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. 2011 May;39(9):3836-51.
doi: 10.1093/nar/gkq1324. Epub 2011 Jan 17.

Loqs-PD and R2D2 define independent pathways for RISC generation in Drosophila

Julia V Hartig  1 , Klaus Förstemann
Affiliations

Loqs-PD and R2D2 define independent pathways for RISC generation in Drosophila

Julia V Hartig et al. Nucleic Acids Res. 2011 May.

Abstract

In Drosophila, siRNAs are classified as endo- or exo-siRNAs based on their origin. Both are processed from double-stranded RNA precursors by Dcr-2 and then loaded into the Argonaute protein Ago2. While exo-siRNAs serve to defend the cell against viruses, endo-siRNAs restrict the spread of selfish DNA in somatic cells, analogous to piRNAs in the germ line. Endo- and exo-siRNAs display a differential requirement for double-stranded RNA binding domain proteins (dsRBPs): R2D2 is needed to load exo-siRNAs into Ago2 while the PD isoform of Loquacious (Loqs-PD) stimulates Dcr-2 during the nucleolytic processing of hairpin-derived endo-siRNAs. In cell culture assays, R2D2 antagonizes Loqs-PD in endo-siRNA silencing and Loqs-PD is an inhibitor of RNA interference. Loqs-PD can interact via the C-terminus unique to this isoform with the DExH/D-helicase domain of Drosophila Dcr-2, where binding of R2D2 has also been localized. Separation of the two pathways is not complete; rather, the dicing and Ago2-loading steps appear uncoupled, analogous to the corresponding steps in miRNA biogenesis. Analysis of deep sequencing data further demonstrates that in r2d2 mutant flies, siRNAs can be loaded into Ago2 but not all siRNA classes are equally proficient for this. Thus, the canonical Ago2-RISC loading complex can be bypassed under certain circumstances.

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Figures

Figure 1.
Figure 1.
Dcr-2 interacts with the C-terminal 22 amino acids specific to Loqs-PD. (A) Top panel: Co-immunoprecipitation of endogenous Dcr-2 with GFP-fusion constructs in S2-cells; bottom panel: α-GFP western blot to determine expression levels and successful immunoprecipitation. Fusion of the Loqs-PC specific amino acids to GFP reduce the expression levels; this appears to be generally the case with GFP-PC constructs (see also Figure 2A). (B) Effect of Loqs isoform overexpression on GFP expression levels of the endo-siRNA cell culture reporter; protein isoforms are depicted on the right (dsRBDs are represented by symbols; Loqs-PD-specific sequence is colored in grey); measurement values represent the mean ± SD (n = 3) and were normalized to a pUC18 transfected control (dashed line).
Figure 2.
Figure 2.
Loqs-PD interacts with the N-terminal helicase domain of Dcr-2 for endo-siRNA silencing. (A) Co-immunoprecipitation from Drosophila S2 cell extract co-expressing GFP-fusion proteins together with either Flag-Dcr-2 or Δhel-Flag-Dcr2; the GFP proteins in this experiment also contained an N-terminal myc-tag that was used for detection, but GFP-trap beads were used for immunoprecipitation. (B) Co-immunoprecipitation from Drosophila S2 cell extract co-expressing myc-tagged Loqs-PD, R2D2 or a pUC18 control together with either Flag-Dcr-2 or Δhel-Flag-Dcr-2; α-Flag agarose was used for IP; myc-GFP served as a control.
Figure 3.
Figure 3.
R2D2 acts as an inhibitor of Loqs-PD in endo-siRNA silencing. (A) Co-immunoprecipitation with Drosophila S2 cell extract prepared after transfection of myc-tagged Loqs isoforms or myc-tagged R2D2; α-Loqs-PD-specific antibody was used to immunoprecipitate endogenous Loqs-PD. The white asterisk marks a faster migrating, myc-tag containing band in the lanes from myc-Loqs-PB transfected cells. This band is found consistently but to a varying extent in transfections with this construct (14); it likely represents a degradation product of the overexpressed Loqs-PB. (B) Northern blot using RNA extracted from Drosophila S2 cells after RNAi treatment with combinations of Loqs isoforms and R2D2; DsRed dsRNA served as a control for RNAi; a DNA probe against the long hairpin-derived endo-siRNA CG4068B (66) and 2′-OMe oligonucleotide probes against bantam miRNA were used; 2S rRNA served as a control for loading. (C) Northern blot using RNA isolated from flies carrying mutant alleles of loqs and r2d2. Note that the particularly strong expression of CG4068B endo-siRNA in r2d2/CyO flies is consistent with deep sequencing data obtained from an independent RNA preparation from the same fly strain (Table 1). (D) Effect of RNAi treatment with combinations of Loqs isoforms and R2D2 on GFP expression of the endo-siRNA cell culture reporter; double-stranded RNA directed against DsRed served as a control, RNAi triggers are indicated below the bars; measurement values represent the mean ± SD (n = 3) and were normalized to a DsRed/DsRed control (dashed line); asterisk: P < 0.005 (two-tailed t-test, unequal variance). (E) Effect of R2D2 and Loqs-PD on exo-siRNA mediated silencing; RNAi triggers are indicated below the bars. GFP fluorescence was calculated relative to a control treatment with dsRNA directed against luciferase during the second RNAi step. The dashed line marks GFP fluorescence levels under control conditions (dsRNA against luciferase, followed by dsRNA against GFP); measurement values represent the mean ± SD (n = 3); *P < 0.02, **P < 0.005 (two-tailed t-test, unequal variance).
Figure 4.
Figure 4.
Analysis of silencing triggered by a white-IR transgene. (A–D) Side-by-side comparison of the eye color obtained for the indicated genotypes; (E) Photometric analysis of extracted eye pigments; Oregon R (OR) and w1118 flies were measured as reference points. The genotypes were:

