The role of PACT in the RNA silencing pathway

Abstract

Small RNA-mediated gene silencing (RNA silencing) has emerged as a major regulatory pathway in eukaryotes. Identification of the key factors involved in this pathway has been a subject of rigorous investigation in recent years. In humans, small RNAs are generated by Dicer and assembled into the effector complex known as RNAinduced silencing complex (RISC) by multiple factors including hAgo2, the mRNA-targeting endonuclease, and TRBP (HIV-1 TAR RNA-binding protein), a dsRNA-binding protein that interacts with both Dicer and hAgo2. Here we describe an additional dsRNA-binding protein known as PACT, which is significant in RNA silencing. PACT is associated with an B500 kDa complex that contains Dicer, hAgo2, and TRBP. The interaction with Dicer involves the third dsRNA-binding domain (dsRBD) of PACT and the Nterminal region of Dicer containing the helicase motif. Like TRBP, PACT is not required for the pre-microRNA (miRNA) cleavage reaction step. However, the depletion of PACT strongly affects the accumulation of mature miRNA in vivo and moderately reduces the efficiency of small interfering RNA-induced RNA interference. Our study indicates that, unlike other RNase III type proteins, human Dicer may employ two different dsRBD-containing proteins that facilitate RISC assembly.

Biochemical mechanisms of the RNA-induced silencing complex

“…siRNA knockdown of TRBP resulted in diminished miRNA production and loss of reportergene silencing in HeLa cells [35,36]. Similar results were observed in similar experiments targeting PACT [37]. However, the precise role of TRBP and PACT in mediating human RISC activity remains poorly understood.…”

“…Transactivating response RNA-binding protein (TRBP) and protein activator of protein kinase R (PACT) each harbor three annotated dsRNA-binding domains and have been shown to interact with Dicer [35][36][37]. siRNA knockdown of TRBP resulted in diminished miRNA production and loss of reportergene silencing in HeLa cells [35,36].…”

Principles and effects of microRNA-mediated post-transcriptional gene regulation

“…In addition, orthologues of R2D2 are important for RNAi and miRNA processing in worms flies and mammals. The miRNA specific fly Dicer-1 requires Loquacious, for proper miRNA processing, Dicer in worms coimmunoprecipitates with RDE-4, a dsRNA-binding protein essential for RNAi initiated with long dsRNA, and human Dicer requires two R2D2 orthologues, TRBP and PACT, for miRNA-mediated gene silencing (Tabara et al, 2002;Forstemann et al, 2005;Haase et al, 2005;Jiang et al, 2005;Saito et al, 2005;Lee et al, 2006). Furthermore, Argonautes seem to interact physically with the miRNA maturation and loading machinery as immunoprecipitated hAgo2 complexes contain functional Dicer activity that can asymmetrically process exogenous pre-miRNAs, generating mature RISC that can cleave a perfect complementary target RNA Maniataki and Mourelatos, 2005;Meister et al, 2005).…”

Principles of micro-RNA production and maturation

“…Nevertheless, just like DGCR8 in the case of Drosha, proteins with double-stranded RNA-binding domains, such as TRBP and PACT in humans (Figure 3), bind to Dicer and contribute to Dicer function Forstemann et al, 2005;Gregory et al, 2005;Haase et al, 2005;Jiang et al, 2005;Maniataki and Mourelatos, 2005;Saito et al, 2005;Lee et al, 2006). Thus, they can enhance the affinity of Dicer for RNAs and participate in the selection of mature miRNA strands and/or the transfer of miRNAs to their final stop, the Argonaute proteins.…”

“…Dicer or its complex interacts with a family of conserved proteins called Argonautes (Bernstein et al, 2001;Hammond et al, 2001;Doi et al, 2003;Tahbaz et al, 2004;Chendrimada et al, 2005;Forstemann et al, 2005;Gregory et al, 2005;Haase et al, 2005;Jiang et al, 2005;Maniataki and Mourelatos, 2005;Saito et al, 2005;Lee et al, 2006). Mature miRNAs are eventually transferred to Argonaute proteins and serve as guides in RNA silencing (Caudy et al, 2002;Hutva´gner and Zamore, 2002;Ishizuka et al, 2002;Mourelatos et al, 2002;Liu et al, 2004;Meister et al, 2004).…”