HelF, a putative RNA helicase acts as a nuclear suppressor of RNAi but not antisense mediated gene silencing

Abstract

We have identified a putative RNA helicase from Dictyostelium that is closely related to drh-1, the 'dicer-related-helicase' from Caenorhabditis elegans and that also has significant similarity to proteins from vertebrates and plants. Green fluorescent protein (GFP)-tagged HelF protein was localized in speckles in the nucleus. Disruption of the helF gene resulted in a mutant morphology in late development. When transformed with RNAi constructs, HelF- cells displayed enhanced RNA interference on four tested genes. One gene that could not be knocked-down in the wild-type background was efficiently silenced in the mutant. Furthermore, the efficiency of silencing in the wild-type was dramatically improved when helF was disrupted in a secondary transformation. Silencing efficiency depended on transcription levels of hairpin RNA and the threshold was dramatically reduced in HelF- cells. However, the amount of siRNA did not depend on hairpin transcription. HelF is thus a natural nuclear suppressor of RNA interference. In contrast, no improvement of gene silencing was observed when mutant cells were challenged with corresponding antisense constructs. This indicates that RNAi and antisense have distinct requirements even though they may share parts of their pathways.

Figure 1

Protein alignment. Alignment of DEAH-box RNA helicase domains from HelF, RrpA (D.discoideum, DDB0191515, DictyBase, ), dcr-1 (C.elegans, NP_498761), DCL1 (A.thaliana, NP_171612) and drh-1 (C.elegans, NP_501018). Identical residues are marked in red, similar residues in blue.

Figure 2

Analysis of HelF⁻ strain. (A) Southern blot of genomic DNA, isolated from wild-type cells and from two HelF disruption clones (no. 5 and 6). DNA was digested with EcoRI and XbaI, separated on an agarose gel, blotted and hybridized with an oligo-labelled probe, resulting in labelled fragments of 7600 and 1037 bp in wild-type in the KO strains, respectively (arrows). (B) RT–PCR of total RNA isolated from wild-type and the HelF knock-out strain was carried out with an oligodT primer. PCR was done on the cDNA using the helF specific primers. 5′-CAATAAACTTTTATCAAATGGTG-3′ (5′ primer) and 5′-CTCTAAATTTTTAATTAAATTATAAATT-3′ (3′ primer). The expected product of 1.500 bp was only detected in the wild-type. As a control, primers for the thioredoxin gene were used on the same cDNA. Similar amounts of PCR products were obtained for cDNA of both strains and only the spliced trx fragment of ∼300 bp was detected, thus confirming the efficient removal of DNA contaminants. C1 and C2 are control PCR with helF and trx primers, respectively but without cDNA template.

Figure 3

HelF⁻ phenotype. Wild-type cells (A and B) and HelF⁻ cells (C–E) were grown in parallel on a lawn of K.aerogenes. Mutant slugs were oversized and aberrant (D and E). After prolonged development, the plaques formed by the mutant strain were covered with a network of dead stalk (C) originating from the long slug tails and a reduced number of mature fruiting bodies. Scale bars represent 0.25 mm.

Figure 4

Localization of HelF. (A) Fluorescence microscope image of a HelF-GFP cells; (a) GFP, (b) DNA stained with DAPI, (c) merge of (a) and (b). (B) Subcellular distribution of non-fusion GFP as a control. Scale bars represent 5 µm.

Figure 5

Silencing by RNAi. RNAi mediated silencing of discoidin (disci), coronin (cori), thioredoxin (trxi) and sp96 (sp96i) is summarized. Bars represent the fraction of clones (in %), showing different silencing efficiencies in wild-type and HelF⁻ background on the level of northern, western and colony blots. ‘Silenced’ was defined as 0 to 10%, ‘partially silenced’ as 10 to 50% and ‘non-silenced’ as more than 50% of the wild-type signal. For colony blots, partially silenced clones were not defined. n = number of clones examined.

Figure 6

Dicer activity is not enhanced in the helF knock-out strain. Dicer activity in cell free extracts of wild-type and helF knock-out strains was examined as described previously (36). A 600 bp dsRNA generated from in vitro transcripts of the discoidin gene was used as a substrate and increasing amounts of extract (1, 5, 10 and 20 µg of protein as determined by Bradford assay) were added. Incubation was for 60 min at room temperature. The dsRNA substrate and the 21mer products are indicated. The marker represents end-labelled DNA fragments (pGEM-3Z digested with Sau3A).

Figure 7

Silencing by asRNA. Antisense RNA mediated silencing of discoidin (discas), coronin (coras) and thioredoxin (trxas) is summarized. Bars represent the fraction of clones (in %), showing different silencing efficiencies in wild-type and HelF⁻ background on the level of northern, western and colony blots. ‘Silenced’ was defined as 0 to 10%, ‘partially silenced’ as 10 to 50% and ‘non-silenced’ as more than 50% of the wild-type signal. For colony blots, partially silenced clones were not defined. n = number of clones examined.

Figure 8

Retroactive silencing. A partially silenced clone (Disci9), transformed with the discoidin RNAi vector was supertransformed with the helF KO construct. After selection on blasticidin, the transformants (Disci9/HelF⁻) were assayed for discoidin expression on western blots (A). Expression in the Disci9 parent clone is shown as a control. Disruption of the helF gene was examined by PCR (B), the band of 1480 bp is indicative for a successful disruption. K- indicates the PCR control without template.

Figure 9

Transcriptional analysis of discoidin. (A) Northern blot analysis of discoidin steady-state expression in silenced (Disci1, Disci2, Disci9/HelF⁻and Disci10/HelF⁻), partially silenced (Disci9) and non-silenced (Disci10) clones, used for run-on assays. Ethidium bromide staining of the large rRNA was used as a loading control. (B) Nuclear run-on assays. The relative ratios of discoidin/actin transcription levels (×100) were calculated for each individual filter, hybridized to labelled run-on transcripts from the different clones. The filters were stripped and re-hybridized to wild-type run-on transcripts. The relative ratio of discoidin/actin15 was subtracted from the overall level of run-on transcripts in the mutant strains. The difference comprised the expression level of the discoidin hairpin construct. This calculation was required since the discoidin in vitro transcripts, applied on the filter, hybridized to both endogenous and hairpin run-on transcripts. Relative transcription levels of discoidin in wild-type cells (WT) are shown for a comparison. Silencing levels are indicated (s = silenced, p.s. = partially silenced, n.s. = non-silenced). (C) Northern blot analysis for discoidin siRNAs. Detection of siRNAs in silenced (s), partially silenced (p.s.) and non-silenced (n.s.) clones carrying the discoidin RNAi construct in the wild-type or HelF⁻ background. M: RNA decade marker (Ambion), shown after short exposure for better resolution. U6 spliceosomal RNA (A. Hinas, P. Larsson, L. Avesson, L.A. Kirsebom, A. Virtanen, F. Söderbom, manuscript submitted) is shown as a loading control.