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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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Multiple siRNAs demonstrate significant Ago2-independent activity. Wild-type and Ago2−/− mouse embryonic fibroblasts (MEFs) were transfected with siRNAs at concentrations ranging from 300 pM to 300 nM as detailed in ‘Materials and Methods’ section. The following day target mRNA reduction was measured by qRT/PCR. Percent inhibition at each treatment concentration is shown. (A) PTEN siRNA activity in WT cells. (B) PTEN siRNA activity in Ago2−/− cells. (C) Il4R-α siRNA activity in WT cells. (D) Il4R-α siRNA activity in Ago2−/− cells.
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gks239-F1: Multiple siRNAs demonstrate significant Ago2-independent activity. Wild-type and Ago2−/− mouse embryonic fibroblasts (MEFs) were transfected with siRNAs at concentrations ranging from 300 pM to 300 nM as detailed in ‘Materials and Methods’ section. The following day target mRNA reduction was measured by qRT/PCR. Percent inhibition at each treatment concentration is shown. (A) PTEN siRNA activity in WT cells. (B) PTEN siRNA activity in Ago2−/− cells. (C) Il4R-α siRNA activity in WT cells. (D) Il4R-α siRNA activity in Ago2−/− cells.

Mentions: We have previously shown that a significant portion of on-target siRNA activity mediated by an siRNA directed against PIK3CB is maintained in mouse Ago2−/− cells and is Ago2 cleavage independent (17). To identify other Ago2 cleavage independent siRNAs, a series of siRNAs directed against Il4R-α or PTEN was screened in wild-type and Ago2−/− mouse fibroblasts. All PTEN siRNAs evaluated had similar activity in wild-type cells each with an IC50 of ∼10 nM (Figure 1A). In Ago2−/− cells, the activity of the same set of siRNAs was more variable (Figure 1B), and the potency and efficacy (maximum target reduction) were much lower than observed in the wild type cells. The siRNAs targeted to Il4R-α displayed a greater variation in activity in the wild-type cells with IC50’s ranging from ∼5–50 nM (Figure 1C). While all, but one Il4R-α siRNA (383266) exhibited some activity in the Ago2−/− cells (Figure 1D), one siRNA, 383281, had similar efficacy and an IC50 of <10 nM in both Ago2- and wild-type cells.Figure 1.


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

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

Multiple siRNAs demonstrate significant Ago2-independent activity. Wild-type and Ago2−/− mouse embryonic fibroblasts (MEFs) were transfected with siRNAs at concentrations ranging from 300 pM to 300 nM as detailed in ‘Materials and Methods’ section. The following day target mRNA reduction was measured by qRT/PCR. Percent inhibition at each treatment concentration is shown. (A) PTEN siRNA activity in WT cells. (B) PTEN siRNA activity in Ago2−/− cells. (C) Il4R-α siRNA activity in WT cells. (D) Il4R-α siRNA activity in Ago2−/− cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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gks239-F1: Multiple siRNAs demonstrate significant Ago2-independent activity. Wild-type and Ago2−/− mouse embryonic fibroblasts (MEFs) were transfected with siRNAs at concentrations ranging from 300 pM to 300 nM as detailed in ‘Materials and Methods’ section. The following day target mRNA reduction was measured by qRT/PCR. Percent inhibition at each treatment concentration is shown. (A) PTEN siRNA activity in WT cells. (B) PTEN siRNA activity in Ago2−/− cells. (C) Il4R-α siRNA activity in WT cells. (D) Il4R-α siRNA activity in Ago2−/− cells.
Mentions: We have previously shown that a significant portion of on-target siRNA activity mediated by an siRNA directed against PIK3CB is maintained in mouse Ago2−/− cells and is Ago2 cleavage independent (17). To identify other Ago2 cleavage independent siRNAs, a series of siRNAs directed against Il4R-α or PTEN was screened in wild-type and Ago2−/− mouse fibroblasts. All PTEN siRNAs evaluated had similar activity in wild-type cells each with an IC50 of ∼10 nM (Figure 1A). In Ago2−/− cells, the activity of the same set of siRNAs was more variable (Figure 1B), and the potency and efficacy (maximum target reduction) were much lower than observed in the wild type cells. The siRNAs targeted to Il4R-α displayed a greater variation in activity in the wild-type cells with IC50’s ranging from ∼5–50 nM (Figure 1C). While all, but one Il4R-α siRNA (383266) exhibited some activity in the Ago2−/− cells (Figure 1D), one siRNA, 383281, had similar efficacy and an IC50 of <10 nM in both Ago2- and wild-type cells.Figure 1.

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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