Inhibition of RNA Interference and Modulationof Transposable Element Expression by Cell Death
in DrosophilaWeiwu Xie,* Chengzhi Liang,†,1 and James A. Birchler*,2
*Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211, yCold Spring Harbor Laboratory,Cold Spring Harbor, New York 11724
ABSTRACT RNA interference (RNAi) regulates gene expression by sequence-specific destruction of RNA. It acts as a defensemechanism against viruses and represses the expression of transposable elements (TEs) and some endogenous genes. We report thatmutations 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 ofthe inhibition. Antiapoptotic transgenes that reverse cell death will also reverse the inhibition of RNAi. Using GFP and a low level of celldeath produced by a heat shock-head involution defective (hs-hid) transgene, the inhibition appears to occur by blocking theconversion of double-stranded RNA (dsRNA) to short interfering RNA (siRNA). We also demonstrate that the mus308 gene andendogenous transposable elements, which are both regularly silenced by RNAi, are increased in expression and accompanied bya reduced level of siRNA, when cell death occurs. The finding that chronic ectopic cell death affects RNAi is critical for an understandingof the application of the technique in basic and applied studies. These results also suggest that developmental perturbations, diseasestates, or environmental insults that cause ectopic cell death would alter transposon and gene expression patterns in the organism bythe inhibition of small RNA silencing processes.
RNA interference (RNAi) uses double-stranded RNA(dsRNA) to target the destruction of the homologous
messenger RNAs via short interfering RNAs (siRNA)(Zamore and Haley 2005). In Drosophila, dsRNA can begenerated by transcribing a sequence from both directionsor from inverted repeats. The ribonuclease Dicer-2 processesthe dsRNA into �21-nt siRNA. Single-stranded siRNA isthen incorporated into the RNA-interference silencing com-plex (RISC) and the latter recognizes and cleaves a targetRNA, which perfectly or nearly perfectly matches the siRNA.This targeted degradation mechanism has been demon-strated as an immunity defense against viruses that havedsRNA in their life cycle (Li and Ding 2005). Discovery of en-dogenous siRNAs homologous to transposable elements (TEs)also suggests this siRNA pathway plays a role in suppressing
transposition (Chung et al. 2008; Czech et al. 2008; Ghildiyalet al. 2008; Kawamura et al. 2008; Tam et al. 2008; Watanabeet al. 2008) in addition to the PIWI-associated small RNA(piRNA) pathway (Aravin et al. 2007; Brennecke et al. 2007).
Cell death is a central part of the immune system of manymulticellular organisms. Cells infected with pathogens cantrigger the apoptotic pathway and cell death to prevent thepathogens from spreading (Postigo and Ferrer 2010). Sucha mechanism is also used to remove damaged cells or extra cellsunused in tissue formation. When diseased or damaged cells areremoved, proliferation signals are generated from the dying cellsto the adjacent cells to stimulate compensatory cell divisions(Huh et al. 2004; Pérez-Garijo et al. 2004; Ryoo et al. 2004).
Deep sequencing of endogenous siRNAs reveals thata significant proportion of them are derived from transcrip-tion of certain sequences from opposite directions, ofhairpin-structured sequences, or of homologous sequences(e.g., genes and their pseudogenes) from which comple-mentary sense and antisense sequences are generated(Chung et al. 2008; Czech et al. 2008; Ghildiyal et al.2008; Kawamura et al. 2008; Okamura et al. 2008; Tamet al. 2008; Watanabe et al. 2008). These siRNAs have been
demonstrated to match important genes and their expres-sion is repressed in oocytes of mice and somatic cells of flies,thus implying a genome-wide regulation role by RNAi.Among these genes, the endogenous mus308 gene in Dro-sophila increases in expression when RNAi is compromised(Czech et al. 2008; Okamura et al. 2008).
Here we report that cell death in Drosophila, inducedeither by apoptotic genes or caused by developmentaldefects, inhibits RNAi. We also demonstrate that cell deathsuppresses the silencing of mus308 and transposable ele-ments. The increased expression is accompanied by siRNAreduction and dsRNA accumulation, suggesting that the pro-cessing of dsRNA to siRNA is impaired.
Materials and Methods
Strains, genetic tests, and microscopy
All eye images were obtained using a dissecting microscopewith ·4 magnification with an attached digital camera. Tento 30 flies of the same genotype were observed and repre-sentative flies photographed.
The RNAi strains with homozygous GMR-wIR insertionson chromosomes X or 3 (Lee et al. 2004) were kindly pro-vided by R. Carthew, Northwestern University, Evanston, IL.These strains were crossed to the multiple balancer strainBasc/Basc; In(2LR)SM1, al2 Cy cn2 sp2/In(2LR)bwV1, ds33K
dpOV bwV1; In(3LR)Ubx130, Ubx130 es/In(3LR)C, Sb; svspa-pol.The eye phenotype was recorded in the F1 with Bar and inthe F2 with svspa-pol/svspa-pol. Also a male with GMR-wIR andB was recovered. A GMR-wIR B strain was generated andtested to confirm that the X chromosome carried w+, B,and GMR-wIR by recombination with a regular w2 X chro-mosome. The Bar effect in the males was also observed byusing another strain y/BsY; tra2ts2 bw/CyO (From B. Taylorat Oregon State University, Corvallis, OR), which carries theBar mutation on the Y chromosome.
The GMR-wIR / B larvae were treated with acetamine asdescribed (Fristrom 1972).
The RNAi stocks were crossed to the following strains PrDr/TM3; Sco/In(2L)Cyt, Cy amosRoi-1; P{w+mc.hs = GawB}elavc155 w* P{ry+t7.2 = neo FRT} 19A; Bc1 EgfrE1/CyO andbic L2/CyO. The eye phenotype of the F1 withDr, Roi (amosRoi-1),Egfr or L combined with GMR-wIR was documented. Thestrain w+; Gla/SM6a; TM3, Ser/MKRS was crossed to astrain carrying GMR-wIR on the X to produce the strain w+
GMR-wIR; Gla/SM6a; TM3, Ser/MKRS. The wgGla-1 (sub-sequently referred to as Gla) effect on RNAi was examinedin the F1. This new strain was crossed to the stocks of ro andhhbar3 and the F2 with heterozygous or homozygous muta-tions and wIR was examined with or without Gla.
The GMR promoting cell death transgenes including thefive inducers and the two inhibitors (Table S1) (Xu et al.2003) were kindly provided by B. Hay, California Institute ofTechnology, Pasadena, CA. These strains, GMR-diap1 on the X,GMR-p35 on the X, GMR-hid on 2, GMR-grim/TM3, Sb, GMR-
rpr/TM3, Sb, GMR-strica/CyO, and GMR-ttk88-myc(19)/CyO,were crossed to the RNAi strains and the F1 phenotype wasrecorded (all the transgenes carry a w+mc marker).
