In Arabidopsis thaliana, functionally diverse small RNA (smRNA) pathways bring about decreased RNA accumulation of target genes via several different mechanisms. Cytological experiments have suggested that the processing of microRNAs (miRNAs) and heterochromatic small interfering RNAs (hc-siRNAs) occurs within a specific nuclear domain that can present Cajal Body (CB) characteristics. It is unclear whether single or multiple smRNA-related domains are found within the same CB and how specialization of the smRNA pathways is determined within this specific sub-compartment. To ascertain whether nuclear smRNA centers are spatially related, we localized key proteins required for siRNA or miRNA biogenesis by immunofluorescence analysis. The intranuclear distribution of the proteins revealed that hc-siRNA, miRNA and trans-acting siRNA (ta-siRNA) pathway proteins accumulate and colocalize within a sub-nuclear structure in the nucleolar periphery. Furthermore, colocalization of miRNA- and siRNA-pathway members with CB markers, and reduced wild-type localization patterns in CB mutants indicates that proper nuclear localization of these proteins requires CB integrity. We hypothesize that these nuclear domains could be important for RNA silencing and may partially explain the functional redundancies and interactions among components of the same protein family. The CB may be the place in the nucleus where Dicer-generated smRNA precursors are processed and assigned to a specific pathway, and where storage, recycling or assembly of RNA interference components takes place.
Assembly of heterochromatin at centromeric DNA regions in the fission yeast Schizosaccharomyces pombe involves an intimate interplay between chromatin modifying complexes and components of the RNAi pathway. The RNA-induced transcriptional silencing (RITS) complex, containing Chp1, Ago1, Tas3, and centromeric siRNAs, localizes to centromeric DNA repeats and is required for the assembly and maintenance of heterochromatin. RITS brings together two types of molecular recognition modules: a chromodomain protein, which binds to lysine 9 methylated histone H3 (H3K9), and Argonaute, which binds to specific sequences by siRNA-directed base-pairing interactions. The RNA-directed RNA polymerase complex (RDRC), composed of Rdp1, the Hrr1 helicase, and the Cid12 Poly(A) polymerase family member, synthesizes doublestranded RNA and creates the substrate for Dicer to generate siRNAs. RDRC physically associates with RITS, and both complexes localize to noncoding centromeric RNAs and centromeric DNA repeats, suggesting that recognition of nascent RNA transcripts may be involved in localization of these complexes to specific chromosome regions. In support of this possibility, tethering of the RITS complex to the transcript of the normally euchromatic ura4 + gene results in siRNA generation and RNAi-and heterochromatin-dependent silencing of the ura4 + gene. Finally, silencing of a subset of endogenous and transgene promoters within heterochromatic DNA domains occurs by RNAi-dependent degradation of nascent transcripts by a mechanism that we have termed co-transcriptional gene silencing (CTGS).
BackgroundPlants have evolved a unique epigenetic process to target DNA cytosine methylation: RNA-directed DNA methylation (RdDM). During RdDM, small RNAs (smRNAs) guide methylation of homologous DNA loci. In Arabidopsis thaliana, the de novo DNA methyltransferase that ultimately methylates cytosines guided by smRNAs in all sequence contexts is DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2). Recent reports have shown that DRM2 requires the catalytic mutated paralog DRM3 to exert its function through a still largely unknown process. To shed light on how DRM3 affects RdDM, we have further characterized its role at the molecular and cytological levels.FindingsAlthough DRM3 is not required for RdDM loci transcriptional silencing, it specifically affects loci’s DNA methylation. Interestingly, DRM3 and DRM2 regulate the DNA methylation in a subset of loci differently.Fluorescence In Situ Hybridization and immunolocalization analyses showed that DRM3 is not required for the large-scale nuclear organization of heterochromatin during interphase, with the notable exception of the 45S ribosomal RNA loci. DRM3 localizes exclusively to the nucleus and is enriched in a round-shaped domain located in the nucleolar periphery, in which it colocalizes with components of the RdDM pathway.ConclusionsOur analyses reinforce the previously proposed chaperone role of DRM3 in RdDM. Overall, our work further demonstrates that DRM3 most likely functions exclusively with DRM2 in RdDM and not with other A. thaliana DNA methyltransferases. However, DRM3’s regulation of DNA methylation is likely target- or chromatin context-dependent. DRM3 hypothetically acts in RdDM either upstream of DRM2, or in a parallel step.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-721) contains supplementary material, which is available to authorized users.
In organisms as diverse as humans, flies, nematodes, fission yeast and plants, miRNAs (micro RNAs) and/or siRNAs (small interfering RNAs) regulate numerous essential processes. These include inactivation of mRNAs in development, viral defense, silencing of transposons/endogenous repeats and proper centromere functioning, as required for accurate chromosome segregation to daughter cells in each round of cell division [1,2,3,4]. In general, siRNAs or miRNAs that are generated by Dicer (DCL) endonucleases are stably associated with Argonaute (AGO) proteins within RNA-induced silencing complexes (RISCs). RISCs can direct RNA-dependent DNA methylation and heterochromatin formation at homologous genes to cause transcriptional silencing or they can mediate translational arrest or cleavage of homologous mRNAs to silence genes posttranscriptionally.Arabidopsis thaliana has an elaborate set of proteins involved in small RNA biogenesis. Many of these proteins function in distinct pathways but there is substantial crosstalk and functional redundancy among components of the pathways. Critical for small RNA production are the four DCL proteins that act in partnership with various additional activities. For instance, DCL1 is primarily responsible for miRNA production while DCL3 is involved in the biogenesis of heterochromatic siRNA that target DNA methylation. These Dicers also contribute to siRNA production associated with transgene silencing or viral defense, along with DCL2 and DCL4 [5]. There is clear collaboration between the DCL1-mediated production of miRNAs and the resulting production of tasiRNAs, which involves DCL4 dicing of precursors that are generated by miRNAdirected cleavage and subsequent RNA-DEPENDENT RNA POLYMERASE 6 (RDR6)-dependent production of double-stranded RNA (dsRNA) [5]. Until recently, it was thought that the DCL3-dependent pathway for heterochromatic siRNA production, involving Nuclear RNA polymerase IV (pol IV), RDR2 and AGO4 was devoted solely to transcriptional gene silencing and RNA-dependent DNA methylation (RdDM) [6]. However, Pol IV and RDR6, but not RDR2, are also required for production of regulatory siRNAs generated by DCL2 from dsRNAs formed where the 3' ends of adjacent gene transcription units overlap [5].Our previous cytological analysis of siRNA-directed DNA methylation pathway strongly suggests a stepwise model for nuclear siRNA biogenesis and target locus chromatin modifications in a process that involves a hypothetical siRNA-processing center (Figure1A) [7] similar to [8] and the periphery of heterochromatic regions. On the other hand, an analogous nucleolar structure for DCL1 has been described, designated as dicing bodies [9], in which all the proteins required for primary miRNA transcript processing are colocalizing with pri-miRNA precursors, suggesting that these nuclear bodies represent miRNA processing centers. Similarly to the DCL3 processing centers we described, DCL1 processing centers/dicing bodies are associated with the nucleolus but is not clear how they are relate...
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