PROJECT SUMMARY
Small interfering RNAs (siRNAs) processed from virus-produced double-stranded RNA
(dsRNA) mediate potent antiviral immunity, often termed as antiviral RNA interference (RNAi), in
fungi, plants and invertebrates. Antiviral RNAi is often initiated by dicer proteins which chop viral
dsRNAs into siRNAs. Argonaut (AGO) proteins recruit dicer-produced siRNAs and use them as
sequence guide to identify and destroy viral RNA transcripts with matching sequence. The destruction
of target viral transcripts is mediated by the slicer activity of AGO proteins. dsRNA binding proteins
(DRBPs) also contribute to antiviral RNAi by facilitating siRNA production or loading into AGO
proteins.
Currently, whether RNAi mediates antiviral immunity in mammals under physiological
conditions is still under hot debate. Although the antiviral activity of mammalian RNAi is active in
non-differentiated stem cells and newborn mice it becomes undetectable in differentiated cells and
fully developed mice. The fact that both dicer and the AGO protein required for siRNA production
remain functionally active in differentiated cells suggest that there is a regulatory mechanism that
actively suppresses the antiviral activity of mammalian RNAi in differentiated cells. Currently, how
this regulatory mechanism works remains largely unknown. Since key mammalian RNAi genes, such
as those encoding dicer and Ago2, are required for the biogenesis or function of miRNAs, which
regulate essential cell differentiation and division, it is impossible to knock out these genes to study the
antiviral function of mammalian RNAi without causing cell growth arrest or lethality.
By co-delivery of human dicer and Ago2, one of the human Ago proteins with slicer activity,
the PI’s lab successfully reconstituted human antiviral RNAi in C. elegans. Importantly, the antiviral
activity is further enhanced in the presence of a human TRBP transgene but appears to be suppressed
in the presence of a human PACT transgene. Both TRBP and PACT are dsRNA-binding proteins
sharing similar domain structure. Previous in vitro studies have demonstrated that whereas TRBP
facilitates the processing of dsRNA by dicer PACT seems to inhibit dicer processing of dsRNA. These
observations together with our finding suggest that TRBP and PACT conversely regulate the antiviral
activity of mammalian RNAi and PACT may dominate the regulation, leading to suppressed antiviral
activity under physiological conditions. Here we propose to study the negative regulation of human
antiviral RNAi by PACT in C. elegans. Findings from the proposed research may not only allow us to
gain insight into the mechanism by which mammalian antiviral RNAi is regulated in differentiated
cells but also facilitate the development of novel therapeutic strategies for viral infection prevention.