To examine the combination of cell death inducers andinhibitors, the multiple balancer stock with GMR-wIR de-scribed above was first mated to males of inducer strains.The F1 males carrying the respective inducer transgene andGMR-wIR were then crossed to virgins of the inhibitorstrains. The phenotype of the female offspring of thesecrosses was then analyzed and documented.
The symmetrically transcribing w RNAi strains w1118;SympUAST-w#8 and w1118; SympUAST-w#23/CyO, andthe long stem-loop ones w1118; pUAST-IRsp-w#32/CyOand w1118; pUAST-IRsp-w#41 were kindly provided by E.Giordano (Giordano et al. 2002), Università di Napoli,Naples, Italy. These strains were first crossed to w+; Gla/SM6a; TM3, Ser/MKRS to replace the w mutant gene on theX chromosome and to balance the transgenes on the secondchromosome and double balance the third chromosome. Totest whether B affects RNAi in these strains, virgins of themultibalancer strain Basc/Basc; In(2LR)SM1, al2 Cy cn2 sp2/In(2LR)bwV1, ds33K dpOV bwV1; In(3LR)Ubx130, Ubx130 es/In(3LR)C, Sb; spapol spapol were crossed to males of y w/y+ w;UAS-EGFP Tub-Gal4/TM6b, and then the Stubble F1 maleswith Tub-Gal4 and Basc were crossed to the balanced trans-genic strains. To simplify the genetic tests, GMR- and act5c-Gal4 trangenes on the second chromosome were chosen torecombine together in one chromosome with the UAS wRNAi transgenes. The new strains were then crossed withthe cell death strains and F1 phenotypes were assayed.
The UAS-wRNAiDS (on the third chromosome) strain(Kalidas and Smith 2002) and the GMR-Gal4 or act5c-Gal4 (on the second chromosome) strain were first crossedto the multibalancer strain w+; Gla/SM6a; TM3, Ser/MKRS.Then the F1 flies were crossed together to produce a straincontaining both transgenes balanced on the second andthird chromosomes. This strain was used to cross with thecell death strains and offspring phenotype was documented.
The EGFP RNAi strain w[1118]; P{w[+mC]=UAS-EGFP.dsRNA.R}142 (Roignant et al. 2003) was crossed with theGFP strain y w/w+; UAS-EGFP Tub-Gal4/TM6b, and the F1females were then crossed to the multibalancer strain yw67c23; Gla/SM6a; TM3, Ser/MKRS. The flies with recom-bined transgenes together on the third chromosome wereselected by additive eye color (the transgenes from bothoriginal strains are marked by mini-white) and weak bodyfluorescence (EGFP silenced). The strain was confirmed tocontain the three transgenes by combining the transgenechromosome (the third) with a wild-type one in femalesand finding that the offspring segregated unrepressed GFPflies due to recombination. In parallel, a strain v; bw; TM3,Ser/MKRS was produced. This strain was crossed to the GFPRNAi strain to make the strain v; bw; Tub-Gal4 UAS-EGFPUAS-EGFPir/MKRS. To combine v, bw, and a cell death genein a strain, the following approach was taken. For B, bal-ancer FM7a was used because it contains both B and v; and
for cell death genes on the second chromosome, they wererecombined together with bw. We also balanced the thirdchromosome constructs with MKRS or TM3, Ser. Finally, theEGFP RNAi strain was crossed to the cell death strains, in-dividually, and the phenotype of the offspring was digitallyphotographed using the GFP dissecting microscope in theUniversity of Missouri (MU) cytology facility.
The transgenic hs-hid stock strains from BloomingtonStock Center ( y1 w*; Bl1 L2/CyO, P{w[+mC]=hs-hid}4 onchromosome 2 and y1 w*; Pr1 Dr1/TM3, P{w[+mC]=hs-hid}14, Sb1 on chromosome 3) were crossed to the strainGMR-wIR to observe color restoration in the eyes. The EGFPRNAi strain v; bw; Tub-Gal4 UAS-EGFP UAS-EGFPir/MKRSwas crossed to the line with the third chromosome insertionor first crossed to the multibalancer strain w+; Gla/SM6a;TM3, Ser/MKRS and then crossed to the second chromo-some insertion to generate the hs-hid and EGFP RNAi strains.The flies were raised at 18�. The third instar larvae werecollected for dissection. For midgut analysis, larvae were col-lected before emerging from the food to avoid degradationthat occurs at later stages (Jiang et al. 1997). Larval GFP wasobserved with the GFP dissecting microscope in the MU cy-tology facility. Dissected tissues were immediately mountedon slides with fluorescence mounting medium without cover-ing and photographed under a Zeiss Universal microscopewith a MagnaFire cooled charge-coupled device camera.
Multiple GMR-wIR strains were produced as follows. Boththe GMR-wIR B strain and the GMR-wIR (on chromosome 3)strain were mated to the strain w+ GMR-wIR; Gla/SM6a;TM3, Ser/MKRS and the offspring was mated with each otherto obtain males with the genotype GMR-wIR B; GMR-wIR/TM3, Ser (or MKRS). These males were mated to females ofGMR-wIR (on chromosome 3) and the phenotypes of 1 (fromF1), 2 and 3 (from F2) copies of GMR-wIR with B werephotographed.
For mitotic recombination in the eyes, strains weregenerated with a cell death inducing transgene (GMR-hid,-ttk, -grim, or -rpr) on the second or third chromosomerecombined in the same chromosome with a correspondingneoFRT transgene (Xu and Rubin 1993). The originalneoFRT strains were modified to maintain w+ on the X chro-mosome and ey . FLP1 on the other autosome. Thesestrains were then crossed to female w+ GMR-wIR; Gla/SM6a; TM3, Ser/MKRS flies and male offspring were col-lected to mate with the neoFRT cell death strains. The phe-notypes of the offspring were documented. For simplicity,GMR-ttk eyes with clear boundaries between GMR-ttk andwild-type cell clones were chosen for illustration.
