Inhibition of RNA interference and modulation of transposable element expression by cell death in Drosophila

Abstract

RNA interference (RNAi) regulates gene expression by sequence-specific destruction of RNA. It acts as a defense mechanism against viruses and represses the expression of transposable elements (TEs) and some endogenous genes. We report that mutations and transgene constructs that condition cell death suppress RNA interference in adjacent cells in Drosophila melanogaster. The reversal of RNAi is effective for both the white (w) eye color gene and green fluorescent protein (GFP), indicating the generality of the inhibition. Antiapoptotic transgenes that reverse cell death will also reverse the inhibition of RNAi. Using GFP and a low level of cell death produced by a heat shock-head involution defective (hs-hid) transgene, the inhibition appears to occur by blocking the conversion of double-stranded RNA (dsRNA) to short interfering RNA (siRNA). We also demonstrate that the mus308 gene and endogenous transposable elements, which are both regularly silenced by RNAi, are increased in expression and accompanied by a reduced level of siRNA, when cell death occurs. The finding that chronic ectopic cell death affects RNAi is critical for an understanding of the application of the technique in basic and applied studies. These results also suggest that developmental perturbations, disease states, or environmental insults that cause ectopic cell death would alter transposon and gene expression patterns in the organism by the inhibition of small RNA silencing processes.

Figure 1

Cell death-dependent inhibition of RNAi silencing of the white gene. (A) At top left, a wild-type (WT) eye with w⁺ is red. The yellow eye (second from top left) indicates that w is silenced by RNAi when the transgene GMR-wIR is present in the genome. Heterozygotes of the noted dominant mutations and the homozygote for the recessive genes were combined with a copy of GMR-wIR. Silencing is partially reversed in eye mutations B, Dr, Gla, ro, and hh^bar3, but not in the mutation Roi (amos^Roi-1). Acetamine treatment of the Bar mutant [B (acetamine)] reduces cell death in the eyes accompanied by reduced color restoration. (B) Induced cell death in the eyes by ectopic expression of grim, hid, rpr, strica, and ttk (all driven by the GMR promoter) caused reversal of RNAi. The first row shows the phenotype of the noted cell death transgenes with wild type w⁺. The second row shows the transgenes combined with GMR-wIR. The red color indicates inhibition of RNAi. The third and fourth rows show the cell death transgenes and GMR-wIR combined with two different inhibitors of cell death as noted. To the extent that cell death is reversed, RNAi is restored. (C) EGFP RNAi is inhibited by cell death. Cell death induced by ectopically expressed grim, strica, and ttk restored the fluorescence to most regions, if not the whole eye. In each comparison, control eyes with GFP silenced but without induced cell death are shown on the upper left. Bars, 0.1 mm.

Figure 2

Cell death caused RNAi inhibition in neighboring cells. Mosaic cell death of GMR-ttk and GMR-hid generated by mitotic recombination. (A). The color is restored by the GMR-ttk cells. Selected examples of sectors of ttk cell groups are circled by black lines, which were indicated by the smooth surfaced areas. Because the GMR-ttk/+ genotype has a dominant phenotype (Figure 1B), the normal sectors are those in which mitotic recombination during development have resolved into +/+ sectors. These wild-type cells show reversal of RNAi when adjacent to GMR-ttk sectors. Surrounding two examples of the death areas, the color that is restored in normal cells is circled by white lines. The strength of inhibition diminishes with the distance from the ttk sectors. (B) Death in homozygous GMR-hid cells did not cause inhibition of w RNAi silencing. Mosaic cell death of GMR-grim (C) and GMR-rpr was generated by mitotic recombination (D). In these cases, w RNAi was occasionally reversed as indicated by the red sectors in the eyes. Bars, 0.1 mm.

Figure 3

Cell death-related inhibition of RNAi occurs in different tissues and organs and the increased expression of the marker gene EGFP is caused by impaired processing of dsRNA into siRNA. (A–F). The default expression of the transgene hs-hid inhibits w silencing in the eye (A) and EGFP silencing in larvae (B), wing disc (C), eye-antennal disc (D), midgut (E), and salivary glands (F). In each image except the eye, controls without hs-hid but with EGFP RNAi are located to the right. A salivary gland showing restored fluorescence and a control pair of glands is shown in the lower right. Bars, 0.1 mm. (G–I). Northern blotting detects altered levels of EGFP mRNA, dsRNA (G), and siRNA (H) in the hs-hid strain. A dilution series (1X, 2X, and 4X) of total RNA or total small RNA was loaded and indicated by the numbers of micrograms. Levels of α-tubulin (Tub) mRNA (G), and 5S rRNA (H) were probed, respectively, as input controls. The patterns of siRNA were not different when probed with either sense or antisense EGFP probes (not shown). Statistical analysis of the EGFP mRNA, dsRNA, and siRNA levels in “hs-hid; EGFPir” larvae is shown in I. Northern results were scanned and analyzed by Image Gauge. The fold difference compared to the samples without hid (“EGFPir”) was normalized by the loading control. The heights of solid bars with error bars indicate the fold difference; *P < 0.05 (no cell death), **P < 0.01, ***P < 0.001.

Figure 4

Cell death upregulates the expression of the endogenous gene mus308 and transposable elements and reduces the siRNA level. (A) mus308 mRNA amount was doubled in the hs-hid strain, as detected by real-time PCR and semiquantitative PCR. The fold difference compared to the samples without hid (“EGFPir”) was normalized by β-tubulin. As noted in the text, the magnitude in affected cells is undoubtedly greater. Levels of statistical significance are designated as described in Figure 2. (B) Expression of transposons 1731, mdg1, 297, BEL, DOC, and S elements in “hs-hid EGFPir” larvae. Semiquantitative RT–PCR results were analyzed by Image Gauge. The fold difference compared to the samples without hid (“EGFPir”) was normalized by the loading control. (C) Northern results show let-7 level remained unchanged but the endogenous esiRNA-sl-1 was reduced to ∼45% in the hs-hid strain. The statistical results for triplicate experiments are shown below. *P < 0.05 (no cell death), **P < 0.01, ***P < 0.001. (D) Distribution of small RNAs matching TEs from deep sequencing data shows a >50% reduction of the 21-nt length in the cell death strain (EGFPir hs-hid) compared to the control. The read number for each sample was normalized by the miRNA counts. We also used 2S rRNA, which is 30 nt long and included in our sequencing data, to normalize the counts with similar results. Biological replications are indicated by #1 and #2.