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As shown in Figure 14B,overexpression of other Loqs isoforms leads to impaired endo-siRNA silencing. By transfecting endo-siRNA reporter cells with increasing amounts of Loqs isoforms or only the Loqs-PB C-terminus (encompassing the third dsRBD of Loqs-PB), I tested for a correlation between the amount of transfected expression plasmid and an increase in GFP-expression (Figure 19A).The expression of the Loqs-PB C-terminus, which is responsible for Dcr-1 interaction, did not have a significant influence on the reporter, nor did Loqs-PD. In contrast, I saw a strong correlation between the quantity of transfected Loqs-PA or -PB with impaired endo-siRNA silencing. The effect of Loqs-PC was considerably weaker; this may however be due to lower expression levels of the construct (see Appendix 4). The de- repression of the reporter was not influenced by the epitope tag, since transfection with Flag-myc-tagged constructs caused a comparable effect in the endo-siRNA cell culture reporter (Figure 19B). Moderate overexpression levels of Loqs-PD here even lead to hyperrepession of GFP levels. Could the dominant-negative effect be explained by multimerization of overexpressed Loqs isoforms together with endogenous Loqs-PD and the formation of dsRBP complexes unsuitable for silencing? I overexpressed Flag-myc-Loqs-PB together with myc-tagged Loqs isoforms or R2D2 and analyzed the bound fraction after α- Flag-IP (Figure 19C, left panel). While recovery of Loqs-PA/-PB/-PC was high, Loqs-PD associated only to a moderate extent with Loqs-PB, and R2D2 was not detected in the bound fraction at all. The result indicates that overexpressed Loqs isoforms can oligomerize, but that R2D2 does not form a complex with Loqs-PB. Analogous co-immunoprecipitation from cells expressing the Flag-myc-Loqs-PDgenomic construct yielded a barely detectable amount of R2D2 in the bound fraction, as well as considerable Loqs-PA and -PC recovery (Figure 19C, right panel; Loqs-PB association was not quantifiable, since myc-Loqs-PB and Flag-myc-Loqs- PD migrate together).

To further analyze the association of myc-tagged Loqs variants with Loqs-PD I used the Loqs- PD-specific antibody to precipitate endogenous Loqs-PD (see Figure 17A, bottom panel). The result confirmed the finding that an interaction of Loqs-PD with all other isoforms as well as R2D2 in Drosophila S2 cells is at least possible. However, these interactions may have been forced by overexpression and might be indirect. I therefore tested for oligomerization of Loqs-PD and other Loqs-isoforms at endogenous levels by immunoprecipitation with our

Loqs-PD specific antibody, followed by Western blotting with α-Loqs monoclonal antibody. This did not reveal any significant recovery of Loqs-PB or Loqs-PA associated with Loqs-PD (Figure 19D, α-Dcr-1 IP was used as a control to mark Loqs-PB precipitation). Taken together, my experiments indicate that endogenous Loqs-PD does not form complexes with other Loqs isoforms to a significant extent but can be induced to do so upon overexpression.

If overexpression of other isoforms impairs endo-siRNA silencing on the level of Loqs- PD/Dcr-2 association, then endo-siRNA biogenesis should be disturbed. Loqs-PD overexpression from both cDNA- and genomic DNA-derived expression constructs did not change the levels of the long hairpin-derived endo-siRNA CG4068 B. Expression of full-length myc-Loqs-PB/-PA or Loqs-PC (either full-length or reconstituted L1L2+PCspec) led to a reduction of mature CG4068 B(Figure 19E).In accordance with my reporter cell experiments (Figure 14B, C) truncated Loqs, lacking only the PD-specific amino acid sequence (L1L2), caused the same impairment of long hairpin endo-siRNA biogenesis as the Loqs-PC isoform (Figure 19E). In summary, multimerization induced by overexpression of individual Loqs isoforms perturbs the balance of the other, endogenous dsRBD-proteins and impairs the function of the small RNA silencing system.

(legend Figure 19 continued)

A) Correlation of GFP fluorescence increase in the endo-siRNA cell culture reporter with the amount of transfected plasmid; the transfected myc-tagged construct is indicated below the bars; cells were transfected with 10 ng, 25 ng, 50 ng, 75 ng and 100 ng of the respective expression plasmid; the values were normalized to a pUC18 control (red line); Loqs-PB C-term. = expression construct for the 3rd dsRBD of Loqs-PB

B) Effect of Flag-myc-tagged Loqs-isoforms on the endo-siRNA cell culture reporter; transfected plasmids are indicated below the bars; mock-transfection (red horizontal line) and an expression construct for the Flag- tag only served as controls; measurement values represent the mean ± SD (n=3)

C) Left panel: α-myc Western blot after immunoprecipitation of Flag-myc-Loqs-PB from Drosophila S2 cell extracts co-expressing myc-Loqs isoforms or myc-R2D2; α-Flag antibody was used for IP; myc-GFP served as a control;

Right panel:α-myc Western blot after immunoprecipitation of Flag-myc-Loqs-PD from Drosophila S2 cell extracts co-expressing myc-Loqs isoforms or myc-R2D2; α-Flag antibody was used for IP; myc-GFP served as a control; note that Flag-myc-Loqs-PD co-migrates with myc-Loqs-PB during SDS-PAGE;

bottom panels show a longer exposure to detect faint R2D2 bands; red arrow indicates co-precipitated R2D2

D) 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

E) 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+PCspec = reconstituted Loqs-PC; myc-loqs-PD (genomic) = expression construct derived from genomic DNA; DNA probes against the long hairpin-derived endo-siRNA CG4068 B (Okamura et al., 2008c) and against bantam miRNA were used; 2S rRNA served as a control for loading