Acridine orange staining, Northern blotting, and RT–PCR
The transgenic hs-hid strains from the Bloomington StockCenter ( y w*; Bl L2/CyO, P{w+mC=hs-hid}4 and y w*; PrDr/TM3, P{w+mC=hs-hid}14, Sb) and Canton-S were usedfor acridine orange (AO) staining. The larvae were raised at18� and collected between the second instar to early thirdinstar. Late third instar larvae were avoided because of nor-
mal degradation of the midguts (Jiang et al. 1997). Midgutswere dissected and stained with AO (Jiang et al. 1997) andobserved for green fluorescence under a ·4 objective lens ofa Zeiss Universal microscope with a MagnaFire cooledcharge-coupled device camera. For positive controls, larvaeat the same age were used after heat shock at 37� in a waterbath for 30 min and then allowed to recover at 18� for .1hr. More than 10 pairs of Canton-S and hs-hid larvae andthree heat shocked hs-hid individuals were analyzed.
A total of 50–80 late third instar larvae were collected forTRIzol RNA isolation according to the manufacturer’s pro-tocol (Gibco BRL Life Technologies). If the RNA was forsiRNA preparation, the RNA precipitation was placed ondry ice for 20 min before centrifugation to ensure that thesmall RNA was precipitated. Isolated RNA was dissolved informamide at 55–65� and stored at 220�. RNA concentra-tion was measured by 10- to 100-fold water dilution witha NanoDrop ND-1000 spectrophotometer.
EGFP mRNA and dsRNA levels were analyzed by North-ern blotting according to Auger et al. (2005). Twenty micro-grams of total RNA of Tub-Gal4 UAS-EGFP/TM6b, Tb (EGFP)and Canton-S (CS) and 5, 10, and 20 mg of total RNA ofTub-Gal4 UAS-EGFP UAS-EGFPir/MKRS (EGFPir) and Tub-Gal4 UAS-EGFP UAS-EGFPir/TM3, hs-hid Sb (hs-hid; EGFPir)were loaded in the gel. EGFP coding sequence amplified byprimers WXo28 (AAG GGC GAG GAG CTG TTC AC) andWXo29 (CCA TGT GAT CGC GCT TCT CG) was cloned intopGEM-T Easy Vector (Promega) and sense and antisenseEGFP probes were transcribed from the plasmid DNA usingMaxiScript kit (Ambion). a-Tubulin exon 2 sequence wasamplified by primers WXo67 (TCT ATC CAT GTT GGTCAG GC) and WXo68 (GGT AGT TGA TGC CAA CCT TG)and cloned into pGEM-T Easy Vector. Antisense probe wasthen transcribed to detect a-tubulin mRNA on the stripedmembranes as input controls.
EGFP siRNA detection by Northern analysis was per-formed according to Pal-Bhadra et al. (2002). Ten micro-grams of total small RNA of Tub-Gal4 UAS-EGFP/TM6b, Tb(EGFP) and 2.5, 5, and 10 mg of total small RNA of Tub-Gal4UAS-EGFP UAS-EGFPir/MKRS (EGFPir) and Tub-Gal4 UAS-EGFP UAS-EGFPir/TM3, hs-hid Sb (hs-hid; EGFPir) wereloaded in the gel. Sense and antisense EGFP probes werefragmented before hybridization. The 5S rRNA sequencewas amplified by WXo89 [TAA TAC GAC TCA CTA TAGGGc caa caa cac gcg gtg t, T7 promoter attached (uppercase)] and WXo90 (gcc aac gac cat acc acg c) from genomicDNA and then purified to transcribe an antisense RNAprobe, which was used to detect 5S rRNA level as inputcontrols. Locked nucleic acid (LNA) probes against miRNAand endogenous siRNAs were designed and synthesized byExiqon with optimal Tm for Northern blots.
All the Northern blotting experiments were carried out atleast three times using RNA samples isolated separately. Toquantify the difference of the EGFP mRNA, dsRNA, andsiRNA with or without hs-hid, the exposed X-ray films werescanned and analyzed by the software Image Gauge (v 3.3)
or exposed and scanned by a phosphorimager (FujifilmGlobal) and analyzed by Multi Gauge. The fold differencebetween the death and no death samples was calculated inthe formula: (Sh/Lh)/(S/L), in which Sh and S are thedetected strength from a Northern blot for hid and no hidsamples, respectively; Lh and L are the corresponding load-ing controls. When a sample with the detected signalstrength was significantly less than the double amount ofthe twofold diluted sample, it was discarded as overex-posed. Samples of death and no death were paired for com-parison with the same amount of RNA transferred on thesame membrane.
Late third instar “hs-hid; EGFPir” larvae raised at 18�were collected and heat shocked at 37� in a water bath for30 min and allowed to recover at 18� for 1 hr and thenfrozen at 280�. Total RNA isolated from “EGFPir” larvaeand heat shocked and not shocked “hs-hid; EGFPir” larvaewas first precipitated from the formamide solution (25–40 mg RNA in 5 ml plus 15 ml 100% ethanol and 0.5 ml5 M NaCl). The RNA was dissolved in 100 ml of nucleasefree water and the concentration was measured with a Nano-Drop spectrophotometer. A total of 10 mg RNA was treatedwith RQ1 DNase (Promega). First-strand cDNA was synthe-sized with SuperScript III reverse transcriptase (Invitrogen)and poly(T) primer. Primer WXo93 (aac aag cgc agc tga acaag) were designed matching the UTS flanking sequence sub-sequent to the hsp70 promoter in the hs-hid transgene(Grether et al. 1995). Primer WXo91 (ga tga act cga cgctac gtc) matches the coding sequence of hid exon 1. Thispair of primers was used to amplify specifically the hs-hidtransgene cDNA. PCR was terminated after 25, 30, and 35cycles, respectively. PCR products were analyzed after gelelectrophoresis and ethidium bromide staining.
Semiquantitative and real-time PCR
To study whether the expression of endogenous genes andtransposable elements was changed, RNAs and cDNAs wereprepared as described above from the larvae of “EGFPir” and“hs-hid; EGFPir” or Canton-S and hs-hid strains from theBloomington Drosophila Stock Center.
At least two pairs of primers were designed for eachmRNA matching close to the 59 end to avoid possible de-tection of degraded 39 fragments with a poly(A) tail, whichcan be reverse transcribed by a poly(T) primer. For semi-quantitative PCR, a series of dilutions of the cDNA (one-,two-, and fourfold) was used as templates for PCR to ensurethat the stop point was within the range of exponentialamplification. Consensus results were observed for differentprimers and for technical and biological replications.
To confirm the mus308 expression change, real-time PCRwas performed in an ABI 7300 system using the manufactor’ssupplied Syber Green reaction master mix and the results wereanalyzed by the provided software. Multiple primer pairs weretested for the amplification efficiency and the most efficientones (59-CTTAATGGCGCTGGAGAAAG-39 and 59-GGCCTGATTCACTTCGTAGG-39) were used. A cDNA concentration range
was used for detecting differences in a dilution series. Threebiological replications were analyzed.
