C. elegans RNA-dependent RNA polymerases rrf-1 and ego-1 silence Drosophila transgenes by differing mechanisms

Abstract

Drosophila possesses the core gene silencing machinery but, like all insects, lacks the canonical RNA-dependent RNA polymerases (RdRps) that in C. elegans either trigger or enhance two major small RNA-dependent gene silencing pathways. Introduction of two different nematode RdRps into Drosophila showed them to be functional, resulting in differing silencing activities. While RRF-1 enhanced transitive dsRNA-dependent silencing, EGO-1 triggered dsRNA-independent silencing, specifically of transgenes. The strain w; da-Gal4; UAST-ego-1, constitutively expressing ego-1, is capable of silencing transgene including dsRNA hairpin upon a single cross, which created a powerful tool for research in Drosophila. In C. elegans, EGO-1 is involved in transcriptional gene silencing (TGS) of chromosome regions that are unpaired during meiosis. There was no opportunity for meiotic interactions involving EGO-1 in Drosophila that would explain the observed transgene silencing. Transgene DNA is, however, unpaired during the pairing of chromosomes in embryonic mitosis that is an unusual characteristic of Diptera, suggesting that in Drosophila, EGO-1 triggers transcriptional silencing of unpaired DNA during embryonic mitosis.

Fig. 1

Silencing of the endogenous gene, pebble, in Drosophila and detection of the primary as well as secondary siRNAs. a Expression of canonical C. elegans RdRps does not noticeably enhance RNAi-mediated silencing of the Drosophila endogenous gene, pebble. For activation of the dsRNA expression, flies harboring the pebble gene dsRNA construct pblRNAi ^4B(3.1) were crossed with the tissue-specific driver en-Gal4 in the presence (b–d) or absence (a) of one of the RdRps, RRF-1 or EGO-1. Compared with the control (a), noticeable changes in pebble gene silencing in either b or c were not observed; however, expression of higher levels of EGO-1 from the UAST vector gave a wild-type phenotype (d) through comparison with WT (e). b Northern hybridization did not detect any production of secondary siRNAs. Probe 1, designed based on the pebble targeted sequence (Fig. S1), successfully detected 1° siRNAs, while probe 2, designed based on the sequence upstream of the targeted sequence, did not detect any 2° siRNA production regardless of the presence of either of the RdRp genes. We used the RNA samples from UASP-ego-1 rather than UAST-ego-1 as da-gal4-activated UAST-ego-1 automatically silenced pblRNAi ^4B(3.1) and gave WT phenotype, i.e., could not suspend the development at pupae stage as occurred in UAST-rrf-1 and UASP-ego-1. c Secondary siRNAs detection with RNAse protection assays. RNAs was prepared from pupae. Lane 1: RNA marker, lane 2: positive control RNA; lanes 3 and 4 are controls for probe 3 with yeast or no RNA; lane 5 is control for probe 4 with RNAse digestion. Lanes 6–8 are the results for detection of 2° siRNAs upstream of the targeted pebble sequence (Fig. S1 and S2) with probe 3; da-Gal4-activated rrf-1 (lane 7) or ego-1 (lane 8) did not generate detectable 2° siRNAs. Lane 9: detection of the primary siRNAs with probe 4, lane 10: no detection in w ¹¹¹⁸

Fig. 2

C. elegans RdRp RRF-1 enhances dsRNA-triggered silencing of exogenous target genes, whereas silencing of exogenous genes by EGO-1 under the control of UAST is dsRNA-independent. Crosses for obtaining the larvae illustrated above are shown in Supplementary Methods S2, online supplement. Each cross was set up in at least for four replicates, and about 12 1° instar larvae from each cross were randomly selected and checked for consistency of the fluorescence signal; three of these were randomly selected for photography. a, h, and n are positive controls for fluorescence from NaChbac.eGFP, syt.eGFP, or Rab4-mRFP, respectively; b and i show a reduction of green fluorescence signals, indicating that the EGFP transgenes were partially silenced with the introduction of the corresponding EGFP dsRNA; c and j show a dramatic further reduction in the green fluorescence signal after combination with the RdRp gene, rrf-1, indicating a dramatic enhancement of silencing of the EGFP gene. Addition of the rrf-1 gene resulted in no silencing enhancement in the absence of the dsRNA trigger for either GFP [d, k or for the RFP line (o)]. In contrast, total silencing of fluorescent protein gene expression was observed in the presence of ego-1, irrespective of whether the EGFP dsRNA hairpin was present (e, l) or not (g, m); the RFP gene was also silenced totally in the absence of any dsRNA trigger (p). Expression of the EGFP-linked NaChBac gene resulted in suspended larval development at the 1° instar stage (k). f is the negative control from w¹¹¹⁸ under a GFP filter (using a Leica MZ11 fluorescence microscope); for the RFP gene a TXR filter was used

