NEW YORK (GenomeWeb) – A Massachusetts Institute of Technology-led research team this month reported new data suggesting that an siRNA targeting a messenger RNA's 3' untranslated region (UTR), rather than its coding sequence (CDS), can harness the activity of multiple Argonaute proteins to better suppress its target.
As such, RNAi-based therapeutics might benefit from targeting 3' UTRs in order to engage Ago proteins that lack any slicing activity into the target knockdown process, the scientists wrote in their study.
When it comes to RNAi in mammals, one Ago protein in particular — Ago2 — has been shown to be a critical component of the RNA-induced silencing complex as it is the only member of this family that is capable of target mRNA cleavage.
Little is known, however, about the functionality of siRNA binding to the other three mammalian Ago proteins, and what data are available are conflicting. For instance, while some studies have shown residual target knockdown in the absence of Ago2, others have reported not seeing any response to siRNA treatment in cells lacking the protein.
Further, all four Ago proteins encoded in the mammalian genome are expressed in most tissues and cultured mammalian cell lines, the MIT group wrote in their study, which appeared in PLOS One. They have also all have been shown to bind microRNAs and siRNA indiscriminately of sequence to interact with a common set of helicases and mRNA-binding proteins, and to localize to P-bodies in mammalian cells with a capability of targeting mRNAs to the general eukaryotic machinery for translation control and mRNA degradation.
To better understand the roles of individual mammalian Ago proteins in the RNAi process, the research group, which included scientists from Alnylam Pharmaceuticals, conducted a systematic analysis of the effect of Ago2 absence on the knockdown of endogenous genes by siRNAs targeting either CDS or 3' UTR in vitro.
They found that, in cells lacking Ago2, 3' UTR-targeting siRNAs retained some silencing activity, while those targeting CDS did not cause any knockdown, even at high doses.
To extend these observations in vivo, the researchers examined the knockdown effect of siRNAs targeting the CDS and 3' UTR of three genes — coagulation factor VII, fatty acid desaturase 1, and Ras-related protein Rab-5C — in mice.
In line with their cell-based experiments, they found a complete loss of activity of the siRNAs targeting CDS regions of the three genes in the absence of Ago2 in mouse liver.
The team further observed a persistence of knockdown by siRNAs targeting 3' UTRs of the same three genes, and discovered that both Ago1 and Ago3 proteins present in physiological amounts contribute to residual knockdown observed in the absence of Ago2 in liver, according to the PLOS One report.
Based on their findings, the researchers suggested those using RNAi for either therapeutic or research applications may benefit from targeting 3'UTRs in order to turn non-slicer Argonautes from siRNA deposit/sequestration platforms into active participants of knockdown.