Trends in Biochemical Sciences
siRNA and miRNA: an insight into RISCs
Introduction
Although small interfering RNA (siRNA; see Glossary) and microRNA (miRNA) were initially discovered in unrelated studies, both types of small RNA are closely related in their biogenesis, assembly into RNA–protein complexes and ability to regulate gene transcripts negatively in diverse eukaryotes 1, 2, 3, 4. Both siRNAs and miRNAs are generated by Dicer (DCR), a multidomain enzyme of the RNase III family. DCR cuts long, double-stranded RNA (dsRNA) into siRNAs and chops short precursor miRNAs with imperfect stem-loop structure into miRNAs.
The nascent siRNAs and miRNAs are double-stranded duplexes. These duplexes need to be unwound before they can be assembled into an RNA-induced silencing complex (RISC). By comparing the thermodynamic stabilities at the two ends, siRNAs can be divided into two classes: symmetric siRNAs and asymmetric siRNAs. A symmetric siRNA has two equally stable ends; thus, both strands of the siRNA are assembled into the RISC with equivalent efficiency [5]. By contrast, an asymmetric siRNA has one end that is less stable than the other. Because it is easier to unwind siRNA from the less stable end, one strand of the siRNA is preferentially incorporated into the RISC complex in a process referred to as the ‘asymmetric assembly of RISCs’ 5, 6. Intriguingly, most miRNAs are highly asymmetric, ensuring efficient asymmetric assembly of a miRISC in cells 5, 6.
In vitro and in vivo biochemical studies have shown that a siRISC can function as a miRISC to repress translation of the target mRNA; similarly, a miRISC can function as a siRISC to cleave the target mRNA. This functional interchangeability between a siRISC and a miRISC argues that siRISCs and miRISCs are highly similar, if not identical 7, 8, 9, 10. Much evidence suggests, however, that siRISCs and miRISCs are distinct types of complex.
First, the biogenesis, maturation and subsequent assembly of siRNAs and miRNAs into silencing complexes are different [1], which can result in RISCs with distinct functions. Second, Argonaute (AGO) proteins, which are principal components of RISCs, are encoded by a multigene family and can be divided into functionally distinct subgroups 11, 12, 13, 14, 15. These functionally different AGOs endow their corresponding RISCs with distinct functions. Third, the complementarity between the small RNAs and their target mRNAs has been proposed to affect the functional mode of RISCs in terms of the regulation of mRNA stability and translation. Nevertheless, RISCs containing small RNAs that are extensively complementary to their target mRNAs do not always specify efficient cleavage of the targets as was previously predicted: some RISCs direct efficient target cleavage 7, 8, 16, 17, whereas others do not [15] (G. Tang et al., unpublished). Fourth, RISCs vary markedly in size, from the smallest ‘core RISC’ of ∼160 kDa to the largest ‘holo RISC’ of 80 Svedberg (80S) 18, 19, 20, 21.
Last, siRNA and miRNA programmed RISCs have distinct targeting functions in cells. Many endogenous miRNAs and their RISCs are genetically programmed to regulate gene expression and thus are important for the growth and development of an organism [22]. By contrast, siRNAs are produced from dsRNAs that are often synthesized in vitro or in vivo from viruses or repetitive sequences introduced by genetic engineering. In addition, dsRNA can be produced from endogenously activated transposons. Thus, siRNAs have been proposed to function in: (i) antiviral defense (despite the fact that viruses develop counter-defense strategies as well 23, 24), (ii) silencing mRNAs that are overproduced or translationally aborted, and (iii) guarding the genome from disruption by transposons 4, 25, 26.
In this review, I focus on RISC assembly by discussing the initial synthesis of siRNA and miRNA by DCRs, the formation of a RISC loading complex (RLC), the transition from an RLC to an active RISC, and target recognition and interaction by the RISC. Finally, I discuss how two general types of RISC can explain the complexity of their functions in cleavage and translational repression of the target mRNA.
Section snippets
Initiation of the RNAi and miRNA pathways: role of DCRs
It is generally accepted that the RNA interference (RNAi) and miRNA pathways are initiated by DCR members of the RNase III enzyme family. DCR was initially identified in Drosophila [27] and has been subsequently found in diverse eukaryotic organisms including humans, plants and fungi (Table 1). Some organisms have a single homolog of DCR 28, 29, 30, 31, 32, 33, 34, 35, whereas others have more 8, 36. In species with several DCRs, different homologs are responsible for producing siRNAs and
siRNA– and miRNA–protein interactions: the RLC
The DCR-cleaved siRNAs and miRNAs are initially double stranded. The transition from double-stranded to single-stranded RNAs during RISC assembly is achieved via RNA–protein and protein–protein interactions. The RLC is the initial RNA–protein complex formed in cells after the production of small RNAs. The small RNAs in the RLC are probably double stranded and ready to be unwound for functional RISC assembly. The RLC has been characterized to some extent in Drosophila and C. elegans, but its
RLC structure and assembly: orientation of the small RNA duplex
In Drosophila, asymmetric RISC assembly requires an asymmetric RLC, which is primarily determined by the structure of the siRNA duplex [41]. The composition of the RLC has not been determined in full. The minimal components of the RLC are thought to be the siRNA duplex and the DCR2–R2D2 heterodimer [41]. It is not known what and how additional cofactors are involved in RLC assembly.
