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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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Il4R-α siRNA activity does not require P-body associated proteins. Ago2−/− MEFs were treated with ASOs targeting Ago1 or with P-body (PB) associated proteins GW182 and DDX6. The following day Ago1 or P-body reduced cells were seeded in 96-well plates then transfected with PTEN or Il4R-α siRNA at concentrations ranging from 300 pM to 300 nM. Messenger RNA reduction was measured after 24 h by qRT/PCR. IC50 curves are plotted versus no siRNA control for each treatment condition. (A) Activity of PTEN siRNA 29592 in WT cells (filled triangle), KO cells (circle), or KO cells reduced in Ago 1 (inverted triangle) or PB (filled circle). (B) Activity of Il4R-α siRNA 383281 in WT cells (filled triangle), KO cells (circle), or KO cells reduced in Ago 1 (inverted triangle) or PB (filled circle).
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gks239-F4: Il4R-α siRNA activity does not require P-body associated proteins. Ago2−/− MEFs were treated with ASOs targeting Ago1 or with P-body (PB) associated proteins GW182 and DDX6. The following day Ago1 or P-body reduced cells were seeded in 96-well plates then transfected with PTEN or Il4R-α siRNA at concentrations ranging from 300 pM to 300 nM. Messenger RNA reduction was measured after 24 h by qRT/PCR. IC50 curves are plotted versus no siRNA control for each treatment condition. (A) Activity of PTEN siRNA 29592 in WT cells (filled triangle), KO cells (circle), or KO cells reduced in Ago 1 (inverted triangle) or PB (filled circle). (B) Activity of Il4R-α siRNA 383281 in WT cells (filled triangle), KO cells (circle), or KO cells reduced in Ago 1 (inverted triangle) or PB (filled circle).

Mentions: Since degradation of a siRNA targeted message may occur in P-bodies even in the absence of Ago2-mediated cleavage (25), the effect of P-body reduction on the activity of the PTEN and Il4R-α siRNAs was next evaluated. Ago2−/− fibroblasts were treated with ASOs targeting Ago1 or with P-body associated proteins GW182 and DDX6. Reduction of the targeted RNAs was confirmed to be >80% by qRT/PCR (data not shown). After 48 h, Ago1/P-body reduced and control Ago2−/− MEF cells were treated with 3–300 nM siRNA. Reduction of either Ago1 or the P-body proteins resulted in a reduction in Ago2-independent activity of PTEN siRNA 29592 (Figure 4A), suggesting that the Ago2-independent activity of this siRNA is RISC dependent and mediated via a P-body dependent degradation pathway. In contrast, the activity of Il4R-α siRNA 383281 neither was affected by Ago1 reduction nor was activity diminished in the P-body reduced cells (Figure 4B). Together, these data suggest a novel siRNA-mediated degradation pathway independent of Ago1-4, RISC, and associated P-body degradation.Figure 4.


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

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

Il4R-α siRNA activity does not require P-body associated proteins. Ago2−/− MEFs were treated with ASOs targeting Ago1 or with P-body (PB) associated proteins GW182 and DDX6. The following day Ago1 or P-body reduced cells were seeded in 96-well plates then transfected with PTEN or Il4R-α siRNA at concentrations ranging from 300 pM to 300 nM. Messenger RNA reduction was measured after 24 h by qRT/PCR. IC50 curves are plotted versus no siRNA control for each treatment condition. (A) Activity of PTEN siRNA 29592 in WT cells (filled triangle), KO cells (circle), or KO cells reduced in Ago 1 (inverted triangle) or PB (filled circle). (B) Activity of Il4R-α siRNA 383281 in WT cells (filled triangle), KO cells (circle), or KO cells reduced in Ago 1 (inverted triangle) or PB (filled circle).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gks239-F4: Il4R-α siRNA activity does not require P-body associated proteins. Ago2−/− MEFs were treated with ASOs targeting Ago1 or with P-body (PB) associated proteins GW182 and DDX6. The following day Ago1 or P-body reduced cells were seeded in 96-well plates then transfected with PTEN or Il4R-α siRNA at concentrations ranging from 300 pM to 300 nM. Messenger RNA reduction was measured after 24 h by qRT/PCR. IC50 curves are plotted versus no siRNA control for each treatment condition. (A) Activity of PTEN siRNA 29592 in WT cells (filled triangle), KO cells (circle), or KO cells reduced in Ago 1 (inverted triangle) or PB (filled circle). (B) Activity of Il4R-α siRNA 383281 in WT cells (filled triangle), KO cells (circle), or KO cells reduced in Ago 1 (inverted triangle) or PB (filled circle).
Mentions: Since degradation of a siRNA targeted message may occur in P-bodies even in the absence of Ago2-mediated cleavage (25), the effect of P-body reduction on the activity of the PTEN and Il4R-α siRNAs was next evaluated. Ago2−/− fibroblasts were treated with ASOs targeting Ago1 or with P-body associated proteins GW182 and DDX6. Reduction of the targeted RNAs was confirmed to be >80% by qRT/PCR (data not shown). After 48 h, Ago1/P-body reduced and control Ago2−/− MEF cells were treated with 3–300 nM siRNA. Reduction of either Ago1 or the P-body proteins resulted in a reduction in Ago2-independent activity of PTEN siRNA 29592 (Figure 4A), suggesting that the Ago2-independent activity of this siRNA is RISC dependent and mediated via a P-body dependent degradation pathway. In contrast, the activity of Il4R-α siRNA 383281 neither was affected by Ago1 reduction nor was activity diminished in the P-body reduced cells (Figure 4B). Together, these data suggest a novel siRNA-mediated degradation pathway independent of Ago1-4, RISC, and associated P-body degradation.Figure 4.

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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