Trends in Biochemical Sciences
ReviewMaking RISC
Section snippets
RISC: the effector complex for small RNAs
The major role that 20- to 30-nt small RNAs play in regulating gene expression has been increasingly recognized. Endogenous and exogenous siRNAs (endo-siRNAs and exo-siRNAs) mediate sequence-specific cleavage of their target mRNAs, a phenomenon termed RNA interference (RNAi) 1, 2, 3. siRNAs have become a powerful research tool that also possesses exciting therapeutic promise. microRNAs (miRNAs) are a phylogenetically conserved family of endogenous small RNAs. They are proposed to regulate over
Argonaute: the core component of RISC
At the core of RISCs is a member of the Argonaute (Ago) family of proteins, which provides a unique platform for target recognition and silencing 1, 2, 3. The association of a small RNA with a specific Ago protein dictates its function. The larger Ago family can be classified into the Ago subfamily, to which siRNAs and miRNAs bind, and Piwi subfamily, to which piRNAs bind. In mammals, each of the four Ago subfamily proteins (AGO1–4) can repress translation of their target mRNAs, but only AGO2
Two steps in RISC assembly: RISC loading and unwinding
siRNAs and miRNAs are born double-stranded: the RNase III enzymes, Drosha and Dicer, process the precursor RNAs into small RNA duplexes called siRNA duplexes and miRNA–miRNA* duplexes 1, 2, 3. Because small RNAs in RISC must anneal to their target RNAs, the two strands of a small RNA duplex must be separated and one strand discarded to form a functional RISC. The discarded strand is called the ‘passenger strand’ whereas the strand retained in RISC is called the ‘guide strand’. RISC assembly can
RISC-loading machinery
Ago proteins can receive single-stranded small RNAs via ‘bypass’ loading 13, 14, 15 (Box 2). However, naked Ago proteins seem unable to incorporate double-stranded small RNAs without the aid of the RISC-loading machinery 4, 16. What factors are needed for ‘canonical’ RISC loading of small RNA duplexes? Dicer-2 (Dcr-2) and its double-stranded (ds) RNA-binding protein partner R2D2 are components of the RISC-loading complex (RLC) for Drosophila Ago2 17, 18, 19. dcr-2 null flies, which lack both
Slicer-dependent unwinding: cleavage of the passenger strand
A pre-RISC that is loaded with a small RNA duplex is similar to a mature RISC bound to a target RNA in that the Ago protein is occupied by two RNA strands – the guide and passenger strands in a pre-RISC and the guide strand and the target RNA in a mature RISC. Hence, the passenger strand can serve as the guide strand's first RNA target. Several groups concurrently examined this interesting idea, and demonstrated that fly Ago2 and human AGO2, which both possess efficient slicer activities,
ATP: the driving force for RISC assembly
RISC assembly has long been known to require ATP (but see Box 4). In 2001, Zamore and colleagues found that siRNA duplexes, whose production is also ATP-dependent, are first incorporated into an ∼360 kDa complex – which is deduced to be the RLC or its core part [18] – in fly embryo lysate [57]. This inactive complex was then converted to a single-stranded guide-containing active complex – the mature Ago2-RISC – in an ATP-dependent manner. Native gel analyses of siRNA–protein complexes also
A structural viewpoint for RISC assembly
Structures of Thermus thermophilus Ago (TtAgo) protein in complex with guide DNA alone or with both guide DNA and target RNA strands provide insight into the molecular actions of Ago proteins 45, 58, 59 (reviewed in Ref. [60]). The structure with a 10-nt guide DNA adopts the most compact form, representing a nearly nucleic acid-free state [45]. When single-stranded guide DNA is incorporated, TtAgo adopts a significantly extended structure with the 5′ end of the guide anchored in the phosphate
Concluding remarks: how is RISC assembled?
Although much is known about RISC assembly, a major question remains unanswered: what factors are necessary and sufficient for RISC assembly? Other than the well-studied fly Ago2-RLC, which contains Dcr-2–R2D2, the identity of RISC-loading machinery remains largely unknown. Proteomic analyses have identified numerous factors associated with Ago proteins including many helicases (e.g. Ref. [62]). What are the functions of these Ago-interacting proteins? What proteins constitute RISC and
Acknowledgements
We thank Hiroshi Sasaki, Hervé Seitz, Phillip D. Zamore and members of the Tomari laboratory for helpful discussions and critical readings of the manuscript. We apologize to our colleagues whose important work is not cited because of space limitations. Research in the Tomari laboratory is supported by Grant-in-Aids for Young Scientists and a Grant-in-Aid for Scientific Research on Innovative Areas (‘functional machinery for non-coding RNAs’) from the Japanese Ministry of Education, Culture,
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