Mechanistic Studies of Human RISC Loading Complex Function in RNA Interference

RNA interference (RNAi) pathways are critical eukaryotic post-transcriptional gene regulatory systems that control the expression of at least one third of all human coding genes. In each of these pathways, short single-stranded RNAs (ssRNAs) bind to cognate messenger RNAs (mRNAs) and direct their endonucleolytic cleavage or translational repression by RNA-Induced Silencing Complexes (RISCs). In the cytoplasmic portion of these pathways, silencing is triggered by long double-stranded RNA (dsRNA) precursors (pre-siRNAs) or hairpin RNAs (pre-miRNAs), which are processed by the RNase III enzyme Dicer to yield short 21-23 nucleotide (nt) duplex RNAs termed short-interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. These short duplex RNAs are then loaded onto Argonaute2 (Ago2), the catalytic component of RISC. Ago2 retains one strand of the duplex (the "guide" strand) for subsequent gene targeting and discards the other. This process of asymmetrically loading siRNA or miRNA duplexes onto RISCs such that one strand is preferentially retained is fundamental to the target specificity of RNAi pathways./nStructural studies of the individual components of the RNAi machinery have proven immensely informative to our mechanistic understanding of the individual steps of RNAi pathways such as substrate cleavage by Dicer and subsequent mRNA targeting by Ago2. What has been missing is a clear picture of how different RNAi components functionally interact to transfer small RNAs from one protein to another in a way that ultimately allows for the selection of the proper guide strand by Ago2. In Drosophila and humans, protein complexes called RISC Loading Complexes (RLCs) have been implicated in facilitating this process. At their core, these complexes are composed of Ago2, Dicer, and a dsRNA binding protein (dsRBP). Two such complexes exist in humans and are defined by their associated dsRBP. One complex contains HIV-1 Trans-activation Response (TAR) RNA Binding Protein (TRBP) and the other contains Protein Activator of PKR (PACT). Here we have used electron microscopy (EM) and single particle reconstruction methods to show that human Dicer has an L-shaped architecture consisting of a long catalytic branch and a shorter DExH/D helicase-containing base branch. Further, we present a three-dimensional (3D) reconstruction of a highly dynamic human RLC containing Ago2, Dicer, and TRBP. TRBP interacts with the distal end of Dicer's helicase branch, whereas Ago2 interacts with Dicer's catalytic branch. The structural information garnered in this study, combined with previous biochemical data, led to a testable model for how human RLCs efficiently transfer Dicer products to Ago2 in an orientation that allows for accurate selection of the targeting strand./nThe preferred guide strand of any given siRNA or miRNA can be predicted with high accuracy based on the thermodynamics of the duplex ends. The RNA strand that has its 5´ end at the less stable end of the duplex is preferentially loaded onto Ago2 as the guide strand. In flies, an RLC subcomplex containing Dicer-2 and the dsRBP R2D2 is involved in sensing the thermodynamics of siRNAs prior to their loading onto Ago2, although it is unclear which protein is the actual sensor of thermodynamic asymmetry. In humans the sensor of siRNA thermodynamics has been unclear, given that Dicer forms a complex with either TRBP or PACT. In plants, some Dicer/dsRBP heterodimers are involved in sensing siRNA thermodynamics while others are not. Using in vitro biochemical experiments that were guided by structural insights, we show that human Dicer products are released and then rebound at a novel site along Dicer's helicase domain in a TRBP- or PACT-dependent manner. This novel binding site allows either Dicer/dsRBP heterodimer to sense siRNA thermodynamic asymmetry such that Dicer binds to the less stable end of the duplex and the associated dsRBP binds to the more stable end. We further demonstrate that Dicer itself is the sensor of siRNA thermodynamics and that this functionality is activated upon association with either TRBP or PACT. These crucial insights into siRNA positioning by human Dicer/dsRBP complexes led to a revised model for how strand-selective RISC loading may be achieved by RLCs in human RNAi pathways./nAlthough structural and biochemical data supports the notion that human RLCs function similarly to the Drosophila Ago2/Dicer-2/R2D2 RLC in strand-selective RISC loading, a number of clear differences exist. For example, in humans it has been shown that Ago2 can bind siRNAs and miRNAs in the absence of Dicer, which is not the case in flies. Furthermore, in certain cases Ago2 alone binds to duplex RNAs strand-selectively. The extent to which human RLCs are actually important for Ago2 loading and strand selection has therefore been controversial. We have used a reconstituted system to determine the degree to which each of the core components of the human RNAi machinery contributes to RISC loading and guide strand selection. We show that Ago2 has intrinsic but substrate-dependent strand selection capabilities. This activity, however, is in many cases enhanced substantially when Ago2 is in complex with Dicer and TRBP or PACT as a functional RLC. Our findings suggest that rather than functioning exclusively in human strand selection, Ago2's binding preferences serve instead as a secondary RISC loading checkpoint that acts in concert with Dicer/dsRBP asymmetry sensors to ensure proper strand selection by RLCs. The specific roles of each component in this process are dictated and fine-tuned by specific duplex parameters such as thermodynamics, 5´ nucleotide identity, and duplex structure. Surprisingly, our results also show that strand selection for some miRNAs is enhanced by PACT-containing complexes but not by those containing TRBP. Furthermore, overall mRNA targeting by miRNAs is disfavored for complexes containing TRBP but not PACT. This key finding reveals the possibility that RLCs containing TRBP may be optimized for the siRNA pathway, whereas RLCs containing PACT are optimized for the miRNA pathway. Overall, the body of work presented herein represents a significant step forward in our understanding of how core components of human RNAi pathways functionally interact within RLCs to achieve proper target specificity through strand-selective RISC loading.

Advisor: Doudna, Jennifer A