Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila

Abstract

RNA interference (RNAi) designates the multistep process by which double-stranded RNA induces the silencing of homologous endogenous genes. Some aspects of RNAi appear to be conserved throughout evolution, including the processing of trigger dsRNAs into small 21-23-bp siRNAs and their use to guide the degradation of complementary mRNAs. Two remarkable features of RNAi were uncovered in plants and Caenorhabditid elegans. First, RNA-dependent RNA polymerase activities allow the synthesis of siRNA complementary to sequences upstream of or downstream from the initial trigger region in the target mRNA, leading to a transitive RNAi with sequences that had not been initially targeted. Secondly, systemic RNAi may cause the targeting of gene silencing in one tissue to spread to other tissues. Using transgenes expressing dsRNA, we investigated whether transitive and systemic RNAi occur in Drosophila. DsRNA-producing transgenes targeted RNAi to specific regions of alternative mRNA species of one gene without transitive effect directed to sequences downstream from or upstream of the initial trigger region. Moreover, specific expression of a dsRNA, using either cell-specific GAL4 drivers or random clonal activation of a GAL4 driver, mediated a cell-autonomous RNAi. Together, our results provide evidence that transitive and systemic aspects of RNAi are not conserved in Drosophila and demonstrate that dsRNA-producing transgenes allow powerful reverse genetic approaches to be conducted in this model organism, by knocking down gene functions at the resolution of a single-cell type and of a single isoform.

FIGURE 1.

Structure of the UAS-IR constructs. (A) Strategy for generation of transgenic RNAi. A portion of the coding sequence of a gene is dimerized in a head-to-head orientation and placed in the pUAST expression vector under the control of UAS transcription elements. (B) Maps of the batman, batman-GFP, and EcR (Talbot et al. 1993) genes depict the arrangement of the exons. Exons common to all three EcR isoforms are numbered 3–6. Specific exons of the EcR isoforms are designated by greek letters. The positions of the cDNA fragments cloned in the UAS-IR constructs for dsRNA expression are indicated by black arrows. The position of the probes used for the RNase protection assays are indicated by solid black bars.

FIGURE 2.

Specific inactivation of batman and batman-GFP. (A) Analysis of small RNAs from da-GAL4/+ (control) and UAS-IR[batman]/da-GAL4 (UAS-IR[batman]) third instar larvae. (Left panel) siRNAs were analyzed by Northern blot using the bat probe (see Fig.1B ▶) of sense or antisense polarity as indicated. (Right panel) siRNAs were analyzed by RNase protection assay using the bat probe of sense polarity. (B) Western blot analysis of Batman in da-GAL4/+ (control) and UAS-IR[batman]/da-GAL4 mid-third instar larvae. Immunodetection of the MBF-1 protein was used as a loading control. (C) Analysis of small RNAs from da-GAL4, UAS-batman-GFP/+ (control) and da-GAL4, UAS-batman-GFP/UAS-IR[GFP] (UAS-IR[GFP]) third instar larvae. RNase protection assays were performed as indicated using the bat, gp1, and gfp2 probes (all of sense polarity, see Fig.1B ▶). (D) Western blot analysis of late-third larval instar extracts from control w¹¹¹⁸ larvae (WT), da-GAL4, UAS-batman-GFP/+ larvae (+), and da-GAL4, UAS-batman-GFP/UAS-IR[GFP] (UAS-IR[GFP]) larvae using an anti-Batman specific antibody (several independent UAS-IR[GFP] transgenic insertions were tested). The Batman-GFP protein (39 kD) was undetectable or strongly reduced, but the levels of the Batman (14 kD) and MBF-1 (16 kD) proteins remained unchanged. (E) GFP fluorescence in da-GAL4, UAS-batman-GFP/+ control larvae (left panel) and da-GAL4, UAS-batman-GFP/UAS-IR[GFP] larvae (right panel).

FIGURE 3.

