From guide to target: molecular insights into eukaryotic RNA-interference machinery

Abstract

Since its relatively recent discovery, RNA interference (RNAi) has emerged as a potent, specific and ubiquitous means of gene regulation. Through a number of pathways that are conserved in eukaryotes from yeast to humans, small noncoding RNAs direct molecular machinery to silence gene expression. In this Review, we focus on mechanisms and structures that govern RNA silencing in higher organisms. In addition to highlighting recent advances, we discuss parallels and differences among RNAi pathways. Together, the studies reviewed herein reveal the versatility and programmability of RNA-induced silencing complexes and emphasize the importance of both upstream biogenesis and downstream silencing factors.

Figure 1

Strategies for RNA recognition and processing a) Human TRBP (gold), the quintessential double-strand RNA binding protein (dsRBP), is comprised of three double-strand RNA binding domains (dsRBDs) connected by linkers. Each dsRBD binds one face of the double helix (grey) and is situated over the major groove with additional interactions along the adjacent minor grooves. The stability of this complex is almost entirely driven by direct and water-mediated hydrogen-bonding interactions (PDBs: 3ADL [119] and 1DI2 [120]). b) The microprocessor complex (PDB: 2YT4 [20]) responsible for processing pri-miRNAs is comprised of the nucleases Drosha and DGCR8/Pasha. The crystal structure of the dsRBDs of human DGCR8 (pink/purple) suggests it recognizes dsRNA in the same manner as TRBP. A-form RNA (grey) was modeled on each dsRBD based on the structure of TRBP in the presence of dsRNA. c) Crystal structures of mammalian Dicer fragments or homologs (helicase domains from P. furiosis, RNaseIII domain from Giardia) are positioned within the electron microscopic map of human Dicer (grey) based on structures from lower organisms and electron microscopic labeling [31]. The PAZ domain (magenta) recognizes the 3′ 2 nucleotide RNA overhang. Opposing RNaseIII domains (purple) perform duplex cleavage. The cleavage product’s length is measured based on the size of the ruler domain between the PAZ and RNaseIII domains. The helicase domain (green) has been implicated in processivity. d) The endonuclease Zucchini (green) (PDB: 4GGJ [44]) recognizes the phosphate backbone of single-stranded (purple; modeled) piRNA precursor substrates via a narrow, positively-charged groove. This positions the scissile phosphate between two opposing active-site histidines (center dashed circle). Additionally, each monomer binds a single zinc atom through a CCCH zinc finger (beige).

Figure 2

Structures and modes of effector step silencing a) The overall structure of human Argonuate2 (composite of PDBs: 4F3T [90] and 4OLB [91]) bound to an RNA guide colored by domain: N-terminal (N) (blue), linker 1 (L1) (grey), PAZ (red), linker 2 (L2) (yellow), MID (green), and PIWI (purple). Upon target recognition (PDBs 4F3T (faded, prerecognition) to 4W5O [93] (solid, postrecognition), substantial conformational changes in the PAZ domain and a helix in the L1 linker are observed (arrows), the latter being essential for duplex accommodation. Additional ordering of the guide RNA also occurs upon target binding. A model of target slicing, based on the structure of RNase H bound to a DNA–RNA hybrid (PDB 1ZBI [121]) and focusing on the active site is shown in the inset. The catalytic tetrad (D597, E637, D669 and H807) coordinates two magnesium ions and mediates the cleavage of the target (scissile phosphate circled in red). When directing silencing without slicing, Ago2 interacts with GW182 family proteins through tryptophan binding sites in the PIWI domain, indicated by arrows. GW182, in turn, recruits downstream silencing machinery. (b) The structure of the binding domains of CNOT1 (blue) and CNOT9 (teal), two CCR4–NOT subunits, in the presence of tryptophan (PDB 4CRV [102]) support that CNOT9 can be directly recruited to GW182 family proteins rich in glycine and tryptophan residues. (c) The MIF4G domain of the CCR4–NOT subunit CNOT1 (green) interacts directly with the DEAD-box helicase DDX6 (yellow; PDB 4CT4 [103]) in addition to the deadenylase CAF1 (burgundy; PDB 4GMJ [122]). Together, these interactions provide a platform to link Ago targets to deadenylation, translational repression and the decapping machinery via GW182.