One of the key issues concerning asymmetric RLC assembly is whether the siRNA duplex produced by the DCR2–R2D2
The transition from RLC to RISC: RNA helicase and AGO proteins
Handing siRNAs from the RLC to the RISC requires an AGO protein and possibly also an RNA helicase (Table 1). Even though Drosophila DCR2 has a DExH helicase domain, it does not unwind siRNA duplexes in vitro [41], which suggests that a specific RNA helicase or other factors might be required during the transition from RLC to RISC. Such a hypothetical RNA helicase is thought to unwind the siRNA duplex, after which the RISC is formed by the recruitment of an AGO protein as the core component.
RISC assembly from RLC: a sequential process
Recent biochemical studies have shown that RISCs vary markedly in size from complexes as small as 160 kDa in humans to ones as large as 80S in Drosophila 12, 18, 19, 20, 21, 56. In vitro RISC assembly in Drosophila indicates that assembly starts with the formation of an RLC that contains DCR2 and R2D2, and ends with a large protein complex the size of 80S ribosome [21]. Furthermore, a series of intermediate complexes has been observed during the transition from RLC to RISC. Notably, the RLC, the
RISC functions: target mRNA cleavage and the ‘slicer’ activity
In RNAi, cleavage of an RNA target by a RISC is defined mainly by the endonuclease or ‘slicer’ activity of the RISC. Much recent evidence has demonstrated that slicer is a subtype of the AGO protein family 13, 15, 59. AGO proteins have been previously shown to be in tight association with siRNAs and miRNAs by affinity purification studies using either tagged AGO protein or tagged siRNA 13, 15, 18, 19. Notably, among the tagged AGO proteins that have been expressed in human cells, only mammalian
Two types of RISC: multiple functions
RISC assembly is most complex in the RNAi and miRNA pathways. It involves small RNA producers (DCRs), small RNA duplex structures and RLC assemblies, and it requires the unwinding of a symmetric–asymmetric RNA duplex and the recruitment of distinct AGO proteins. Different AGO proteins on RISCs have distinct functions that are most probably determined by the PIWI domain of the AGO protein. Moreover, some PIWI domains confer slicer activity, whereas others do not. On the basis of the type of AGO
Interactions between the RISC and other pathways
RISCs function in the control of specific pathways primarily by negatively regulating gene expression. Sometimes, the RISC regulates its targeting pathways by interacting with proteins from these pathways. For example, immunoprecipitation and sucrose gradient fractionation of RISCs have identified not only the common components of an active holo siRISC [21], but also proteins from other pathways 54, 58, 73. These RISC interacting partners include ribosome proteins from the protein translation
Concluding remarks
In summary, RISC assembly is central to the RNAi and miRNA pathways. Biochemical and genetic studies have identified many putative RISC components, RISC interacting proteins and also the structural features of the siRNAs and miRNAs. The various components of RISCs belong to families of proteins, and each family member specifies a RISC with distinct functions. The discovery of distinct AGO proteins and the identification of slicer identity on these proteins have established two general types of
Acknowledgements
I thank Phillip D. Zamore, in particular, for his support and for critically reading the article. Thanks also to Craig Mello, Gad Galili, Eric Lai, Qinghua Liu, Jian-Kang Zhu, Shou-wei Ding, Zuoshang Xu, Nick Rhind, Benjamin Burr, Christian Matranga and Fengan Yu for constructive comments on the article; and colleagues for sharing unpublished results. G.T. is a Charles A. King Trust Research Fellow of the Medical Foundation with funding from the Charles A. King Trust and the June Rockwell Levy
Glossary
- Argonaute (AGO):
- A large protein family that constitutes key components of RISCs. AGO proteins are characterized by two unique domains, PAZ and PIWI, whose functions are not fully understood. Current evidence suggests that the PAZ domain binds the 3′-end two-nucleotide overhangs of the siRNA duplex, whereas the PIWI domain of some AGO proteins confers slicer activity. PAZ and PIWI domains are both essential to guide the interaction between the siRNA and the target mRNA for cleavage or
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