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Isis Team Reports on Identification of Argonaute-, RISC-Independent siRNAs

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Researchers from Isis Pharmaceuticals this week reported in vitro evidence on the existence of a new class of siRNA, which can promote target RNA reduction independent of both Argonaute and RISC.

The authors of the study, which appeared in Nucleic Acids Research, caution that much work remains to be done before the significance of these siRNAs can be established, however.

Most siRNAs silence their targets through an mRNA cleavage and degradation process mediated by Ago2 and RISC. However, recent studies suggest that siRNA-mediated off-target mRNA reduction occurs through Ago2-independent processes, directing target reduction through deadenylation and degradation of target transcripts through a process likely involving P-bodies, the Isis team explained in Nucleic Acids Research.

“More recently, it has been demonstrated that at least a portion of some siRNA on-target activity is also Ago2 independent,” they noted.

As part of an effort to identify new Ago2 cleavage-independent siRNAs, the Isis investigators screened a number of siRNAs against interleukin-4 receptor-alpha and PTEN in both wild-type and Ago2-lacking mouse fibroblasts.

They discovered several of the small RNAs with significant activity, including one that was equally active in both wild-type cells and those without Ago2. All of the PTEN siRNAs displayed similar activity in the normal cells, but variable activity in the Ago2-/- cells. Meantime, the IL4R-alpha siRNAs exhibited variable and limited activity in both sets of cells except for one, which had similar efficacy in both.

Follow-on testing with control siRNAs confirmed that the inhibition was sequence-specific, and that the IL4R-alpha was being inhibited by the active siRNA.

“Since we had previously shown that Ago2-independent siRNA activity may be mediated by Ago1, the effect of Ago1 reduction on the activity of [the siRNA] was next evaluated,” the team wrote. “Ago2-/- fibroblasts were treated with [antisense oligos] targeting Ago1, Ago3, or Ago [and after 48 hours], Ago-reduced and wild-type cells were treated.”

The IL4R-alpha siRNA was again found to have similar activity in both wild-type and Ago2-/- cells, while an siRNA against PTEN was primarily active in wild-type cells only.

The Ago2-independent activity of [the PTEN siRNA] was not affected by reducing Ago3 or Ago4, however reduction of Ago1 in the Ago2-/- cells resulted in a complete ablation of activity,” according to the paper. “In contrast, the activity [of the IL4R-alpha] siRNA … was not affected by reduction of any of the Ago proteins.”

Because siRNA target degradation may occur in P-bodies even in the absence of Ago2-mediated cleavage, the researchers treated Ago2-/- fibroblasts with antisense oligos against Ago1 or with P-body-associated proteins GW182 and DDX6.

After 48 hours, both the Ago1/P-body reduced cells and control cells were treated with siRNAs against PTEN and IL4R-alpha. The team found that cutting Ago1 or P-body proteins inhibited the Ago2-independent activity of the PTEN siRNA, suggesting its activity is RISC-dependent and mediated via a P-body-dependent degradation pathway. In contrast, the activity of the IL4R-alpha was unaffected by either Ago1 or P-body reduction.

“Together, these data suggest a novel siRNA-mediated degradation pathway independent of Ago1-4, RISC, and associated P-body degradation,” the researchers wrote.

Additional experimentation showed that the IL4R-alpha siRNA does not compete with other siRNAs for free RISC, reinforcing the notion that it functions in a RISC-independent manner, and that modifications preventing Ago binding do not hinder its activity.

Noting that the IL4R-alpha siRNA binds a position of the IL4R-alpha mRNA that overlaps a putative polyA signal, the Isis researchers hypothesized that the siRNA may be interfering with the polyadenylation of the pre-mRNA.

“The siRNA target site was shifted relative to this polyA site [and] … activity of the shifted siRNAs was assessed by qRT/PCR in wild-type and Ago2-/-” fibroblasts, according to the paper.

The investigators concluded that the siRNA is “capable of redirecting a small amount of polyadenylation to a downstream polyA site. However, degradation of the message appears to be the result of inhibition of polyadenylation or deadenylation of the transcript.”

They added that siRNAs targeted to the polyA sites of certain other RNAs were ”also capable of promoting target degradation in an Ago and P-body independent manner.”

Overall, the data support a model in which “siRNA targeted to the polyA signal results in deadenylation or inhibition of polyadenylation and subsequent degradation of the immature mRNA transcript,” they wrote, noting that this model requires the presence of the siRNA in the nucleus.

Additional work remains to answer questions including where and how the IL4R-alpha siRNA is unwound, and how the antisense strand makes its way into the nucleus.

“Finally, it is not clear how important or broadly applicable the proposed mechanism is,” the authors stressed. “Clearly, more screening will be required in order to determine if this is a rare process limited to few mRNA targets, or is more ubiquitous.”

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