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RNAi-MEDIATED PATHWAYS IN THE NUCLEUS Marjori A. Matzke, James A. Birchler Nature Rev. Genetics Vol.6, No 1, P. , 2005 | |
RNA interference (RNAi) is an evolutionarily conserved mechanism that uses short antisense RNAs that are generated by 'dicing' dsRNA precursors to target corresponding mRNAs for cleavage. However, recent developments have revealed that there is also extensive involvement of RNAi-related processes in regulation at the genome level. dsRNA and proteins of the RNAi machinery can direct epigenetic alterations to homologous DNA sequences to induce transcriptional gene silencing or, in extreme cases, DNA elimination. Furthermore, in some organisms RNAi silences unpaired DNA regions during meiosis. These mechanisms facilitate the directed silencing of specific genomic regions. Рис.1. | RNA-directed DNA methylation. Рис.2. | RNA interference-mediated heterochromatin assembly. Рис.3. | RNA interference (RNAi) and DNA elimination in Tetrahymena thermophila Рис.4. | Silencing of unpaired DNA during meiosis.
| BoxesBox 1 | Multigene families that encode components of the RNAi machinery Although the fission-yeast genome contains only one copy of each of the genes that encode the core proteins of the RNA interference (RNAi) machinery (Dicer, RNA-dependent RNA polymerase (RdRP) and Argonaute), these proteins are often encoded by multigene families in other organisms. Dicer Proteins of the Dicer family are ribonuclease III enzymes that process dsRNA to produce small RNAs. There are four such proteins in Arabidopsis thaliana, two in Drosophila melanogaster, two in Neurospora crassa, one in mammals and one in Caenorhabditis elegans. RNA-dependent RNA polymerases These synthesize dsRNA from an ssRNA template to initate or amplify the RNAi process. There are four in C. elegans, six in A. thaliana (at least three of which are expressed) and three in N. crassa, but there are none in D. melanogaster or mammals. Argonaute Members of this family provide the small RNA-binding component of silencing-effector complexes. There are 27 in C. elegans, 10 in A. thaliana, 8 in humans, 5 in D. melanogaster and 2 in N. crassa106. The proliferation of these gene families in multicellular eukaryotes indicates a subfunctionalization of the encoded proteins into diverse processes, and provides the possibility of separate nuclear or cytoplasmic distributions in the cell. Although much remains to be learned about the extent of functional diversification and/or redundancy of gene-family members, distinct roles in RNA-mediated gene-silencing pathways for some individual representatives of these families have been discovered. Some of these are mentioned in the text and more examples are provided below. In A. thaliana, nuclear DICER-LIKE 1 (DCL1)107 is required for processing microRNA (miRNA) precursors19 and DCL2 is required for producing viral small interfering RNAs (siRNAs)15. In D. melanogaster, Dicer-1 (DCR1) produces miRNAs whereas DCR2 generates siRNAs108. The two Dicers in N. crassa are functionally redundant in RNAi109. In N. crassa, meiotic silencing of unpaired DNA (in the nucleus) and RNAi (in the cytoplasm) require distinct RdRPs and Argonaute proteins93. In A. thaliana, RdRP6 is needed for RNAi that is triggered by sense transgenes110. In D. melanogaster, Argonaute 1 (AGO1) and AGO2 are implicated in miRNA and siRNA-mediated silencing, respectively111. AGO2 in humans contains an RNaseH-like domain that catalyses mRNA cleavage in the RNAi process6,7. Box 2 | Cytosine demethylation, imprinting and RNA CG methylation is lost passively during successive rounds of DNA replication if it is not maintained by DNA methyltransferases112. However, there is growing evidence for active demethylation without DNA replication in animals and plants, possibly through the activity of DNA glycosylases; enzymes that normally function in In Arabidopsis thaliana, DNA glycosylase domains are present in two large proteins — REPRESSOR OF SILENCING 1 (ROS1) and DEMETER (DME) — that have been implicated in the activation of transcriptionally silenced genes, presumably through the removal of methylated cytosines115-118 (Рис. 1). DME is specifically required for the demethylation and activation of the maternal alleles of the imprinted genes MEDEA (which encodes a polycomb-group protein that regulates endosperm development)117 and FWA (which encodes a homeodomain transcription factor that functions in the seed endosperm)54. Whether the demethylation of target genes by DME or ROS1 is guided by RNA is unknown, but RNA-directed DNA methylation (RdDM) has been implicated in establishing methylation of FWA (Ref. 46) and of a transgene promoter that is demethylated by ROS1 (Ref. 115). Intriguingly, in vertebrate cells, RNA that is complementary to the methylated DNA strand was reported to be required for active demethylation by DNA glycosylases119. In addition, the chicken DNA glycosylase associates tightly with an RNA helicase, p68 (Ref. 120). In Drosophila melanogaster, the p68 orthologue is required for efficient RNA interference (RNAi)121. If the p68 orthologue in chicken is also needed for RNAi, a connection would be established between demethylation by DNA glycosylases and the RNAi machinery in vertebrates. Box 3 | Does RNA-directed DNA methylation operate in mammals? Non-CG methylation, a hallmark of RNA-directed DNA methylation (RdDM) in plants, has been detected in DNA of mammalian embryonic stem cells122 and in human L1 retrotransposons123, but it is unknown whether this methylation is triggered by RNA. One issue is whether mammals have the molecular machinery needed for RdDM. Two of the DNA methyltransferases required for RdDM in plants, DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and DNA methyltransferase MET1, have mammalian counterparts (DNMT3A/B and DNMT1, respectively), although the catalytic domain of DRM2 is rearranged relative to DNMT3A/B and there are some differences in their amino termini104. The plant-specific DNA methyltransferase CHROMOMETHYLASE 3 (CMT3) is important for CNG methylation38, 39, but whether this enzyme is an absolute requirement for RdDM is not clear. Another plant-specific factor of the RdDM machinery is the SNF2-like protein DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1). The closest non-plant homologues of DRD1 are members of the conserved Rad54/ATRX subfamily of SNF2-like proteins48. ATRX (α-thalassaemia, mental retardation, X-linked) is needed for CG methylation of some repetitive sequences in mammals124. Whether this methylation is triggered by RNA is unknown, but ATRX could conceivably substitute for DRD1 in the RdDM pathway in mammalian cells. RdDM clearly takes place in the nucleus, whereas the mRNA degradation step of RNAi occurs typically in the cytoplasm. Are small RNAs that trigger RdDM likely to be present in nuclei of mammalian cells? Plant cells have DICER-LIKE activities that are localized in the nucleus, such as Arabidopsis thaliana DCL3 (Ref. 15) and DCL1 (Ref. 107), which can produce small RNAs in the nucleus. Moreover, in plant cells, small RNAs that initiate RNAi in the cytoplasm can move to the nucleus to induce methylation of homologous DNA125. Although the single Dicer of mammals is present in the cytoplasm126, a mechanism for translocating small RNAs to the nucleus of mammalian cells is indicated by the presence of mature miRNAs in nuclear as well as cytoplasmic cellular fractions7. Small RNAs produced in the cytoplasm might also be able to interact with DNA when the nuclear envelope breaks down during cell divisions57. So, mammals seem to have much of the machinery for RdDM, but whether this process occurs regularly in mammals remains to be determined. LinksDATABASESNCBI Taxonomy: FURTHER INFORMATION |