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siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs.

Vickers TA, Crooke ST - Nucleic Acids Res. (2012)

Bottom Line: While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies.In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC.These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA. tvickers@isisph.com

ABSTRACT
While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

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Exosome reduction leads to specific inhibition of Il4R-α siRNA 383281 activity. Ago2−/− MEFs were treated with antisense ASOs targeting exosomal proteins Exosc9 and SKIV2l. The following day cells were seeded in 96-well plates, then transfected with si383281. Cells were harvested the following day and expression of Il4R-α mRNA evaluated by quantitative RT/PCR. (A) Concentration response curves for si383281 in control (solid line) or exosome-reduced (dashed line) Ago2−/− MEF cells. (B) Gene expression in exosome-reduced relative to untreated control cells at 48 h post-transfection.
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gks239-F9: Exosome reduction leads to specific inhibition of Il4R-α siRNA 383281 activity. Ago2−/− MEFs were treated with antisense ASOs targeting exosomal proteins Exosc9 and SKIV2l. The following day cells were seeded in 96-well plates, then transfected with si383281. Cells were harvested the following day and expression of Il4R-α mRNA evaluated by quantitative RT/PCR. (A) Concentration response curves for si383281 in control (solid line) or exosome-reduced (dashed line) Ago2−/− MEF cells. (B) Gene expression in exosome-reduced relative to untreated control cells at 48 h post-transfection.

Mentions: Degradation of messenger RNA initiated by deadenylation has previously been demonstrated to be mediated by the exosome, a 3′-5′-exoribonuclease complex that functions in both the nucleus and cytoplasm (35). In the nucleus, the RNA exosome complex is involved in the elimination of mRNAs with processing defects, thereby limiting or excluding their export to the cytoplasm. We evaluated the effect of exosome reduction on the activity of Il4R-α si383281. MEFs were treated with 75 nM each of ISIS 308299, an ASO targeting PmSCl-75 (Exosc9), a non-catalytic component of the RNA exosome complex which has 3′ → 5′ exoribonuclease activity, and ISIS 454373, an ASO targeting SKIV2L, a putative RNA helicase, which has been shown to mediate 3′–5′ RNA degradation by the exosome. The following day cells were seeded in 96-well plates, then transfected with si383281. Cells were harvested the following day and expression of Il4R-α mRNA evaluated by quantitative RT/PCR. Reduction of these exosomal proteins resulted in a significant decline in the activity of Il4R-α siRNA 383281 (Figure 9A), suggesting that ago-independent reduction by IL4 targeting siRNA is at least partially mediated by the exosome. The expression of several other transcripts was also evaluated in the same cells (Figure 9B). Interestingly, exosome reduction resulted in an increase in the levels of Il4R-α, but not the other transcripts evaluated. However, more extensive screening using PCR arrays of control and exosome reduced cells identified several transcripts also affected by exosome reduction (Supplementary Figure S4). siRNAs were designed to target the poly A sites of three of these transcripts: GATA3, WAF1 and STAT2. Only the siRNA directed to the polyA site of WAF1 demonstrated comparable activity in both wild-type and Ago2−/− MEF cells (Supplementary Figure S5). The targeted polyA signal is the only one known to be used for WAF1 mRNA. We therefore performed 3′ RACE and qRT/PCR to compare the activity to that of siRNA 383281. Wild-type and Ago2−/− MEFs were treated with WAF1 and Il4R-α siRNA and corresponding full MOE ASOs at 100 nM. The following day 3′ RACE was performed using total RNA purified from the treated cells. As previously observed, treatment with both the siRNA and MOE ASO resulted in a significant decrease in polyadenylated Il4R-α mRNA in WT and Ago2−/− cells (Figure 10A). Quantitative PCR was performed using oligo dT-primed cDNA. Amplification with a primer/probe set specific to the region upstream of the targeted polyA site indicated that treatment with both the Il4R-α siRNA and MOE ASO resulted in a strong decrease in mRNA polyadenylated at site 1 (Figure 10B). However, when a primer/probe set specific to the region downstream of the targeted polyA was used, treatment with the MOE ASO resulted in an increase in mRNA polyadenylated at site 2 or 3. In contrast, treatment with the corresponding siRNA resulted in a decrease in both long and short mRNA, suggesting that the full MOE and siRNA work via different mechanisms.Figure 9.


siRNAs targeted to certain polyadenylation sites promote specific, RISC-independent degradation of messenger RNAs.