Sequencing and analysis of small RNAs
Total RNA from “EGFPir” and “EGFPir hs-hid” third instarlarvae was first size separated by polyacrymide gel electro-phoresis and 15–40 nt molecules were collected for con-structing a cDNA library and then sequenced by a Solexasystem in the MU DNA core facility. Primers (59-CGACAGGTTCAGAGTTCTACAGTCCGACGATC (N)n TCGTATGCCGTCTTCTGCTTG-39) from both ends in the raw readswere removed and only the sequences with sizes between19 and 31 were selected. Trimming was performed at eitheror both ends of the read for all or part of the primer/adaptersequence above. If the 39 end of the read matched all or partof the left end of the right primer then that matched partwas removed from the read. Similarly, if the 59 end of theread matched all or part of the right end of the left primerthen that matched part was removed from the read. Nomismatches were allowed in any of the trimming.
The reads for each sample are mapped to Drosophilaknown miRNAs (from miRBase, http://microrna.sanger.ac.uk/sequences/index.shtml), Drosophila transposons (fromflybase, http://flybase.org/) using BLAT (James 2002) withwhich the parameters used allow maximum sensitivity. AfterBLAT search, however, more stringent criteria were used toparse the results to retain only the highly confident align-ments. For miRNAs, only exact matches (same query/targetsequence length and 100% sequence alignment identity)were counted for normalization. For si/piRNAs mapped totransposons, only fully mapped sequences with 100% iden-tity were counted. To further verify the si/piRNAs, anno-tated pi/siRNAs were downloaded from GEO datasets(GPL4738 and GPL6664) (Brennecke et al. 2007; Ghildiyalet al. 2008) and all cleaned sequences were mapped onthem with BLAT (only the exact matches were counted).
Results
Eye mutations producing cell death restore eye colorotherwise reduced by white RNAi
The Drosophila compound eye develops from an eye-antennalimaginal disc, which originates from a cluster of ectodermalcells invaginated during embryogenesis. The cells are pro-liferated through the first, second, and early third larvalinstars, but remain undifferentiated. Morphogenesis andpattern formation begin with the morphogenetic furrow(MF) sweeping from the posterior to the anterior of theeye disc in the mid- and late third instar and continuingthrough the late pupal stage (Reifegerste and Moses1999). The morphogenetic furrow defines a clear boundaryat which the cells undergo differentiation (Heberlein et al.1993). Undifferentiated sections in mutants with impairedMF movement undergo cell death at the late third instar andare not included in the adult eye (Fristrom 1969; Heberleinet al. 1993; Mozer 2001). Cell death also regularly occurs
from 24 to 36 hr after pupal formation to remove the extraaccessory cells, namely, the secondary and tertiary pigmentcells (Brachmann and Cagan 2003).
An RNAi construct (GMR-wIR) (supporting information,Figure S1) repressing the expression of the white (w) genereduces the eye color from brick red to light yellow (Figure1A) (Lee et al. 2004). In the process of manipulation of thisconstruct, we crossed the RNAi stock to laboratory strainscarrying dominant or recessive marker genes causing eyemorphological abnormalities. Surprisingly, the normal redeye color was partially restored in Bar (B) and Drop (Dr)flies, and weakly restored in the presence of the Gla andsparkling-poliert (svspa-pol) mutations (Figure 1A and TableS1). Because the eye color change occurs in living cells andis adjacent to the missing portions in the B mutant eyes, wehypothesized that the inhibition of RNAi is caused by adja-cent tissue that has undergone cell death.
Bar and Drop are gain-of-function mutants, ectopicallyexpressing homeobox genes BarH1/BarH2 or muscle seg-ment homeobox (msh), respectively, in the eye disc. Bar pre-maturely stops the MF movement beginning from thedorsal–ventral center and spreading laterally (Heberleinet al. 1993). Cell death occurs anterior to the arrested MF(Fristrom 1969) causing the adult phenotype. In the w RNAiand B flies, the restored eye color gradually diminishes pos-teriorly and laterally (Figure 1A), suggesting it may becaused by cell death in the adjacent region.
When B mutant larvae are raised on food containing 1–2% acetamine, the mutant effect can be largely (but notcompletely) reversed by prolonging development, which sig-nificantly reduces cell death in the late third instar (Fristrom1972). When female animals with B and GMR-wIR on the Xchromosomes were treated with acetamine, reversal of RNAiis diminished as expected (Figure 1A).
The hedgehog mutation bar3 (hhbar3), which also arreststhe MF and causes a similar eye phenotype (Heberlein et al.1993), likewise caused inhibition of w RNAi and restoredthe color around the indented anterior portion of the eye butappearing more focused than with B (Figure 1A). In theDrop mutants, the MF is initiated but defective in movement(Heberlein et al. 1993). Cells in the MF undergo normaldevelopment and grow to a few rows of regular ommatidia.The remaining cells in the anterior region are maintained inan undifferentiated state and degraded later by cell death(Mozer 2001). In the w RNAi strain, the differentiated om-matidia showed restored red color (Figure 1A). As detailedin Table S1, the Gla, ro (rough), and svspa-pol mutations alsocause cell death and inhibit RNAi weakly, whereas the Egfr(Epidermal growth factor receptor), and Roi (amosRoi-1)mutations, which do not cause cell death, do not reverseRNAi (Figure 1A). Thus, the RNAi inhibition is only associ-ated with mutations that condition cell death and is nota secondary consequence of eye morphological changes.The L2 (Lobe) mutant induces abnormal cell death before
Figure 1 Cell death-dependent inhibition of RNAi silenc-ing of the white gene. (A) At top left, a wild-type (WT) eyewith w+ is red. The yellow eye (second from top left) indi-cates that w is silenced by RNAi when the transgene GMR-wIR is present in the genome. Heterozygotes of the noteddominant mutations and the homozygote for the recessivegenes were combined with a copy of GMR-wIR. Silencingis partially reversed in eye mutations B, Dr, Gla, ro, andhhbar3, but not in the mutation Roi (amosRoi-1). Acetaminetreatment of the Bar mutant [B (acetamine)] reduces celldeath in the eyes accompanied by reduced color restora-tion. (B) Induced cell death in the eyes by ectopic expres-sion of grim, hid, rpr, strica, and ttk (all driven by the GMRpromoter) caused reversal of RNAi. The first row shows thephenotype of the noted cell death transgenes with wildtype w+. The second row shows the transgenes combinedwith GMR-wIR. The red color indicates inhibition of RNAi.The third and fourth rows show the cell death transgenesand GMR-wIR combined with two different inhibitors ofcell death as noted. To the extent that cell death is re-versed, RNAi is restored. (C) EGFP RNAi is inhibited by celldeath. Cell death induced by ectopically expressed grim,strica, and ttk restored the fluorescence to most regions, ifnot the whole eye. In each comparison, control eyes withGFP silenced but without induced cell death are shown onthe upper left. Bars, 0.1 mm.
the initiation of differentiation (Singh et al. 2006) and doesnot affect w silencing, suggesting the duration of the signalfrom cell death is temporally limited.
