Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing

Abstract

In plants, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effectors of RNA silencing, a process involved in defense through RNA interference (RNAi) and in development. Plant viruses are natural targets of RNA silencing, and as a counterdefensive strategy, they have evolved highly diverse silencing suppressor proteins. Although viral suppressors are usually thought to act at distinct steps of the silencing machinery, there had been no consensus system so far that allowed a strict side-by-side analysis of those factors. We have set up such a system in Arabidopsis thaliana and used it to compare the effects of five unrelated viral silencing suppressors on the siRNA and miRNA pathways. Although all the suppressors inhibited RNAi, only three of them induced developmental defects, indicating that the two pathways are only partially overlapping. These developmental defects were remarkably similar, and their penetrance correlated with inhibition of miRNA-guided cleavage of endogenous transcripts and not with altered miRNA accumulation per se. Among the suppressors investigated, the tombusviral P19 protein coimmunoprecipitated with siRNA duplexes and miRNA duplexes corresponding to the primary cleavage products of miRNA precursors. Thus, it is likely that P19 prevents RNA silencing by sequestering both classes of small RNAs. Moreover, the finding here that P19 binds siRNAs and suppresses RNAi in Hela cells also suggests that this factor may be useful to dissect the RNA silencing pathways in animals. Finally, the differential effects of the silencing suppressors tested here upon other types of Arabidopsis silencing-related small RNAs revealed a surprising variety of biosynthetic and, presumably, functional pathways for those molecules. Therefore, silencing suppressors are valuable probes of the complexity of RNA silencing.

Figure 1.

The P1-HcPro, P19, and P15 Proteins, but Not the P38 or P25 Proteins, Induce Developmental Defects in Arabidopsis. (A) Wild-type Arabidopsis ecotype Col-0. (B) to (D) P1-HcPro–transgenic Arabidopsis of class I (B), class II (C), and class III (D). (E) Compared leaf morphology among the class I, II, and III P1-HcPro plants. (F) Compared levels of P1-HcPro transcripts in flowers of class I, class II, and class III P1-HcPro plants, as assessed by RNA gel blot analysis. rRNA, ethidium bromide staining of rRNA. (G) Flower from wild-type Arabidopsis ecotype Col-0. The sepals were opened to allow observation of the internal floral whorls. (H) A flower from a P1-HcPro plant of class II. Note the loose flower structure, narrow sepals and petals, and partially unfused carpel (arrow). (I) Flower from a P1-HcPro plant of class I. The petals were removed to allow observation of the carpel and narrow sepals. (J) Close-up view of the unfused carpel in the flower depicted in (I). (K) P19-transgenic Arabidopsis. (L) Compared rosette leaf morphology in wild-type and P19 plants. (M) and (N) Altered flower morphology in P19 plants (M) as compared with wild-type plants (N). Note the narrow sepals and petals and overall loose structure of the flower. (O) P15-transgenic Arabidopsis. (P) Compared rosette leaf morphology in wild-type and P15 plants. (Q) Flower defects in P15-expressing plants, as compared with wild-type plants. Note the narrow sepals and petals and overall loose aspect of the flower. (R) and (S) P38 transformants (R) and P25 transformants (S) do not exhibit a noticeable developmental phenotype.

Figure 2.

Suppression of CHS-RNAi by P1-HcPro, P19, P38, P25, and P15. (A) One day after light induction, total RNA was extracted from leaves of wild-type plants or of line CHS-RNAi crossed or not with the silencing suppressor–expressing lines. Fifteen micrograms of this RNA was subjected to RNA gel blot analysis using a CHS cDNA probe. Anthocyanins were extracted in parallel and quantified by spectrophotometry. (B) RNA gel blot analysis of low molecular weight RNA (15 μg) extracted before light induction. The hybridization was with a CHS cDNA probe. nt, nucleotides. (C) Twenty-five micrograms of the RNA used in (B) was treated with RNase A, deproteinized, heat denatured, and subjected to RNA gel blot analysis using a CHS cDNA probe.

Figure 3.

siRNA Coimmunoprecipitate with P19 in Arabidopsis and in Mammalian Cells. (A) Immunodetection of P19:HA and P19m:HA (anti-HA antibody) in seedlings of CHS-RNAi plants that had been transformed either with the P19:HA or with the P19m:HA construct. The proteins accumulate to similar levels in the two lines shown here. Coomassie blue staining of the immunoblot indicates equal protein loading. (B) Anthocyanin accumulation and RNA gel blot analysis of the low molecular weight RNA fraction extracted from the P19:HA and P19m:HA seedlings. Hybridization was with a CHS cDNA probe. nt, nucleotides. (C) Total proteins were extracted from seedlings. P19:HA and P19m:HA were immunoprecipitated with an anti-HA antibody, and the presence of each protein was assayed by protein gel blot analysis of the immunoprecipitated (IP) fractions (top panel). After deproteinization, nucleic acids extracted from the IP fractions were subjected to RNA gel blot analysis using a CHS cDNA probe (bottom panel). (D) Dual-LUC assay performed in human Hela cells transfected with pGL3-CMV (encoding the firefly LUC) and pRL-CMV (encoding the renilla LUC) together with pSG5m (mock) or with pSGP19 (P19), psGP19HA (P19:HA), or psGP19mHA (P19m:HA). Twenty-four hours later, cells were supertransfected with 0 (−) or 300 ng (+) of siRNAs directed against the firefly LUC mRNA. The renilla LUC mRNA is not targeted by these siRNAs and is therefore used as a reference in this assay. For each treatment, the luminescence ratio firefly/renilla was calculated. This ratio was then normalized to the luminescence values from a transfection experiment performed in parallel, in which anti-LUC siRNAs were omitted. This provided a relative LUC activity. For each treatment, results of two independent assays are presented. Values from each assay were from duplicate independent transfections. (E) Human Hela cells were transfected with pSGP19:HA together with anti-LUC siRNAs. Two days later, P19:HA was immunoprecipitated with an anti-HA antibody, and the presence of the protein was assayed by protein gel blot analysis (top panel). After deproteinization, nucleic acids were extracted from the immunoprecipitated fractions (IP) and subjected to RNA gel blot analysis using radiolabeled anti-LUC siRNAs as probe (bottom panel). nt, nucleotides. (F) Same experimental set up as in (D) but performed with pSG5m expression vectors for the P25, P38, and P15 proteins. The values in each bar are from three independent experiments conducted in triplicate.

