Recombinant human Argonaute2 alone is capable of acquiring a guide RNA strand by associating with siRNAs or microRNAs, triggering targeted cleavage of transcripts in vitro, according to a paper published last month.
However, this is true only in certain cases, and Dicer and trans-activation response RNA-binding protein — TRBP — or protein activator of PKR — PACT — are often “essential” for achieving maximal levels of strand selection, the report, appearing in RNA, notes.
“All of this revolves around one of the significant debates in the RNAi field, which regards the extent to which human RISC-loading complexes are, in fact, bona fide RISC-loading complexes,” Cameron Noland, a former researcher at the University of California, Berkeley, and lead author of the paper, told Gene Silencing News.
“Some people have thought that Ago2 in humans can load double-stranded RNAs strand-selectively … on its own,” he said. “Others, including us, have thought that Dicer, TRBP, and PACT are involved in that.”
In a 2011 paper, he and his colleagues in the lab of UC Berkeley researcher Jennifer Doudna examined Dicer and TRBP and PACT, its double-stranded RNA-binding protein partners, to see if they sense the thermodynamics of duplex RNAs in human cells the same way they do in flies.
“Basically, we showed that Dicer is the physical sensor of thermodynamic asymmetry of siRNA duplexes in humans,” he said.
But questions remains for Noland in light of research from a group at the University of Tokyo indicating that, in some cases, mammalian Dicer is dispensable for asymmetric RISC loading, both in vivo and in vitro.
“That raised a lot of questions as to whether this Dicer asymmetry-sensing system in humans was really relevant at all,” he said. The University of Tokyo team, however, only looked at a “very limited set” of duplex RNAs with “very particular parameters in terms of the end nucleotides and terminal mismatches” — all of which could have affected their findings.
As such, Noland decided to try to recapitulate those findings with an expanded set of RNAs in order to test other parameters. By controlling for end nucleotide identities and thermodynamics, the UC Berkeley team could “really start to pick apart the thermodynamic and structural parameters involved in strand selection,” he said.
In the end, the investigators found that Ago2 could properly select a guide strand on its own in some cases.
“It was previously concluded that Dicer is dispensable for asymmetric RISC loading, but our expanded set of duplex RNAs has demonstrated that this conclusion is true only for certain types of duplex RNAs such as those with unfavorable 5’ nucleotides on the passenger strand or with terminal mismatches,” they wrote in RNA.
“Furthermore, even when Ago2 alone is capable of robust strand selection, Dicer and TRBP or PACT, in some cases, enhance that selection, as is the case with the miRNAs tested here,” they added.
Overall, the findings “indicate the presence of a hierarchy of parameters that dictate the necessity of human Dicer and its associated dsRBPs for strand-selection enhancement that considers thermodynamic, 5’ nucleotide, and other structural contributions,” the study’s authors wrote.
To Noland, such parameters could prove useful in the creation of effective siRNAs for research and therapeutic applications, offering guidelines to “systematically test the efficacies of different RNAs with different modifications, and how that relates to the overall silencing efficacy and strand selection.”