BIOLOGICAL OVERVIEW

Because of its ability to digest double-stranded RNA (dsRNA) into uniformly sized, small RNAs, this enzyme has been named Dicer. Dicer contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi (RNA interference). RNAi is a mechanism through which double-stranded RNAs silence cognate genes. In plants and animals, this can occur at both the transcriptional and the post-transcriptional levels. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC (RNA induced silencing complex), to destroy specific messenger RNAs. Dicer, an enzyme that can produce putative guide RNAs, is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain (involved in unwinding RNA) and dual RNase III motifs (Bernstein, 2001).

Post-transcriptional gene silencing (PTGS) can be distinguished from RNAi by the fact that the target mRNA is not degraded but is translationally silenced. Biochemical studies have suggested that PTGS is accomplished, in part, by a mechanism similar to RNAi in that involves a nuclease that targets RNAs for degradation. Subsequently, an enzyme complex similar to RISC, targets mRNA for translational silencing. The specificity of this complex may derive from the incorporation of a small guide sequence that is homologous to the mRNA substrate. These ~22-nucleotide RNAs, originally identified in plants that were actively silencing transgenes, have been produced during RNAi in vitro using an extract prepared from Drosophila embryos. Putative guide RNAs can also be produced in extracts from Drosophila S2 cells. To investigate the mechanism of RNAi and PTGS, both biochemical fractionation and candidate gene approaches have been performed to identify the enzymes that execute each step of the related processes (Bernstein, 2001).

The enzyme complex RISC, an effector nuclease for RNAi [Hammond, 2000: see Characterization of RISC (RNA-induced silencing complex) in Drosophila] was isolated from Drosophila S2 cells in which RNAi had been initiated in vivo by transfection with double-stranded RNA (dsRNA). First, an investigation was carried out to discover whether the RISC enzyme, and the enzyme that initiates RNAi through processing of dsRNA into 22-nucleotide sequences, are distinct activities. RISC activity can be largely cleared from extracts by high-speed centrifugation (100,000g for 60 min), whereas the activity that produces 22-nucleotide sequences remains in the supernatant. This simple fractionation indicates that RISC and the 22-nucleotide sequence-generating activity may be separable. However, it seems probable that these enzymes interact at some point during the silencing process, and it remains possible that initiator and effector enzymes share common subunits (Bernstein, 2001).

RNase III family members are among the few nucleases that show specificity for dsRNA. Analysis of the Drosophila and Caenorhabditis elegans genomes revealed several types of RNase III enzymes: (1) the canonical RNase III, which contains a single RNase III signature motif and a dsRNA-binding domain (dsRBD); (2) a class represented by Drosha, a Drosophila enzyme that contains two RNase III motifs and a dsRBD (CeDrosha in C. elegans); (3) a class that contains two RNase III signatures and an amino-terminal helicase domain (for example, Drosophila CG4792 (Dicer 1) and CG6493 (Dicer 2) ; C. elegans K12H4.8), which have been proposed as potential RNAi nucleases. Representatives of all three classes were tested for the ability to produce discrete RNAs of ~22 nucleotides from dsRNA substrates (Bernstein, 2001).

To test the dual RNase III enzymes, variants of Drosha and CG4792 (subsequently called Dicer-1) tagged with the T7 epitope were prepared. These were expressed in transfected S2 cells and isolated by immunoprecipitation using antibody-agarose conjugates. Treatment of the dsRNA with the CG4792 immunoprecipitate yielded fragments of about 22 nucleotides, similar to those produced in either the S2 or embryo extracts. Neither the activity in extract nor that in immunoprecipitates depended on the sequence of the RNA substrate, since dsRNAs derived from several genes were processed equivalently. Negative results were obtained with Drosha and with immunoprecipitates of a DExH box helicase (Homeless). Western blotting confirmed that each of the tagged proteins was expressed and immunoprecipitated similarly. Thus, it is concluded that CG4792 may carry out the initiation step of RNAi by producing guide sequences of about 22 nucleotides from dsRNAs (Bernstein, 2001).