Fig. 3

Partial silencing of NaChBac:eGFP fusion gene releases suspension of larval development at the 1° instar larvae stage. a Left panel fluorescence images; right panel bright light images of the same larvae. Partial silencing of the EGFP-linked NaChBac gene by introduction of the EGFP dsRNA hairpin gene (1), allows larval development beyond the 1° instar stage whereas development is suspended in larvae expressing EGFP-linked NaChBac (2). Introduction of rrf-1 in the absence of the dsRNA hairpin had no effect on larval development (3). However, the partial silencing achieved by introduction of the EGFP dsRNA is insufficient for full development to adult stage. The green fluorescence signal is decreased (1) compared with no such silencing in 2 and 3. b Schematic model proposed for the genetic interactions observed for EGFP silencing. (i) The general driver da-Gal4 activates the NaChBac.eGFP fusion gene, resulting in the suspension of Drosophila development at the 1° instar stage. (ii) Activation by the general driver da-Gal4 of the EGFP dsRNA hairpin gene, as well as of the NaChBac.eGFP fusion gene, results in partial silencing of the latter and a reduction of the NaChBac.eGFP protein product, partially releasing the developmental arrest, and allowing some larvae to develop further, to the 2° instar stage. (iii) When RRF-1 was introduced, EGFP dsRNA-dependent silencing of the NaChBac.eGFP was dramatically enhanced, allowing larvae to go through full development towards the adult fly stage. In contrast, EGO-1 directly and completely silences the NaChBac.eGFP transgene, independent of the corresponding dsRNA, allowing full development of Drosophila to occur. c Schematic model proposed for the genetic interactions observed for pebble silencing. (i) The general driver da-Gal4 activates the pblRNAi ^4B(3.1)dsRNA construct, whose expression silences the pebble gene, resulting in development being suspended at the pupal stage. (ii) General activation of pblRNAi ^4B(3.1)as well as EGO-1 (in UAST), resulted in pblRNAi ^4B(3.1) expression being repressed by EGO-1, which in turn released pebble silencing imposed by pblRNAi ^4B(3.1) and subsequently led to full development

Fig. 4

Northern blot and hybridization for detection of the primary and secondary siRNAs in flies with the transgene egfp RNAi. A diagram illustrating the probes and target sequence is provided in Fig. S3 and S4. a Detection of the syt anti-sense 2° siRNAs with syt sense probe, compared with control (EGFP.dsRNA + syt.eGFP) without either rrf-1 or ego-1, in which the 2° siRNAs (blue) were produced by endogenous non-canonical RdRp activity, the 2° siRNA production in the presence of RRF-1 (red) was dramatically increased, while siRNA production in the presence of EGO-1 was dramatically reduced and difficult to see with naked eye (yellow), indicating an enhanced transitive RNAi pathway in the presence of RRF-1 but not EGO-1. The detection of syt sense 2° siRNAs in c is consistent with the results shown in a. In comparison, in b, EGFP anti-sense 1° siRNAs without RdRp (blue) and with RRF-1 (red) were detected at a similar signal strength, but production of these siRNAs was dramatically decreased (yellow) when EGO-1 was present, indicating an independent, direct silencing of the EGFP dsRNA hairpin transgene by EGO-1, which is consistent with the EGFP sense 1° siRNAs detection in d. In addition, independent silencing by EGO-1 was achieved not through producing 1° or 2° sense or anti-sense siRNAs (green boxes in all panels)

Fig. 5

Silencing by unpaired DNA mechanism. The crosses used to generate the flies are provided in the Supplementary Methods S3. The flies were checked for genotype and the consistency of EGFP expression/non-expression. Three representative flies were then selected for photography under the same conditions and the same fluorescence microscope parameter. EGFP was silenced in the heterozygous flies (a) but not in the homozygous ones (b)

Fig. 6

Methylation detection and rearrangement of the promoter region of the transgenes. a Southern hybridization detected the cutting pattern unchanged in the EGFP coding region, and altered in the transgenes’ promoter regions, respectively. DNA samples in lane 1: w; da-Gal4/+; UAST-syt.eGFP/+ cut with HpaII; lane 2: w; da-Gal4/+; UAST-syt.eGFP/+ cut with HhaI; lane 3: w; da-Gal4/+; UAST-syt.eGFP/T-ego-1 cut with HpaII; lane 4: w; da-Gal4/+; UAST-syt.eGFP/T-ego-1 cut with HhaI. The cutting pattern in EGFP coding region was not affected by expression of the EGO-1 gene (left); in contrast; the pattern in transgenes’ promoter regions was altered by the co-expression of EGO-1 (right). b Bisulfite sequencing analysis in the promoter regions of the transgenes in the presence or absence of EGO-1. Sample S4: syt.eGFP only without ego-1, S12, S20: Syt.eGFP with ego-1, S29, S33, S34: ego-1 only. “N stands for any nucleotide and “H” is either A, C, or T