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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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siRNA microwalk around Il4R-α polyA site. (A) Position of siRNAs relative to Il4R-α upstream poly A site. PolyA site is shown in grey. Wild-type (B) and Ago2−/− (C) MEF cells 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. (D) Ago2−/− fibroblasts were transfected with siRNAs 100 nM. Western blot analysis was performed 24 h post-transfection as detailed in ‘Materials and Methods’ section. A total of 50 µg protein was loaded in each lane.
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gks239-F7: siRNA microwalk around Il4R-α polyA site. (A) Position of siRNAs relative to Il4R-α upstream poly A site. PolyA site is shown in grey. Wild-type (B) and Ago2−/− (C) MEF cells 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. (D) Ago2−/− fibroblasts were transfected with siRNAs 100 nM. Western blot analysis was performed 24 h post-transfection as detailed in ‘Materials and Methods’ section. A total of 50 µg protein was loaded in each lane.

Mentions: Il4R-α siRNA 383281 binds at position 3555–3575 of the Il4R-α mRNA sequence (ACC. NM_001008700). Since this target site overlaps a putative polyA signal (position 3568–3573), we hypothesized that this siRNA may be interfering with polyadenylation of the pre-mRNA. The siRNA target site was shifted relative to this polyA site as depicted in Figure 7A. Activity of the shifted siRNAs was assessed by qRT/PCR in wild-type and Ago2−/− MEFs as previously detailed. In wild-type cells, siRNAs 459728 and 459732 were less active than the parent siRNA 383281 (Figure 7B), however a comparable level of activity was maintained in the Ago2−/− cells (Figure 7C), suggesting that in both cell types the activity is primarily Ago2 independent. siRNA 459730 was significantly less active, even though it covers the entire polyA signal. This may reflect a directionality requirement for hybridization at the site or may be the result of the reduced binding affinity of si459730 relative to si383281. Ago2−/− MEFs were also treated at a single concentration of 100 nM of the same series of siRNAs, followed 24 h later by western blot analysis. Each of these siRNAs also effectively reduced expression of Il4R-α protein, although there was less discrimination in activity at the concentration used in this experiment. Still, as for the reduction of RNA, si459730 appears to be less active than the others.Figure 7.


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

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

siRNA microwalk around Il4R-α polyA site. (A) Position of siRNAs relative to Il4R-α upstream poly A site. PolyA site is shown in grey. Wild-type (B) and Ago2−/− (C) MEF cells 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. (D) Ago2−/− fibroblasts were transfected with siRNAs 100 nM. Western blot analysis was performed 24 h post-transfection as detailed in ‘Materials and Methods’ section. A total of 50 µg protein was loaded in each lane.
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gks239-F7: siRNA microwalk around Il4R-α polyA site. (A) Position of siRNAs relative to Il4R-α upstream poly A site. PolyA site is shown in grey. Wild-type (B) and Ago2−/− (C) MEF cells 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. (D) Ago2−/− fibroblasts were transfected with siRNAs 100 nM. Western blot analysis was performed 24 h post-transfection as detailed in ‘Materials and Methods’ section. A total of 50 µg protein was loaded in each lane.
Mentions: Il4R-α siRNA 383281 binds at position 3555–3575 of the Il4R-α mRNA sequence (ACC. NM_001008700). Since this target site overlaps a putative polyA signal (position 3568–3573), we hypothesized that this siRNA may be interfering with polyadenylation of the pre-mRNA. The siRNA target site was shifted relative to this polyA site as depicted in Figure 7A. Activity of the shifted siRNAs was assessed by qRT/PCR in wild-type and Ago2−/− MEFs as previously detailed. In wild-type cells, siRNAs 459728 and 459732 were less active than the parent siRNA 383281 (Figure 7B), however a comparable level of activity was maintained in the Ago2−/− cells (Figure 7C), suggesting that in both cell types the activity is primarily Ago2 independent. siRNA 459730 was significantly less active, even though it covers the entire polyA signal. This may reflect a directionality requirement for hybridization at the site or may be the result of the reduced binding affinity of si459730 relative to si383281. Ago2−/− MEFs were also treated at a single concentration of 100 nM of the same series of siRNAs, followed 24 h later by western blot analysis. Each of these siRNAs also effectively reduced expression of Il4R-α protein, although there was less discrimination in activity at the concentration used in this experiment. Still, as for the reduction of RNA, si459730 appears to be less active than the others.Figure 7.

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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