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. 2002 Feb;13(2):445-53.
doi: 10.1091/mbc.01-04-0211.

RNAi in Dictyostelium: the role of RNA-directed RNA polymerases and double-stranded RNase

Henrik Martens  1 , Jindrich Novotny, Jürgen Oberstrass, Theodore L Steck, Pamela Postlethwait, Wolfgang Nellen
Affiliations

RNAi in Dictyostelium: the role of RNA-directed RNA polymerases and double-stranded RNase

Henrik Martens et al. Mol Biol Cell. 2002 Feb.

Abstract

We show that in Dictyostelium discoideum an endogenous gene as well as a transgene can be silenced by introduction of a gene construct that is transcribed into a hairpin RNA. Gene silencing was accompanied by the appearance of sequence-specific RNA about 23mers and seemed to have a limited capacity. The three Dictyostelium homologues of the RNA-directed RNA polymerase (RrpA, RrpB, and DosA) all contain an N-terminal helicase domain homologous to the one in the dicer nuclease, suggesting exon shuffling between RNA-directed RNA polymerase and the dicer homologue. Only the knock-out of rrpA resulted in a loss of the hairpin RNA effect and simultaneously in a loss of detectable about 23mers. However, about 23mers were still generated by the Dictyostelium dsRNase in vitro with extracts from rrpA(-), rrpB(-), and DosA(-) cells. Both RrpA and a target gene were required for production of detectable amounts of about 23mers, suggesting that target sequences are involved in about 23mer amplification.

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Figures

Figure 1
Figure 1
Constructs used for β-gal gene silencing and their efficiency. In the first two lanes, the construct introduced into the cells is described. In the third lane, the average β-gal activity and SD of randomly chosen tested clones are given in units per milligram of total protein. The last lane shows how many clones have been tested and how many of them exhibited gene silencing. As (a) a control an empty vector was introduced; (b) an antisense copy (800-bp fragment) of the transgene was introduced (V18 promoter), (c) an additional copy (800-bp fragment) of the transgene controlled by the V18 promoter was introduced (equivalent to cosuppression approaches in plants), (d) sense and antisense RNA both under the control of the V18 promoter were expressed from different gene constructs in the same vector, (e) the 800-bp gene fragment was inserted between two opposing promoters (V18 and actin15), (f) an 800-bp inverted repeat with a 500-bp linker, corresponding to β-gal gene sequences, was fused to the V18 promoter, and (g) an 800-bp fragment was inserted between two T7 promoters and transformed bacteria (strain BL21) were used to feed Dictyostelium cells. Only in e was silencing of the transgene observed.
Figure 2
Figure 2
(A) Western blot with a discoidin-specific antibody on protein isolated from wild-type AX2 cells (control) and from four of nine randomly chosen clones silenced by discoidin RNAi (top). mcRNAi1 and 2 display complete silencing, and mcRNAi 3 and 4 display partial silencing. A Northern blot with RNA from the same cell lines, hybridized with a discoidin-specific probe, is shown in the middle. As a control for equal loading, ethidium bromide-stained large rRNA is shown in the bottom. The RNAi construct was introduced into cells via a multicopy vector (mc). (B) Protein and RNA were isolated from an AX2 strain carrying the G418 resistance gene under control of the actin 6 promoter (A6P-NPT) and from the discoidin RNAi strain (mcRNAi2) expressing the RNAi from the same promoter grown in axenic medium to a density of 2 × 106 cells/ml (I), grown in bacterial suspension to a density of 3 × 106 cells/ml (II), grown in bacterial suspension to a density of 3 × 106 cells/ml and then developed in shaking culture for 5 h (III), and grown in axenic culture to a density of 2 × 106 cells/ml after passage through bacterial growth and development for 5 h (IV). First line, cellular protein separated by SDS-PAGE, blotted, and probed for discoidin. Second line, Northern blot probed for discoidin mRNA. Third line, slot blot probed for NPT mRNA. Fourth line, ethidium bromide-stained large rRNA as a loading control.
Figure 3
Figure 3
(A) Schematic comparison of the Dictyostelium RrpA and DosA genes with RdRP genes from C. elegans and N. crassa and of a Dictyostelium dicer homologue gene with dicer from Drosophila and C. elegans. Shaded boxes exhibit at least 65% similarity to the Dictyostelium genes. Overall similarities to the corresponding Dictyostelium genes are given in percentages. The alignments are not exactly drawn to scale. Complete alignments are available as supplementary material on the web. Characteristic homology boxes in the RNaseIII domain (R), the helicase domain (H), and the RdRP domain (P) are marked; amino acid positions refer to the Dictyostelium proteins RrpA and dicer homologue: R1, LGDS domain, AA 907–969; R2, EALIG domain, AA 975–994; R3, DAVL domain, AA 1063–1103; H1, PRIL domain, AA 187–197; H2, VLEE domain, AA 441–500; P1, SGSD domain, AA 1306–1370; P2: SDQY domain, AA 1415–1454. (B) RT-PCR reactions with total RNA from AX2 wild type, RrpA−, and RrpB− cells. The resulting PCR products were separated on a 0.8% agarose gel before and after ClaI cleavage.
Figure 4
Figure 4
A discoidin Western blot is shown from four of six independent clones, each of the RrpA−, RrpB−, and DosA− transformants carrying the discoidin RNAi construct. Clones in lane 1 and 2 display complete silencing in RrpB− and DosA−, whereas clones in lanes 3 and 4 show only partial silencing. No significant reduction of discoidin levels was observed in RrpA− transformants.
Figure 5
Figure 5
(A) Low molecular weight RNAs from silenced and nonsilenced strains. Low molecular weight RNA was isolated from the strains indicated, blotted, and hybridized with a 32P-labeled β-gal sense probe. The position of ∼23mers is indicated by an arrowhead. (B) 32P-Labeled dsRNA was generated by in vitro transcription of a PSV-A gene fragment and incubated with a partially purified dsRNase preparation from Dictyostelium. Reaction products were separated by PAGE in parallel with small RNA isolated from RNAi-silenced β-gal cells. RNA was blotted and hybridized with a 32P-labeled β-gal probe. The blot was exposed for 1 wk (left) and over night (right).
Figure 6
Figure 6
In vitro assays with dsRNase preparations from various strains on a 32P-labeled in vitro prepared PSV-A dsRNA gene fragment and a 32P-labeled β-gal dsRNA fragment. Crude extract (350 μg) was incubated with 13 ng of PSV-A or 40 ng of β-gal dsRNA. ∼23mers are indicated by an arrow. ∼23mers were quantified from four independent assays by the TINA program (Raytest,). Averages are given in pixels per square millimeter below the lanes.
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