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Review
. 2005 May 1;387(Pt 3):561-71.
doi: 10.1042/BJ20041822.

Exploring the functions of RNA interference pathway proteins: some functions are more RISCy than others?

Katarzyna Jaronczyk  1 , Jon B Carmichael, Tom C Hobman
Affiliations
Review

Exploring the functions of RNA interference pathway proteins: some functions are more RISCy than others?

Katarzyna Jaronczyk et al. Biochem J. .

Abstract

PPD (PAZ Piwi domain) proteins and the Dicer family have been the subjects of intense study over the last 6 years. These proteins have well-established roles in RNAi (RNA interference), a process that relies on siRNAs (small interfering RNAs) or miRNAs (microRNAs) to mediate specificity. The development of techniques for applying RNAi as a laboratory tool and a molecular therapeutic technique has rapidly outpaced our understanding of the biology of this process. However, over the last 2 years, great strides have been made towards elucidating how PPD proteins and Dicer regulate gene-silencing at the pre- and post-transcriptional levels. In addition, evidence is beginning to emerge that suggests that these proteins have additional siRNA-independent roles as cell-cycle regulators. In the present review, we summarize the well-known roles of these two classes of proteins in gene-silencing pathways, as well as explore the evidence for novel roles of PPD and Dicer proteins.

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Figures

Figure 1
Figure 1. PPD protein and Dicer domain structures
PPD proteins are classically defined by the presence of PAZ and Piwi domains. The PAZ domain binds to siRNAs, whereas the Piwi domain serves as the binding site for Dicer. The Piwi domains of some PPD proteins possess endonuclease activity. The two ribonuclease III domains of Dicer dimerize to form the catalytic centre that is responsible for cleaving long dsRNA. Some Dicers, such as Schiz. pombe Dcr1, A. thaliana DCL4 and D. melanogaster Dcr1 do not have recognizable PAZ domains. One or more nuclear localization signal can be found in A. thaliana DCL1 and DCL4, D. melanogaster Dcr1, and mouse and human Dicers.
Figure 2
Figure 2. Model of RISC assembly in D. melanogaster
The principal siRNA-generating enzyme complex in D. melanogaster is composed of Dcr2 and R2D2. Although R2D2 does not affect the ability of Dcr2 to cleave long dsRNA, it is important for binding to the siRNA product after cleavage. R2D2 binds to the 3′ end of the guide strand (red) of the siRNA duplex, while Dcr2 binds to the 3′ end of the passenger strand. R1 forms after Dcr2-mediated RNA cleavage of long dsRNAs. The R2 complex may be an intermediate that derives from R1 and ultimately matures into R3. Alternatively, it has been suggested that R3 may form directly from R1. The R3 complex is large (80 S) and associates with ribosomes (not shown). Ago2 displaces the Dcr2–R2D2 complex from the siRNA duplex by binding to the 3′ end of the guide siRNA strand. Formation of this complex is dependent upon ATP. The siRNAs in the R3 complex are single-stranded, having possibly been unwound by the helicase Armitage. R3 binds the cognate mRNA target, which is then cleaved by the Ago2 subunit. It is possible that there are other unidentified proteins (?) associated with each of the intermediate complexes.
Figure 3
Figure 3. Multiple functions of PPD and Dicer proteins
PPD proteins and Dicer are proposed to function in three different pathways. On the right-hand side of the diagram, si/miRNA-dependent functions of Dicer and PPD proteins are illustrated. si/miRNA-directed mRNA cleavage or translational repression does not require Chp1 and Tas1-like proteins and is proposed to occur in the cytoplasm. In contrast, the heterochromatin-specific functions take place in the nucleus. On the left-hand side, we outline a model to account for the cell cycle functions of Dicer and PPD proteins. DNA damage or inhibition of replication results in a cascade of signalling events that lead to cell-cycle arrest via a Cdc2-dependent checkpoint. Although the exact points of PPD or Dicer proteins action along this pathway are unknown (red arrows), it is thought that these functions are independent of si/miRNAs. In contrast, RISC-mediated mRNA degradation and translational suppression (green arrows) require Dicer-dependent si/miRNA production and their inclusion into RISC or RISC-like complexes. Likewise, heterochromatin formation (purple arrows) requires the production and inclusion of si/miRNAs into a specialized complex named RITS. Association of RITS with chromatin results in methylation of histone H3 Lys9, followed by Swi6 binding and subsequent spreading of silencing to neighbouring regions.
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