To rule out the possibility that cell death affects w expres-sion as the basis of the above results, we crossed Dr or B tohypomorphic w mutants including point mutations wa2 andwa3 and insertion mutations, w-blood (wbl) and w-apricot(wa) (Zachar and Bingham 1982) and found no impact (Fig-ure S2). These results indicate that modulation of the whitegene itself is not the basis for the suppression. Another GMR-wIR transgene inserted in chromosome 3, was tested with Band showed similar inhibition of RNAi (data not shown),indicating that the location of the RNAi transgene also doesnot affect the outcome.
Proapoptotic transgenes inhibit w RNAi depending oncell death
To test further the role of cell death signaling for theinhibition of RNAi, cell death was induced by overexpressionof apoptosis promoting genes. The proapoptotic genes grim,head involution defective (hid, or Wrinkled, W) and reaper(rpr), the caspase gene strica (or dream) and the transcriptionfactor tramtrack (ttk) have been shown to cause extensive celldeath and irregular eye phenotypes when ectopicallyexpressed by the GMR promoter (Figure 1B) (for example,Xu et al. 2003). When w RNAi was also introduced into thesestrains, the expression of the w gene can be assayed in theremaining cells and was partially restored, as indicated bydispersed red spots or a centered dorsal–ventral strip of in-creased color in female ttk flies, without exception, when celldeath occurred (Figure 1B, second row).
The double homozygous mutations of vermilion (v) andbrown (bw) are epistatic to w+ and show pale-yellow eyes,thus resembling the w RNAi phenotype. When the cell deathinducer transgenes were combined with these mutations, nopigment accumulation was found (Figure S3). This controlindicates that the increase in pigment with the cell deathtransgenes has white RNAi as a target rather than beinga consequence of altered eye morphology (e.g., closer ar-rangement of pigment cells).
The possibility that the antiapoptotic transgenes restoreRNAi directly can be excluded by the following experiments.The cell death phenotypes can be variably suppressed,except for the overexpression of ttk, by simultaneous expres-sion of the apoptosis inhibitors Drosophila inhibitor of apo-ptosis 1 (diap1 or thread, th) or baculovirus p35 (Figure 1B)(Xu et al. 2003; Hay et al. 2004). When cell death is reversedfully (as indicated by the eye shape and ommatidia arrange-ment returning to normal), RNAi of w is restored; when thecell death was reversed partially, inhibition still occurs (Fig-ure 1B, hid+diap1). The transgenes diap1 and p35 do notsuppress the ttk induced cell death; in this combination,RNAi is still suppressed, excluding the possibility that theseantiapoptotic genes themselves restore the RNAi silencing.Also, crosses that introduce only the antiapoptotic trans-genes show no impact on w RNAi.
It is known that cells overexpressing hid will producea proliferation signal to neighboring cells, a process not af-fected by the suppression of cell death by p35 (Huh et al.2004; Pérez-Garijo et al. 2004; Ryoo et al. 2004). Indeed,the expression of p35 suppressed both cell death and theRNAi inhibition, indicating that this proliferation signal isindependent of any signal affecting RNAi. In contrast, an-other proliferation signal produced by dying cells is affectedby p35 and diap1 (Fan and Bergmann 2008), and thus itcannot be ruled out as coincident with a potential RNAisuppression signal.
Cell death inhibits w RNAi induced by diverse constructs
Different RNAi constructs were used to generate dsRNAs ofthe w gene (Giordano et al. 2002; Kalidas and Smith 2002)(Figure S1). These transgenes utilize an upstream activatingsequence (UAS) for expression and silence the w gene tovarying degrees when the transcription factor Gal4, whichtargets the UAS, is expressed by the promoters GMR, actin5c(act5c), or a-tubulin (Tub) (Giordano et al. 2002; Kalidasand Smith 2002) (Figure S4). To confirm that the RNAiinhibition is not limited to certain constructs, we crossedthese strains to the eye mutations B, Dr, and Gla, as wellas to cell death strains overexpressing grim, hid, rpr, strica,and ttk. Eye color restoration was observed in all the testedstrains with strica, and in all but one construct with Gla, Dr,and grim (Figure S4 and Table S2). However, in some celldeath and RNAi combinations, no clear restoration wasdetected, especially with the constructs that alone showstronger silencing. Weaker pigment restoration was also ob-served when the silencing is achieved by a stronger pro-moter for Gal4, and different degrees of restoration wereobserved with different transgenic strains from the sameRNAi construct (Figure S4 and Table S2). These results sug-gest that the strength of the silencing plays a role in themagnitude of RNAi reversal. Moreover, because differentpromoters were used in these experiments, they demon-strate that downregulation of the GMR promoter of GMR-wIR by cell death signaling is not the basis of the restoredeye color in the original observations.
Cell death suppresses GFP RNAi
To test the generality of the cell death inhibition, RNAi ofGreen Fluorescent Protein (GFP) was examined within thecell death strains. GFP fluorescence can be detected in theeye only in the absence of the normal pigment (Plautz et al.1996; Berghammer et al. 1999). To overcome this obstacle,GFP was combined with the brown and vermilion mutations,which together eliminate the pigments. Under these circum-stances, we observed strong green fluorescence of the EGFPtransgene driven by Tub-Gal4 in the eyes. Weaker fluores-cence is found after EGFP is silenced by the transgene EGFPir(Figure S1 and Figure S3) as previously noted in larvae(Roignant et al. 2003). In this EGFP RNAi background, thecell death mutations B, Dr, and transgenes GMR-grim, -hid,-rpr, -strica or -ttk were introduced. Stronger and global
restoration of GFP was observed in the eyes of grim, strica,and ttk (Figure 1C and Figure S5). Compared to the celldeath control, which did not cause detectable autofluores-cence in this assay (Figure S5), weaker and area-restrictedrestoration occurred in the Dr, hid, and rpr eyes.
