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, 11 (4), 459-69

Decay of mRNAs Targeted by RISC Requires XRN1, the Ski Complex, and the Exosome

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Decay of mRNAs Targeted by RISC Requires XRN1, the Ski Complex, and the Exosome

Tamas I Orban et al. RNA.

Abstract

RNA interference (RNAi) is a conserved RNA silencing pathway that leads to sequence-specific mRNA decay in response to the presence of double-stranded RNA (dsRNA). Long dsRNA molecules are first processed by Dicer into 21-22-nucleotide small interfering RNAs (siRNAs). The siRNAs are incorporated into a multimeric RNA-induced silencing complex (RISC) that cleaves mRNAs at a site determined by complementarity with the siRNAs. Following this initial endonucleolytic cleavage, the mRNA is degraded by a mechanism that is not completely understood. We investigated the decay pathway of mRNAs targeted by RISC in Drosophila cells. We show that 5' mRNA fragments generated by RISC cleavage are rapidly degraded from their 3' ends by the exosome, whereas the 3' fragments are degraded from their 5' ends by XRN1. Exosome-mediated decay of the 5' fragments requires the Drosophila homologs of yeast Ski2p, Ski3p, and Ski8p, suggesting that their role as regulators of exosome activity is conserved. Our findings indicate that mRNAs targeted by siRNAs are degraded from the ends generated by RISC cleavage, without undergoing decapping or deadenylation.

Figures

FIGURE 1.
FIGURE 1.
Generation of RNAi reporters for Drosophila cells. (A) Schematic representation of the reporters. White boxes, exons; gray boxes, sequences derived from vector pAc5.1b (Invitrogen) or pRmHa, respectively; black boxes, sequences complementary to the dsRNAs; IN, introns. The fragments of the transcript detected by the 5′ or 3′ probes used in this study are indicated. (BD) S2 cells expressing the indicated reporter constructs were treated with the corresponding dsRNAs. Total RNA samples were isolated and analyzed by Northern blot using probes specific for adh (3′ probe), NXF1 (3′ probe), and rp49 mRNAs. In lanes 13, dilutions of total RNA samples isolated from untreated cells were loaded to assess the efficiency of RNAi. The levels of the reporters were quantitated in at least three independent experiments and normalized (norm.) to those of rp49 mRNA. These values were set to 100% in untreated cells. Mean values ± standard deviations (SD) are shown.
FIGURE 2.
FIGURE 2.
Reporter mRNAs are degraded through the RNAi pathway. (A,B) S2 cells expressing Mtn-adh were treated with adh dsRNA. Expression of adh was induced for 45 min in treated and untreated cells. Following induction, transcription was inhibited by actinomycin D (5 μg/mL) for the times indicated above the lanes. Total RNA samples were isolated and analyzed as described in Figure 1. (C) The levels of adh mRNA normalized to those of rp49 mRNA shown in panels A and B are plotted as a function of time. (D) S2 cells constitutively expressing adh mRNA were treated with the dsRNAs indicated above the lanes. Total RNA samples were isolated and analyzed as described in Figure 1. The levels of the reporters were quantitated in at least three independent experiments and normalized (norm.) to those of rp49 mRNA. These values were set to 1 in cells treated with GFP and adh dsRNAs. Mean values ± SD are shown.
FIGURE 3.
FIGURE 3.
The 3′ fragments are degraded by XRN1. (AC) S2 cells expressing 5C-adh, Mtn-adh, or Mtn-NXF1 were treated with the dsRNAs indicated above the lanes. Total RNA samples were analyzed as described in Figure 1. Northern blots were hybridized with probes complementary to the 3′ ends of the reporters as shown in Figure 1A. In lane 1 of panels A and C, 20% of an RNA sample isolated from untreated cells was loaded to assess the efficiency of RNAi. The levels of the 3′-intermediate (3′-int.) were normalized to those of the full-length mRNA in at least three independent experiments. These values were set to 1 in control cells treated with GFP and the specific (adh or NXF1) dsRNAs. Mean values ± SD are shown.
FIGURE 4.
FIGURE 4.
Decay rates of the 3′ fragments in cells depleted of XRN1. (A,B) S2 cells expressing Mtn-adh were treated with the indicated dsRNAs. Expression of adh was induced for 45 min. Transcription was inhibited by actinomycin D (5 μg/mL) for the times indicated above the lanes. Total RNA samples were isolated and analyzed as described in Figure 1. Northern blots were hybridized with a 3′ probe (see Fig. 1A). (C) The levels of the 3′ fragments normalized to those of rp49 mRNA shown in panels A and B are plotted as a function of time.
FIGURE 5.
FIGURE 5.
The 5′ fragments are degraded by the exosome. (AC) S2 cell-lines expressing 5C-adh, Mtn-adh, or Mtn-NXF1 were treated with the dsRNAs indicated above the lanes. Total RNA samples were analyzed by Northern blot using probes detecting the 5′ decay intermediates (see Fig. 1A). The levels of the 5′ fragments were normalized to those of the full-length mRNA in at least three independent experiments. These values were set to 1 in control cells treated with GFP dsRNA and dsRNAs targeting the reporters. Mean values ± SD are shown. In lane 1 of panels A and C, a dilution of an RNA sample isolated from untreated cells was loaded to assess the efficiency of RNAi.
FIGURE 6.
FIGURE 6.
Degradation of the 5′ decay intermediates. (A,B) S2 cell-lines expressing Mtn-adh were treated with adh and RRP4 dsRNAs. Expression of adh mRNA was induced for 45 min. Next, transcription was inhibited with actinomycin D (5 μg/mL) for the times indicated above the lanes. Total RNA samples were isolated and analyzed by Northern blot with a 5′ probe. (C) The levels of the 5′ fragments normalized to those of rp49 mRNA shown in panels A,B are plotted as a function of time. (D) S2 cell lines expressing 5C-adh were transfected with the dsRNAs indicated above the lanes. RNA samples were analyzed by Northern blot, as described in Figure 1. The steady-state levels of the 5′ decay intermediates were quantitated and normalized to those of the full-length transcript.
FIGURE 7.
FIGURE 7.
The 5′ intermediates are capped, whereas the 3′ intermediates are polyadenylated. Cells expressing the inducible adh reporter were codepleted of SKI2 and XRN1. In (A,B), total RNA samples were subjected to oligo(dT)-targeted RNase H cleavage. In (C,D), total RNA samples were co-immunoprecipitated with anti-cap antibodies. Northern blots were hybridized with 5′ or 3′ probes as indicated. The positions of the 3′ and 5′ intermediates are shown. In C, one-tenth of the inputs and supernatants and 100% of the immunoprecipitates were analyzed by Northern blot. In D, 100% of the inputs, supernatants, and pellets was loaded. The levels of the intermediates relative to those of the full-length transcript are indicated below the lanes.
FIGURE 8.
FIGURE 8.
Degradation of the 5′ fragments requires ongoing translation. (A,B) Cells expressing 5C-adh were transfected with adh (and GFP) dsRNA as indicated (lanes 3,4). Translation was inhibited with cycloheximide (CHX, 100 μg/mL) for 45 min (lanes 2,4). Total RNA samples were isolated and analyzed by Northern blot with probes detecting the 5′ or the 3′ decay intermediates. Lanes 1 and 2 show that the intermediates are not observed in treated (CHX+) or untreated (CHX-) cells in the absence of adh dsRNA. (C) The levels of the intermediates were normalized to those of the rp49 mRNA in at least three independent experiments. These values were set to 1 in untreated cells (-CHX). Mean values ± SD are shown.

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