOSpro-NPTII gene was assessed by analyzing the kanamycin resistance of seedlings in the presence of the hemizygous 35Spro-NOSpro locus. (A) Before introducing the Cre locus, 100% of the seedlings were resistant, even though 50% contained the NOSpro DR. (B) Following the cross with a plant homozygous for the Cre locus, 50% of the seedlings were sensitive to kanamycin; these were ones that inherited the NOSpro IR (resistant seedlings did not inherit the 355pro-NOSpro locus). (C) Control cross with the target locus showing that the Cre locus alone did not weaken kanamycin resistance.

Fig. 5. Methylation of the target NOSpro and production of NOSpro dsRNA following conversion of a transcribed NOSpro DR into an IR by Cre recombinase. (A) Target NOSpro methylation (digests as in Figure 3C). Before introduction of the Cre locus and in the presence of the NOSpro DR [–Cre, +NP(DR)], the target NOSpro was not methylated, as indicated by the conversion to the smaller fragment after addition of SacII (second lane of panels 7 and 8). After the Cre cross [+Cre, +NP(IR)], target NOSpros became methylated, as indicated by lack of conversion to the smaller fragment (panels 1–4). This methylation was not induced by the Cre locus alone [+Cre, –NP(DR)] (panels 5 and 6). (B) PCR detection of the 19S promoter unique to the NOSpro DR/IR locus. The NOSpro IR was confirmed by cloning and sequencing a PCR fragment (data not shown). The bottom panels show a PCR control for the HPT gene that was present in all plants examined. (C) RNase One-resistant NOSpro dsRNA was only detected in plants containing a NOSpro IR and a methylated target NOSpro (panels 1–4).

Fig. 6. Presence of 35Spro-NOSpro-Sense or -Antisense constructs does not trigger methylation of the target NOSpro. The intact sense construct was integrated in three independent tobacco transformants (A, left) and the antisense construct in four (A, right) as indicated by the presence of EcoRI (E) fragments of the expected sizes (arrowheads; first lanes of each panel). The SacII (S) site in the NOSpro was largely unmethylated in the sense plants (A, left), as indicated by the conversion to the smaller fragment after an ES double digest (second lanes of each panel). The SacII site was slightly methylated in several antisense plants (A, right), as indicated by the less than complete conversion to the smaller fragment following an ES double digest (second lanes of each panel). The significance of this methylation is not known. (B) The target NOSpro, which was the same in all lines, remained unmethylated in the presence of the transcribed sense (B, left) and antisense (B, right) constructs. Digests and explanations as in Figure 3C. Size markers are shown to the right.

Fig. 7. Production of NOSpro sense (S) and antisense (AS) RNAs. The three sense and four NOSpro antisense lines shown in Figure 6 were analyzed by RNase protection for the respective NOSpro RNAs. A fourth sense line described previously (H_7NP; Mette et al., 1999) was included in the analysis. The size of the sense transcripts varies depending on the presence (longer) or absence (shorter) of leader sequences (see T-DNA constructs, Materials and methods).

Fig. 8. Detection of small RNAs in silenced plants. Total RNA from the indicated plants was prepared using a procedure to enrich for small RNAs (Hamilton and Baulcombe, 1999). This RNA was electrophoresed on 15% acrylamide gels, electroblotted onto a nylon membrane, and probed with NOSpro sense (S) and antisense (AS) RNA probes. (A) Small NOSpro sense (lane 5) and antisense (lane 9) RNAs ranging in size from 23 to 25 nt were detected only in silenced plants. These small RNAs were not present in the original NOSpro target line (lanes 3 and 7), in non-silenced plants that synthesized polyadenylated single-stranded NOSpro RNA (lanes 4 and 8), or in silenced plants following the introduction of the 271 locus (lanes 6 and 10), which disrupted synthesis of NOSpro dsRNA (Figure 2). As size and polarity controls, NOSpro sense (lanes 2 and 12) and antisense (lanes 1 and 11) DNA 23mers were used. The larger band visible in lane 2 is probably a multimer of the oligonucleotide present in the commercial preparation. Larger bands in lanes 7–10 are due to non-specific hybridization of the RNA probe. (B) Detection of NOSpro small RNAs in plants containing a transcribed NOSpro IR created in planta by Cre recombinase (lanes 1–4). NOSpro small RNAs were not detected in plants containing a transcribed NOSpro DR before conversion by Cre recombinase (lanes 5–8). Identical results were obtained with a NOSpro-S probe (not shown). The eight plants shown here are the same as those shown in Figure 5, panels 1–8. AS and S lanes show control antisense and sense DNA 23mers, respectively.