The results of cell death induced inhibition of w and EGFPRNAi are summarized in Table S2. Greater cell death iscaused by overexpression of hid and rpr than that of grim,strica, and ttk (as shown by the eye size); yet there is lessRNAi reversal for both w and EGFP. The strica constructrestored EGFP expression to the strongest level as was alsothe case with restoration of w expression. Thus, in general,the relative strength of reversal of RNAi is related to themode of cell death but is independent of the genes (w orEGFP) involved.
RNAi inhibition is quantitatively related to the levelof RNAi
As noted above, a difference in expression of an RNAitransgene affects the amount of restored w expression. Wealso noticed a weaker restoration observable with lesser ex-pression of Gal4 (e.g., GMR-Gal4) driving an RNAi constructbut not with more strongly expressed cases (e.g., Tub-Gal4).This finding suggests that a stronger RNAi effect is above thethreshold of suppression. To test this possibility, we crossedmultiple GMR-wIR transgenes with B and Gla. B and Glashow an additive effect on RNAi suppression. The resultsindicate that the restored color is negatively related to thecopy number of the GMR-wIR transgene (Figure S6).
The RNAi inhibition can be nonautonomous
The reversal of white RNAi in cells adjacent to the cell deathsectors of Bar, bar3, and Dr (Figure 1A) provided an indica-tion of the nonautonomy of RNAi inhibition. As noted above,all these mutants cause a defect of the movement of the MF(Heberlein et al. 1993). The MF determines the fate of thecells: those cells encompassed by it will differentiate andremain alive in the adult eye and the remaining cells willbe removed by cell death and will not be present in the adulteye. The color reversal observed in the adult eyes must belocated in living cells, which did not undergo cell death.Therefore, the inhibition of w RNAi in adult eyes must havebeen induced nonautonomously by the death of adjacentcells.
To examine this feature of inhibition by a second method,we produced mosaic cell death in the eyes. Mitotic re-combination by FLP–FRT (Golic 1991; Xu and Rubin 1993;Stowers and Schwarz 1999) was conducted in the eye imag-inal discs to resolve the zygotic heterozygous genotype intohomozygous sectors of proapoptotic transgenes and normalcells using a promoter for FLP that acts before cell death,which was induced by the GMR-driven expression of thedeath inducer genes in the late third instar (see Materialsand Methods). This technique produces mosaic eyes com-posed largely of two kinds of homozygous lineages: thosefor the death inducer construct and those for wild-type chro-
mosomes (Stowers and Schwarz 1999). After the adultseclosed, cell death sectors in the eyes can be observed asan irregular arrangement of ommatidia. The most useful in-volves the overexpression of ttk, which has a dominant phe-notype and causes a smooth eye surface due to cell death ofspecific cell types within the ommatidia. Areas with smoothsurfaces were observed in the mosaic eyes, which allowsectors homozygous or occasionally heterozygous for thettk construct to be identified definitively. The adjacentregions are homozygous for normal chromosomes; other-wise they would show a smooth eye surface. The ttk sectorswere accompanied by color restoration spreading from twoto several lines of ommatidia, depending on the size of thecell death containing areas (Figure 2A and Figure S7). Thus,the inhibition of RNAi is occurring in the homozygous nor-mal cells, consistent with the pattern of color restoration inthe B, hhBar3, and Dr eyes. The color intensity in the normalsectors progressively decreases with the distance from thedeath sectors. Collectively, these data suggest the existenceof a signal to suppress RNAi initiates from the region of celldeath and travels a limited distance into regions with nor-mal cells.
In contrast, cell death induced by the hid transgene in ananalogous mitotic recombination experiment did not invokeRNAi inhibition in most cases, potentially indicating celldeath does not inhibit RNAi under certain conditions (Figure2B). Given that the same transgene is able to inhibit RNAiwhen heterozygous (Figure 1B) and the mitotic recombina-tion is effective at homozygosis at .90% (Stowers andSchwarz 1999), we suggest this difference is due to therapid elimination of the dying cells in the case of the homo-zygous transgene. Thus, there is possibly a required thresh-old of duration of the dying cells to provoke substantialinhibition of RNAi.
Mosaics of rpr or grim produced by the FLP–FRT methodexhibit colored sectors with the size usually not over anommatidium in all eyes examined (Figure 2, C and D). Inthis case, the eyes from experiments involving homozygosisof rpr and grim transgenes do exhibit RNAi inhibition, pos-sibly because rpr and grim transgenes cause less severe celldeath effects than hid.
Cell death inhibits RNAi in different tissues
To investigate the possibility of cell death inhibition of RNAiin tissues other than the eye, we tested strains with a heat-shock promoter transgene hs-hid in the chromosomal bal-ancers CyO or TM3, respectively. When the flies are grownat 18� or room temperature without heat shock, w RNAi isslightly reversed, showing red spots in the eye (Figure 3A).This result indicates that the constitutive expression of thetransgenic hid is strong enough to cause some RNAi inhibi-tion. AO staining, which is used to detect cell death (Jianget al. 1997), is also observed in the transgenic larvae (FigureS8A). The expression of the hid transgene was confirmedby RT–PCR (Figure S8B). A further confirmation that the re-stored color results from a low level of cell death conditioned
by the hs-hid transgene was that suppression of RNAi wasreversed by adding the p35 transgene to the genotype (FigureS8C). Collectively, these results demonstrate that the trans-gene was expressed and caused sporadic cell death.
To analyze the transgenic hid effect on EGFP RNAi, thirdinstar larvae were directly examined for green fluorescenceor following dissection into tissues and organs. StrongerEGFP expression was obvious in larvae over all and in midg-uts, salivary glands, brains, and imaginal discs (Figure 3, B–F). Similar results were observed when hs-hid was locatedon either chromosome 2 or 3 balancers (Figure S9). Thisresult indicates that the phenotype is indeed caused by thehs-hid gene and not by other factors on the balancer chro-mosomes, which without the transgene have no effect onRNAi. Thus, we conclude that cell death inhibition of RNAioperates in different tissues and developmental stages.
RNAi is inhibited by blocking the processing of dsRNAinto siRNA
The expression change of EGFP in multiple larval tissuesprovides an opportunity to investigate mRNA, dsRNA, andsiRNA changes during suppression of RNAi. As expected, theEGFP mRNA level in hs-hid transgenic animals increasesabout twofold (Figure 3, G and I). This change probablyreflects a mixture of affected and unaffected cells, so thechange in the affected cells is likely of much greater magni-tude. Interestingly, we detected more than a threefold in-
crease of the dsRNA in the hid transgenic strain (Figure 3, Gand I). This accumulation is likely caused by impairment ofEGFP dsRNA processing. Consequently, we expected to seean siRNA decrease. Indeed, EGFP siRNA is reduced �40% inthe transgenic strain (Figure 3, H and I). The results suggestthe target step of RNAi suppression is the conversion ofdsRNA to siRNA.