Specific inactivation of EcR isoforms. (A) Pharate adult from a cross of the da-GAL4 driver line with the UAS-IR[EcR-A] construct line (left panel) and late-third instar larvae from a cross of the da-GAL4 driver line with the UAS-IR[EcR-B1] construct line (right panel). Arrowheads point to necrotic tissues. (B) Analysis of small RNAs from da-GAL4/+ (control), da-GAL4/UAS-IR[EcR-A] (UAS-IR[EcR-A]), and da-GAL4/UAS-IR[EcR-B1] (UAS-IR[EcR-B1]) third instar larvae. RNase protection assays were performed using the indicated sense probes (see Fig.1B ▶). (C) Western blot analysis of mid-third larval instar extracts from control da-GAL4/+ (+) and transgenic lines expressing the UAS-IR[EcR-A] and UAS-IR[EcR-B1] constructs. MBF-1 level was unchanged in the samples.

FIGURE 4.

UAS-IR[batman] induces cell-autonomous inactivation of the batman gene. (A) Confocal analysis of GFP (green) and Batman (red) expression in wing discs from en-GAL4, UAS-GFP/UAS-IR[batman] larvae (a) or in wing discs (b), eye-antennal discs (c), and leg discs (d) from dll-GAL4, UAS-GFP/UAS-IR[batman] larvae. Adult structures in control and inactivated flies are shown in the right panels. The size of the posterior compartment of the wings is reduced in en-GAL4, UAS-GFP/UAS-IR[batman] flies and contains a bubble indicative of abnormal adhesion between dorsal and ventral epithelial sheets (a). In dll-GAL4, UAS-GFP/UAS-IR[batman] flies, wings are abnormally curved (b), third antennal segments are reduced in size, unpigmented, and devoid of bristles (c, black arrow), the size of aristas is greatly reduced (c, white arrow), and the size of the bristles is greatly reduced on the legs (d). (B) Confocal analysis of GFP (green) and Batman (red) in FLP/FRT mediated cell clones coexpressing the UAS-GFP and UAS-IR[batman] transgenes. Clones are shown in a wing disc (a), the fat body (b), the gut (c), and the malpigian tubules (d).

FIGURE 4.

UAS-IR[batman] induces cell-autonomous inactivation of the batman gene. (A) Confocal analysis of GFP (green) and Batman (red) expression in wing discs from en-GAL4, UAS-GFP/UAS-IR[batman] larvae (a) or in wing discs (b), eye-antennal discs (c), and leg discs (d) from dll-GAL4, UAS-GFP/UAS-IR[batman] larvae. Adult structures in control and inactivated flies are shown in the right panels. The size of the posterior compartment of the wings is reduced in en-GAL4, UAS-GFP/UAS-IR[batman] flies and contains a bubble indicative of abnormal adhesion between dorsal and ventral epithelial sheets (a). In dll-GAL4, UAS-GFP/UAS-IR[batman] flies, wings are abnormally curved (b), third antennal segments are reduced in size, unpigmented, and devoid of bristles (c, black arrow), the size of aristas is greatly reduced (c, white arrow), and the size of the bristles is greatly reduced on the legs (d). (B) Confocal analysis of GFP (green) and Batman (red) in FLP/FRT mediated cell clones coexpressing the UAS-GFP and UAS-IR[batman] transgenes. Clones are shown in a wing disc (a), the fat body (b), the gut (c), and the malpigian tubules (d).

FIGURE 5.

UAS-IR[EcR-B1] and UAS-IR[Pcaf] induce cell-autonomous RNAi. (A) Fat body specific inactivation of EcR-B1 in late-third instar larvae. A Lsp2-GAL4 driver line that expresses GAL4 specifically in the third larval instar fat body was crossed with the w¹¹¹⁸ control line (upper panel) or a UAS-IR[EcR-B1] transgenic line (lower panel). EcR-B1 antibody staining (brown) of late-third larval instar tissues showed that EcR-B1 isoform was undetectable in the fat body (FB) but remained at the same level in salivary glands (SG) as well as other larval tissues (not shown). (B) Confocal analysis of GFP (green) and P/CAF (red) expression in leg discs (a), and wing discs (b) from distal-less-GAL4, UAS-GFP/UAS-IR[Pcaf] larvae.