  1. P{wIR}/yw; loqsko/CyO; P{loqs-PB}/+

  2. P{wIR}/yw; loqsko/loqsf00791; +/+

  3. P{wIR}/yw; loqsko/loqsf00791; P{loqs-PB}/+

  4. P{wIR}/yw; loqsko/loqsko; P{loqs-PB}/+

  5. yw/yw; loqsko/loqsko; P{loqs-PB}/+

(note that the P{wIR} transgene is carried on an X-chromosome with a wild-type w gene; sample 5 is therefore a slightly underestimated reference point).
Figure 5.
Figure 5.
Dominant-negative effects on endo-siRNA biogenesis by overexpression of Loqs isoforms. (A) Detection of endogenous Loqs protein from S2-cell extract after immunoprecipitation with α-Dcr-1, α-Loqs-PB C-terminus and α-Loqs-PD; rb IgG and α-R2D2 served as controls; α-Loqs monoclonal antibody was used for detection of endogenous Loqs isoforms. (B) Northern blot from Drosophila S2 cell extract overexpressing Loqs isoforms; transfection with pUC18 served as a control; L1L2 = Loqs truncation lacking the PD-specific amino acid sequence, L1L2:L3PC = reconstituted Loqs-PC; myc-loqs-PD (genomic) = expression construct derived from genomic DNA; DNA probes against the long hairpin-derived endo-siRNA CG4068B (66) and against bantam miRNA were used; 2S rRNA served as a control for loading; the two parts were extracted from a single blot using the same exposure and image processing settings.
Figure 6.
Figure 6.
Analysis of strand asymmetry and read length distribution in deep sequencing data. (A) We calculated the difference in the free energy of duplex formation at either end of the presumed siRNA precursor for each sequence read using the nearest neighbor method (52), then calculated the difference (ΔΔG0′). A positive value indicates that on average the 5′-ends of the reads were less stably base paired than the opposite ends. The thermodynamic asymmetry was calculated for transposon-matching endo-siRNAs of the indicated genotypes; n.a.: not available in the series. (B) Read length distribution of transposon-matching endo-siRNAs in deep sequencing libraries from r2d2/CyO, r2d2/r2d2 and ago2414/ago2414 mutant fly heads. Left side: sequencing libraries from untreated RNAs; right side: sequencing libraries prepared from oxidized RNA to enrich for 2′-O-methyl modified RNA species. (C) Calculation of the thermodynamic asymmetry for white-IR derived siRNAs from the indicated genotypes.
Figure 7.
Figure 7.
Distinct biogenesis pathways for exo- and endo-siRNAs. Loqs-PD and R2D2 both contain two N-terminal double-stranded RNA binding domains (labeled 1 and 2) and associate via their C-terminal domain with the N-terminal helicase domain of Dcr-2. This is analogous to the situation of Dcr-1/Loqs-PB and of human Dicer/TRBP or PACT. Despite the similar architecture, the two Drosophila complexes are functionally distinct: Our results indicate that R2D2 and Loqs-PD compete for association with Dcr-2, and that parallel pathways for processing and loading of siRNAs exist. Analogous to the situation for miRNA biogenesis, they appear to be uncoupled after the dicing step. Thus, it is possible that a siRNA is processed by Dcr-2/Loqs-PD and loaded by Dcr-2/R2D2, but this sequence of events is not obligatory. R2D2 may not be required for the dicing reaction; since competition for Dcr-2 exists we propose that the presence of “free” Dcr-2 unlikely. Two recent publications demonstrate that endogenous siRNAs can also be loaded into Ago1 to some extent (49,50).
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