Adenosine deaminase acting on RNA (ADAR), an RNAediting enzyme, has been shown to compete for dsRNA asa substrate and thus inhibit RNAi (Scadden and Smith 2001;Wang et al. 2005; Heale et al. 2009). ADAR edits the dsRNA(Carpenter et al. 2009) and makes it unfavorable as a sub-strate for Dicer-2, the enzyme component of RNAi thatprocesses dsRNA (Scadden and Smith 2001). Thus, we ex-amined the possibility that ADAR might be induced by celldeath signaling and compete with RNAi for dsRNA substrates.However, this possibility was excluded because there is noincrease in expression of ADAR accompanying cell death,and when ADAR expression is reduced by RNAi, cell death-mediated RNAi inhibition was not affected (Figure S10).
Cell death inhibition of RNAi regulates endogenousgene and transposable element expression
Recent work shows that siRNAs exist in somatic cells andmatch selected endogenous genes and transposable ele-ments. Among those genes, mus308, encoding an enzymewith DNA polymerase and helicase function, has beendemonstrated to be repressed by RNAi (Czech et al. 2008;Okamura et al. 2008). To investigate whether cell deathupregulates mus308 by its inhibition of RNAi, RT–PCR wasperformed to detect the expression level of this gene in thehs-hid transgenic larvae. A doubling of the expression wasobserved by semiquantitative PCR, which was confirmed byreal-time PCR (Figure 4A). This result is similar to the EGFPupregulation and suggests that endogenous genes underRNAi control can be affected in the same manner.
When RNAi is compromised, previous studies haveshown that a severalfold increase of expression for someTEs was detected in S2 cells (Czech et al. 2008; Ghildiyalet al. 2008; Kawamura et al. 2008) and in adult flies (Chunget al. 2008). We examined five of them, 1731, mdg1, 297,BEL, DOC, and S element to analyze the possible effect fromcell death. It was demonstrated that dicer-2 knockout didnot significantly affect 1731 and mdg1 expression in adultflies, whereas 297 and BEL mRNAs are increased, althoughboth mdg1 and 297 siRNA levels are reduced (Chung et al.2008). Our results also show BEL and 297 expression in-creased approximately twofold (P , 0.05) in the presenceof hs-hid–caused cell death, whereas mdg1 and 1731 expres-sion is not significantly changed, and DOC and S may beslightly upregulated (P, 0.1) (Figure 4B). These results areconsistent with the hypothesis that cell death reduces siRNAsynthesis and thus impairs RNAi.
To further confirm the impact of hs-hid on siRNAsand miRNA, LNA probes were synthesized (Exiqon) for de-tecting let-7, miR-1, and esiRNA-sl-1 (the siRNA molecule
Figure 2 Cell death caused RNAi inhibition in neighboring cells. Mosaiccell death of GMR-ttk and GMR-hid generated by mitotic recombination.(A). The color is restored by the GMR-ttk cells. Selected examples ofsectors of ttk cell groups are circled by black lines, which were indicatedby the smooth surfaced areas. Because the GMR-ttk/+ genotype hasa dominant phenotype (Figure 1B), the normal sectors are those in whichmitotic recombination during development have resolved into +/+ sec-tors. These wild-type cells show reversal of RNAi when adjacent to GMR-ttk sectors. Surrounding two examples of the death areas, the color that isrestored in normal cells is circled by white lines. The strength of inhibitiondiminishes with the distance from the ttk sectors. (B) Death in homozy-gous GMR-hid cells did not cause inhibition of w RNAi silencing. Mosaiccell death of GMR-grim (C) and GMR-rpr was generated by mitotic re-combination (D). In these cases, w RNAi was occasionally reversed asindicated by the red sectors in the eyes. Bars, 0.1 mm.
complementary to mus308 mRNA; Kawamura et al. 2008)by Northern blotting (Chung et al. 2008). A change in theamount of a control miRNAwas not detected (let-7 in Figure4C). However, esiRNA-sl-1 was reduced to �45% of thecontrol as also found for the EGFP siRNA (Figure 4C).
To determine whether any global change occurred for TEsiRNAs, we sequenced the small RNAs (19–40 nt) from thehs-hid transgenic and control strains. The read numbers ofsequenced RNAs matching TEs were first normalized by to-tal miRNA reads, then plotted on the basis of their size(Figure 4D, original data see Table S3). Peaks for siRNA(21 nt) and for piRNA (23–29 nt) were clearly evident.The siRNA peak is reduced to �43% (averaged) in cell deathsamples. When the 21-mer set (excluding miRNAs) wascompared for homology to the published siRNA/miRNAdataset from fly heads (Ghildiyal et al. 2008), we observed�40% reduction of matched siRNAs with cell death (620 to372 reads). The 21-mer siRNA molecules derived from geneCG4068, which were shown to silence mus308 (Okamuraet al. 2008), are also reduced (35 to 13 reads) (see Table S3for total siRNA number derived from CG4068), matchingthe Northern results. There is also possibly a weaker reduc-tion in total piRNA amount (Figure 4D and Table S3). How-
ever, we were not able to detect a significant reduction ofEGFP siRNA level in the hs-hid strain by this method (TableS3) for unknown reasons, although this might be the case ifthe high expression of EGFP is at saturating amounts. Over-all, our results indicate that the expression change ofmus308 and TEs is caused by a genome-wide reduction ofsiRNAs.
Discussion
We demonstrate in this study that cells undergoing deathinhibit RNAi. This process occurs in different stages of thelife cycle, in different tissues, and for selected genes silencedby RNAi. Various constructs that generate dsRNA were allfound to be effective, suggesting this step is not affected.Indeed, we observed accumulation of dsRNA and reductionof siRNA, indicating that the processing of dsRNA to siRNAis the step impaired. Either modification of dsRNA or Dicer-2and/or its cofactors may be the target of the cell deathsignaling. However, we were not able to detect a change ofDicer-2 expression at either the transcriptional or proteinlevel (Figure S11). The phenotype of the eye color suggeststhis is not an all-or-none regulation, but rather the restored
Figure 3 Cell death-related inhibition of RNAioccurs in different tissues and organs and theincreased expression of the marker gene EGFPis caused by impaired processing of dsRNA intosiRNA. (A–F). The default expression of thetransgene hs-hid inhibits w silencing in theeye (A) and EGFP silencing in larvae (B), wingdisc (C), eye-antennal disc (D), midgut (E), andsalivary glands (F). In each image except theeye, controls without hs-hid but with EGFPRNAi are located to the right. A salivary glandshowing restored fluorescence and a controlpair of glands is shown in the lower right. Bars,0.1 mm. (G–I). Northern blotting detects alteredlevels 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 wasloaded and indicated by the numbers of micro-grams. Levels of a-tubulin (Tub) mRNA (G), and5S rRNA (H) were probed, respectively, as inputcontrols. The patterns of siRNA were not differ-ent when probed with either sense or antisenseEGFP probes (not shown). Statistical analysis ofthe EGFP mRNA, dsRNA, and siRNA levels in“hs-hid; EGFPir” larvae is shown in I. Northernresults were scanned and analyzed by ImageGauge. The fold difference compared to thesamples without hid (“EGFPir”) was normalizedby the loading control. The heights of solidbars with error bars indicate the fold difference;*P , 0.05 (no cell death), **P , 0.01, ***P ,0.001.
color in the eye is quantitatively reversed relative to thesilencing strength of w RNAi.