Vickers TA, Crooke ST - Nucleic Acids Res. (2012)

Exosome reduction leads to specific inhibition of Il4R-α siRNA 383281 activity. Ago2−/− MEFs were treated with antisense ASOs targeting exosomal proteins Exosc9 and SKIV2l. The following day cells were seeded in 96-well plates, then transfected with si383281. Cells were harvested the following day and expression of Il4R-α mRNA evaluated by quantitative RT/PCR. (A) Concentration response curves for si383281 in control (solid line) or exosome-reduced (dashed line) Ago2−/− MEF cells. (B) Gene expression in exosome-reduced relative to untreated control cells at 48 h post-transfection.
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gks239-F9: Exosome reduction leads to specific inhibition of Il4R-α siRNA 383281 activity. Ago2−/− MEFs were treated with antisense ASOs targeting exosomal proteins Exosc9 and SKIV2l. The following day cells were seeded in 96-well plates, then transfected with si383281. Cells were harvested the following day and expression of Il4R-α mRNA evaluated by quantitative RT/PCR. (A) Concentration response curves for si383281 in control (solid line) or exosome-reduced (dashed line) Ago2−/− MEF cells. (B) Gene expression in exosome-reduced relative to untreated control cells at 48 h post-transfection.
Mentions: Degradation of messenger RNA initiated by deadenylation has previously been demonstrated to be mediated by the exosome, a 3′-5′-exoribonuclease complex that functions in both the nucleus and cytoplasm (35). In the nucleus, the RNA exosome complex is involved in the elimination of mRNAs with processing defects, thereby limiting or excluding their export to the cytoplasm. We evaluated the effect of exosome reduction on the activity of Il4R-α si383281. MEFs were treated with 75 nM each of ISIS 308299, an ASO targeting PmSCl-75 (Exosc9), a non-catalytic component of the RNA exosome complex which has 3′ → 5′ exoribonuclease activity, and ISIS 454373, an ASO targeting SKIV2L, a putative RNA helicase, which has been shown to mediate 3′–5′ RNA degradation by the exosome. The following day cells were seeded in 96-well plates, then transfected with si383281. Cells were harvested the following day and expression of Il4R-α mRNA evaluated by quantitative RT/PCR. Reduction of these exosomal proteins resulted in a significant decline in the activity of Il4R-α siRNA 383281 (Figure 9A), suggesting that ago-independent reduction by IL4 targeting siRNA is at least partially mediated by the exosome. The expression of several other transcripts was also evaluated in the same cells (Figure 9B). Interestingly, exosome reduction resulted in an increase in the levels of Il4R-α, but not the other transcripts evaluated. However, more extensive screening using PCR arrays of control and exosome reduced cells identified several transcripts also affected by exosome reduction (Supplementary Figure S4). siRNAs were designed to target the poly A sites of three of these transcripts: GATA3, WAF1 and STAT2. Only the siRNA directed to the polyA site of WAF1 demonstrated comparable activity in both wild-type and Ago2−/− MEF cells (Supplementary Figure S5). The targeted polyA signal is the only one known to be used for WAF1 mRNA. We therefore performed 3′ RACE and qRT/PCR to compare the activity to that of siRNA 383281. Wild-type and Ago2−/− MEFs were treated with WAF1 and Il4R-α siRNA and corresponding full MOE ASOs at 100 nM. The following day 3′ RACE was performed using total RNA purified from the treated cells. As previously observed, treatment with both the siRNA and MOE ASO resulted in a significant decrease in polyadenylated Il4R-α mRNA in WT and Ago2−/− cells (Figure 10A). Quantitative PCR was performed using oligo dT-primed cDNA. Amplification with a primer/probe set specific to the region upstream of the targeted polyA site indicated that treatment with both the Il4R-α siRNA and MOE ASO resulted in a strong decrease in mRNA polyadenylated at site 1 (Figure 10B). However, when a primer/probe set specific to the region downstream of the targeted polyA was used, treatment with the MOE ASO resulted in an increase in mRNA polyadenylated at site 2 or 3. In contrast, treatment with the corresponding siRNA resulted in a decrease in both long and short mRNA, suggesting that the full MOE and siRNA work via different mechanisms.Figure 9.

Bottom Line: While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies.In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC.These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Inc. 2855 Gazelle Court, Carlsbad, CA 92010, USA. tvickers@isisph.com

ABSTRACT
While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

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