An antiserum directed against the carboxy terminus of the Dicer protein immunoprecipitates a nuclease activity (from either the Drosophila embryo extracts or from S2 cell lysates) that produces RNAs of about 22 nucleotides from dsRNA substrates. The putative guide RNAs that are produced by the Dicer enzyme precisely co-migrate with 22-nucleotide sequences that are produced in extract, and with 22-nucleotide sequences that are associated with the RISC enzyme. The enzyme that produces guide RNAs in Drosophila embryo extracts is ATP dependent. Depletion of ATP results in a roughly sixfold reduction of dsRNA cleavage rate and in the production of RNAs with a slightly lower mobility. Of note, both Dicer immunoprecipitates and extracts from S2 cells require ATP for the production of ~22-nucleotide sequences. The accumulation of lower-mobility products was not observed in these cases, although ~22-nucleotide sequences were routinely observe in ATP-depleted embryo extracts. The requirement of Dicer nuclease for ATP is an unusual property, and may indicate that unwinding of guide RNAs by the helicase domain is required for the enzyme to act catalytically (Bernstein, 2001).

For efficient induction of RNAi in C. elegans and in Drosophila, the initiating RNA must be double-stranded and must also be several hundred nucleotides in length. Similarly, Dicer is inactive against single-stranded RNAs regardless of length. The enzyme can digest both 200- and 500-nucleotide dsRNAs, but is significantly less active with shorter substrates. In contrast, Escherichia coli RNase III can digest to completion dsRNAs of either 35 or 22 nucleotides. This suggests that the substrate preferences of the Dicer enzyme may contribute to, but not wholly determine, the size dependence of RNAi (Bernstein, 2001).

These results indicate that the process of RNAi can be divided into at least two distinct steps. Initiation of RNAi would occur upon processing of a dsRNA by Dicer into ~22-nucleotide guide sequences, although the possibility that another Dicer-associated nuclease may participate in this process cannot be formally excluded. These guide RNAs would be incorporated into a distinct nuclease complex (RISC) that targets single-stranded mRNAs for degradation. An implication of this model is that the guide sequences are themselves derived directly from the dsRNA that triggers the response. In accord with this model, it has been shown that ³²P-labelled, exogenous dsRNAs that have been introduced into S2 cells by transfection are incorporated into the RISC enzyme as 22-nuclotide sequences (Bernstein, 2001).

With the identification of Dicer as a potential catalyst of the initiation step of RNAi, the biochemical basis of this unusual mechanism of gene regulation is now open to investigation. It is now important to determine whether the conserved family members from other organisms, particularly mammals, also have a function in dsRNA-mediated gene regulation (Bernstein, 2001).

PROTEIN STRUCTURE

Amino Acids - 2249

Structural Domains

Dicer contains two RNase III signatures and an amino-terminal helicase domain. A notable feature of the Dicer family is its evolutionary conservation. Homologs are found in C. elegans (K12H4.8), Arabidopsis (for example, CARPEL FACTORY, T25K16.4 and AC012328_1), mammals (Helicase-MOI) and Schizosaccharomyces pombe (YC9A_SCHPO). In fact, the human Dicer family member is capable of generating ~22-nucleotide RNAs from dsRNA substrates: this indicates that these structurally similar proteins may all share similar biochemical functions. Exogenous dsRNAs can affect gene function in early mouse embryos, and these results suggest that this regulation may be accomplished by evolutionarily conserved RNAi machinery (Bernstein, 2001).

In addition to RNase III and helicase motifs, searches of the PFAM database indicate that each Dicer family member also contains a PAZ domain. This sequence was defined on the basis of its conservation in the Zwille/ARGONAUTE/Piwi family that has been implicated in RNAi by mutations in C. elegans (Rde-1) and Neurospora (Qde-2). Although the function of this domain is unknown, it is notable that this region of homology is restricted to two gene families that participate in dsRNA-dependent silencing. Both the ARGONAUTE and Dicer families have also been implicated in common biological processes, namely the determination of stem-cell fates. A hypomorphic allele of carpel factory, a member of the Dicer family in Arabidopsis, is characterized by increased proliferation in floral meristems. This phenotype and a number of other characteristic features are also shared by Arabidopsis ARGONAUTE (ago1-1) mutants. These genetic analyses provide evidence that RNAi may be more than a defensive response to unusual RNAs, but may also have integral functions in the regulation of endogenous genes (Bernstein, 2001).

date revised: 5 June 2002

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