We demonstrate that cell death inhibits RNAi nonauton-omously in neighboring normal cells. The inhibition isconditionally triggered in that when strong cell death wasinduced, little or no RNAi inhibition was observed. Weassume that a threshold of duration of the cells undergoingcell death is required for the effect. This time-lag hypothesisis consistent with the fact that processing of dsRNA to siRNAis reduced and thus caused the RNAi inhibition. Therefore,the lag time may be related to the generation and perduranceof the siRNA molecules. This hypothesis also explains threeother observations. First, we did not observe GFP RNAiinhibition during normal embryo development in whichprogrammed cell death occurs. Second, when the hs-hidtransgenic larvae were heat shocked, they died within 1day. During this period of time, we were not able to detectany GFP expression difference in the GFP RNAi animals com-pared to the controls that were not heat shocked. Third, wealso failed to detect a GFP expression change in the eye discswith EGFP RNAi in the late third instar when proapoptoticgenes were expressed by the GMR promoter, that is, imme-diately after ectopic cell death was induced. However, it isimportant to note that when reversal of RNAi occurred, it didso in normal cells as evidenced from the results from eyemutations and the somatic recombination mosaic analysis.
One possibility for the existence of cell death inducedinhibition of RNAi might be that RNAi acts as a first line ofdefense against viruses (Li and Ding 2005). If this fails and
cell death results from viral proliferation, signaling mightoccur to neighboring cells to induce processes that modifydsRNA as a second line of defense, making them inaccessiblefor processing to siRNA, so that they cannot enter the RNAipathway leaving the integrity of the target mRNA intact,analogous to the interferon response in mammals (Kawaiand Akira 2007; Randall and Goodbourn 2008). This hy-pothesis can account for the collective data that indicatea quantitative negative relationship between the cell deathsignal and the effectiveness of RNAi. Such a second line ofdefense would be selected if RNAi regularly failed to stopviral infection, which is obviously the case, and would pro-vide a different means of attacking dsRNA.
Apoptosis is known to play a role in immunity to virusesin insects in that the apoptotic response can severely limitviral replication and thus many viruses encode antiapoptoticgenes (reviewed in Clarke and Clem 2003; Clem 2005).Recent data on viral infection in Drosophila show that longdsRNAs but not the siRNAs stimulate whole-body immunityto homologous virus infection (Saleh et al. 2009). Thus, theexistence of cell death induced inhibition of RNAi causinglocal maintenance of dsRNA could possibly be beneficial forviral defense by RNAi throughout the body if localized path-ogen-induced cell death occurs.
Knocking down of Dicer-2 or Argonaute-2, the corecomponents of the siRNA pathway, has been shown toincrease the expression of many transposable elements,indicating that this pathway plays a role in repressing TEsin addition to the piRNA pathway (Chung et al. 2008; Czech
Figure 4 Cell death upregulates the expressionof the endogenous genemus308 and transpos-able elements and reduces the siRNA level. (A)mus308 mRNA amount was doubled in the hs-hid strain, as detected by real-time PCR andsemiquantitative PCR. The fold difference com-pared to the samples without hid (“EGFPir”)was normalized by b-tubulin. As noted in thetext, the magnitude in affected cells is undoubt-edly greater. Levels of statistical significance aredesignated as described in Figure 2. (B) Expres-sion of transposons 1731, mdg1, 297, BEL,DOC, and S elements in “hs-hid EGFPir” larvae.Semiquantitative RT–PCR results were analyzedby Image Gauge. The fold difference comparedto the samples without hid (“EGFPir”) was nor-malized by the loading control. (C) Northernresults show let-7 level remained unchangedbut the endogenous esiRNA-sl-1 was reducedto �45% in the hs-hid strain. The statisticalresults for triplicate experiments are shown be-low. *P , 0.05 (no cell death), **P , 0.01,***P , 0.001. (D) Distribution of small RNAsmatching TEs from deep sequencing datashows a .50% reduction of the 21-nt lengthin the cell death strain (EGFPir hs-hid) comparedto the control. The read number for each sam-ple 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 areindicated by #1 and #2.
et al. 2008; Ghildiyal et al. 2008; Kawamura et al. 2008;Okamura et al. 2008). Our findings suggest that chronicectopic cell death inhibition of RNAi also elevates TE expres-sion. This finding suggests that chronic diseases or long-term exposure to pathogens, which might cause cell deathin the stressed tissue, may cause activation of endogenousTEs in the adjacent normal cells. It has been documented inyeast cells under stress, that the retrotransposon Ty5 willrandomize its integration, which otherwise mostly targetsheterochromatin, and thus become mutagenic (Dai et al.2007). Similarly, the activation of TEs in cells surroundingcell death may increase the somatic mutation frequency.Therefore, the inhibition of RNAi induced by chronic ectopiccell death may provide insight for the genesis of certain typesof diseases, such as some cancers, in which persistent inflam-mation and somatic mutations are implicated (Cooper 1995;Coussens and Werb 2002; Copeland and Jenkins 2009).
Lastly, RNAi is widely used in genetic analysis toeliminate specific gene function and many practical appli-cations of this tool have been suggested (Aigner 2007). Thefinding that chronic ectopic cell death alters the pattern ofRNAi is critical for an understanding of the application of thetechnique in basic and applied studies.
Acknowledgments
We thank R. Carthew, B. Hay, E. Giordano, C. Antoniewski,T. Zars, and C. Tan and the Bloomington Drosophila StockCenter for providing fly strains. We thank Ryan Donohue forperforming some PCR experiments, Sean Blake for sequenc-ing small RNAs, and William G. Spollen for generating andtrimming the sequence data. We also thank Kyungju Chin,Robert Gaeta, and Louis Mega for advice.
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