The presence of double-stranded RNA in most eukaryotic cells triggers a sequence-specific gene silencing response known as RNA interference. The dsRNA trigger of this process can be derived from exogenous sources or transcribed from endogenous non-coding RNA genes that produce microRNAs. These and similarly structured silencing triggers feed into a common biochemical pathway, which can result in the sequence-specific suppression of gene expression through one of several effector mechanisms. RNAi begins with the stepwise conversion of the dsRNA silencing triggers into small RNAs of ~21-26 nt in length. This is accomplished by the processing of triggers by specialized members of the RNAseIII family of nucleases, Dicer and Drosha. Resulting small RNAs join the effector complex, known generically as RISC (RNA-Induced Silencing Complex). This occurs though a concerted assembly process in which the RISC-Loading Complex (RLC) acts in an energy-dependent manner to place one strand of the small RNA into RISC. Asymmetry is sensed within an apparently symmetric small dsRNA using thermodynamic criteria; the strand with the less stable duplex at its 5' end is preferentially incorporated into RISC. RISC complex contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. RISC functions through several distinct mechanisms: mRNA cleavage and degradation, translational repression, and chromatin modification.
RNAi first came to fame for its now well-known utility as an exprimental tool for specific gene silencing. With increasing understanding of this amazing process, the research community realized that RNAi provides an important level of regulation of gene expression in physiological processes including cell proliferation, apoptosis, differentiation and development. The role of RNAi in endogenous processes was brought to light by the discovery of hundreds of small endogenous noncoding RNAs, microRNAs. These endogenous triggers of RNAi incorporate into Ago-containing RISC complexes and potentially can regulate thousands of target genes.
The best-studied mode of RISC action is mRNA cleavage and degradation. When programmed with a small RNA that is fully or near fully complementary to the substrate RNA, RISC catalyzes an endonucleolytic cleavage of the target. We recently showed the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for mRNA cleavage activity. A nuclease domain within Argonaute contributes the `Slicer` activity to RISC and provides the catalytic engine for RNAi. However, the majority of mammalian miRNAs do not pair perfectly with their targets and thus are unable to direct the cleavage. Nevertheless, miRNAs still silence the expression of their target genes, presumably at the translational level. We recently demonstrated that mammalian Argonaute proteins localized to specific cytoplasmic foci known as processing bodies (P/GW-bodies) regardless of their catalytic activity. Moreover, reporter mRNAs targeted for translational repression by endogenous or exogenous miRNAs became concentrated in P-bodies in a miRNA-dependent manner. We also demonstrated that the integrity of P-bodies is critical for the RNAi silencing by the small RNA. We are in the process of understanding the precise nature of the functional link between cytoplasmic P-bodies and the ability of a microRNA to repress expression of its target mRNA. The translation apparatus is spatially organized within the cell. Delivery of mRNAs to P-bodies through RISC could provide the compartmentalization that is important for miRNA-regulated repression.
P-bodies were first considered only as sites for mRNA degradation. Our new miRNA study suggests P-bodies also function as storage sites for translationally repressed mRNAs. New studies from Roy Parker's group demonstrated that, in S. cerevisiae, translationally repressed mRNAs stored in the P-bodies could return to the translation machinery through regulated mechanisms. This raised an interesting question regarding the fates of miRNA target mRNAs in the P-body. Degradation or storage of the mRNAs provides irreversible or reversible control on the gene expression, respectively. We are interested in understanding how these different processes are regulated.
Our principal research interest is to understand the regulation of mammalian P-body and how miRNAs mediate gene silencing. We are also interested in understanding the potential connection between RNAi factors/P-body and cancer; how RNAi plays a role in transcriptional gene silencing (TGS) in mammalian system.