Figure 4.

The Effects of Silencing Suppressors on miRNA Accumulation. (A) miRNA accumulation in flowers of wild-type, P19, P1-HcPro (HC), P15, P38, and P25 transgenic plants, as assessed by RNA gel blot analysis. The probes used were labeled oligonucleotides complementary to the miRNA indicated at the top left corner of each filter. Two labeled oligoribonucleotide standards were used as size markers (21 nucleotides and 24 nucleotides). rRNA, ethidium bromide staining of rRNA. The filters on the right and the left panels correspond to two separate RNA preparations, hence the use of separate wild-type samples for internal reference. See also Supplemental Data 3 online for analysis of additional miRNAs. nt, nucleotides. (B) High-resolution RNA gel blot analysis of miR156 and miR164 from the P19 samples used in (A) reveals a mobility shift because of the accumulation of a nucleic acid species with the apparent electrophoretic mobility of a 19- to 20-nucleotide RNA, as assessed with a labeled oligoribonucleotide.

Figure 5.

The P19 Protein Binds to miRNA/miRNA^* Duplexes. (A) Accumulation of miR171 in seedlings of line CHS-RNAi expressing or not P19mHA and P19HA, as described in Figure 3. Hybridization was with a labeled oligonucleotide complementary to miR171. Total proteins were extracted from the same tissues, and P19:HA and P19m:HA were immunoprecipitated (IP) with an anti-HA antibody (data not shown). Upon deproteinization, nucleic acids extracted from the immunoprecipitated fractions were subjected to RNA gel blot analysis using a labeled oligonucleotide complementary to miR171 as probe (bottom panel). nt, nucleotides. (B) Predicted secondary structure of the miR171 precursor transcript. The sequence of miR171 is highlighted in red. The predicted cleavage product of DCL-1 is the miRNA/miRNA^* duplex, of which only one strand (corresponding to miR171) is incorporated into the RISC for target cleavage. The other strand (corresponding to miR171^*) is unstable, presumably because of rapid degradation. (C) The membrane in (A) (bottom panel) was stripped and rehybridized with a labeled oligonucleotide complementary to the sequence of the predicted miR171^*. This small RNA is present in the P19HA but not in the P19mHA immunoprecipitates. (D) and (E) The membrane in (A) (top panel) was stripped and rehybridized with an oligonucleotide complementary to the sequence of the predicted miR171^*. There is strong enhancement of the accumulation of miR171^* in the P19HA samples, which also occurs in inflorescences (E) of the P19-expressing plants depicted in Figure 1K. (F) High-resolution RNA gel blot analysis of the RNAs extracted in (E) reveals a migration shift for miR171^* because of the accumulation of a nucleic acid species with the apparent electrophoretic mobility of a 19- to 20-nucleotide RNA, as assessed with a labeled oligoribonucleotide.

Figure 6.

Effects of the Silencing Suppressors on miRNA-Mediated Cleavage of Target mRNAs. (A) Two possible outcomes of miRNA-guided cleavage of endogenous transcripts in plants. In the first case (1), both the 5′ and 3′ cleavage fragments are degraded, whereas in the second case (2), the 3′ cleaved fragment remains stable. The miRNA is indicated in red. (B) and (C) Fifteen micrograms of total RNA extracted from inflorescences of the various suppressor-expressing lines were subjected to RNA gel blot analysis using cDNA probes specific for the CUC1 (B) or the SCL6-IV and ARF10 mRNAs (C). The size of the predicted 3′ cleavage products of SCL6-IV and ARF10 [SCL6-IV(b) and ARF10(b), respectively] is indicated. nt, nucleotides.

Figure 7.

Diversity and Complexity of the Arabidopsis Silencing-Related Small RNAs. (A) RNA gel blot analysis of RNAs extracted from leaves and stems of the plants that were used to produce the inflorescence data presented in Figure 4. RNA gel blot conditions were the same. See also Supplemental Data 3 online for analysis of additional miRNAs. nt, nucleotides. (B) Total RNA was extracted from inflorescences of wild-type, P1-HcPro class I, and P1-HcPro class III plants and subjected to RNA gel blot analysis. The probes used were labeled oligonucleotides complementary to miR156, miR160, and miR171, respectively. rRNA, ethidium bromide staining of rRNA. (C) Accumulation of the 24-nucleotide miR163 in leaves and flowers of the suppressor lines. (D) and (E) Accumulation of small RNA 96 (D) and of small RNAs of the AtSN1 retroelement (E) in flowers from the various suppressor-expressing lines. (F) Flower-specific accumulation of the 21-nucleotide small RNA2 in inflorescences of the various suppressor-expressing lines. The probes used in (C) to (F) were labeled oligonucleotides complementary to the corresponding small RNAs.