Validating RNAi phenotypes in Drosophila using a synthetic RNAi-resistant transgene

Abstract

RNA interference (RNAi) is a powerful and widely used approach to investigate gene function, but a major limitation of the approach is the high incidence of non-specific phenotypes that arise due to off-target effects. We previously showed that RNAi-mediated knock-down of pico, which encodes the only member of the MRL family of adapter proteins in Drosophila, resulted in reduction in cell number and size leading to reduced tissue growth. In contrast, a recent study reported that pico knockdown leads to tissue dysmorphology, pointing to an indirect role for pico in the control of wing size. To understand the cause of this disparity we have utilised a synthetic RNAi-resistant transgene, which bears minimal sequence homology to the predicted dsRNA but encodes wild type Pico protein, to reanalyse the RNAi lines used in the two studies. We find that the RNAi lines from different sources exhibit different effects, with one set of lines uniquely resulting in a tissue dysmorphology phenotype when expressed in the developing wing. Importantly, the loss of tissue morphology fails to be complemented by co-overexpression of RNAi-resistant pico suggesting that this phenotype is the result of an off-target effect. This highlights the importance of careful validation of RNAi-induced phenotypes, and shows the potential of synthetic transgenes for their experimental validation.

Figure 1. Phenotypic effects of pico RNAi constructs.

A) Gene map showing long (pico-RA) and short (pico-RB) transcripts. Untranslated regions are shown with hatched boxes, coding exons are shown with open boxes. pico-RA and pico-RB share identical 3′ exons but differ at their 5′ ends. The position of oligonucleotide primers used to amply either pico-RA, pico-RB or both pico transcripts (for analysis of expression levels shown in panel B) are indicated with arrows. Magnified image of one of the coding exons shows the position of the inverted repeat constructs: picoRNAi^R2/picoRNAi^R3 (grey rectangle), picoRNAi^IR4 (black rectangle). B) Ectopic expression of inverted repeat contructs using MS1096-GAL4 results in knockdown of pico mRNA levels in the wing imaginal disc. RNA was extracted from wing imaginal discs that had been dissected from larvae expressing UAS-picoRNAi^IR4 (MS1096>picoRNAi^IR4), or UAS-picoRNAi^R2 and UAS-picoRNAi^R3 (MS1096>picoRNAi^{R2, R3}), and was analysed by qRT-PCR. Levels of picoRA, picoRB and all pico transcripts are shown as a percentage of the expression in wing discs from a control strain (w¹¹¹⁸). C) Phenotypic effect of ectopic inverted repeat contructs on wing development. Flies carrying UAS-inverted repeat constructs alone (picoRNAi^IR4 or picoRNAi^R2 and picoRNAi^R3) resemble wild type wings. Ectopic overexpression of picoRNAi^IR4 (MS1096>picoRNAi^IR4) resulted in a significant reduction of adult wings size (p<0.001) without any loss of morphology. In contrast ectopic picoRNAi^R2, picoRNAi^R3 (MS1096>picoRNAi^R2, picoRNAi^R3) resulted in severe loss of wing morphology as evidenced by crumpled wings. D) Ectopic co-overexpression of picoRNAi^R2 and picoRNAi^R3 using MS1096-GAL4 results in the knockdown of at least 4 predicted off-targets as determined by qRT-PCR. Levels of the predicted off targets (CG32082, CG31753, CG7467, CG31160, CG6369) in MS1096>picoRNAi^R2,R3 wing discs are shown as a percentage of the expression in wing discs from a control strain (w¹¹¹⁸).

Figure 2. Sequence comparison of wild type and synthetic pico genes.

Shown is a sequence alignment of the first 493 bp of the synthetic pico transgene (in black) and the equivalent region of the endogenous pico gene (in red). Identical bases in the two sequences are highlighted in yellow. The regions corresponding to picoRNAi^IR4 is underlined. The region overlapping with that of picoRNAi^R2/R3 is in italics.

Figure 3. A synthetic RNAi-resistant pico transgene rescues the phenotypic effect of ectopic picoRNAi^IR4 but not picoRNAi^R3.

A) Expression of a synthetic, Venus-tagged, pico transgene is not affected by co-expression of picoRNAi constructs. Confocal images of wing imaginal discs from flies expressing UAS-Venus-pico^r alone (MS1096>Venus-pico^r), or together with UAS-picoRNAi constructs (MS1096>Venus-pico^r, picoRNAi^R3 or MS1096>Venus-pico^r, picoRNAi ^IR4) are shown. DNA staining with TO-PRO-3 (magenta in the merged image) reveals that each image is of a similar section through the disc, whilst the Venus signal (green in the merged image) reveals that the levels of Venus-pico^r are largely unaffected by co-expression with the pico inverted repeat constructs. B) Ectopic expression of Venus-pico^r rescues the growth defect resulting from picoRNAi^IR4, but not the wing dysmorphology phenotype displayed by picoRNAi^R3. Ectopic Venus-pico^r under the control of MS1096-GAL4 (MS1096>Venus-pico^r) results in an increase in wing size. Ectopic picoRNAi^R3 (MS1096> picoRNAi^R3) results in a crumpled wing (similar to the effect of co-expressing picoRNAi^R2 and picoRNAi^IR3, as in Figure 1C), and this is not suppressed by Venus-pico^r (MS1096>Venus-pico^r, picoRNAi^R3). In contrast, coexpression of Venus-pico^r and picoRNAi^IR4 (MS1096>Venus-pico^r, picoRNAi^IR3) resembles the effect of Venus-pico^r alone.