Intersection of Small RNA Pathways in Arabidopsis thaliana Sub-Nuclear Domains

Abstract

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.

Nonrandom domain organization of the Arabidopsis genome at the nuclear periphery

“…6). This correlates with observations that many small RNA pathway components are concentrated around nucleoli in Arabidopsis (Li et al 2008;Pontes et al 2013). From a spatial point of view, our results provide insights into how these two silencing pathways might collaborate to regulate TEs (Zemach et al 2013), as well as how certain components of one pathway interact with each other (e.g., a recently reported positive feedback loop between Pol IV-dependent small RNA biogenesis and DRM2-dependent CHH methylation (Li et al 2015)).…”

Accessible DNA and Relative Depletion of H3K9me2 at Maize Loci Undergoing RNA-Directed DNA Methylation

“…A well-studied example is RNA-directed DNA methylation (RdDM), which was initially discovered as a response to viral infection in tobacco (Nicotiana tabacum) (Wassenegger et al, 1994) and involves a process related to RNA interference that directs de novo DNA methyltransferase to specific loci (reviewed in Matzke and Mosher, 2014). The link between RdDM and gene silencing has led some authors to refer to the short RNA molecules involved in RdDM as heterochromatic short interfering RNAs (siRNAs) (Axtell, 2013;Pontes et al, 2013;Shin et al, 2013;Bologna and Voinnet, 2014;Creasey et al, 2014). RdDM is primarily associated with the repression of transposons, but it can indirectly affect gene expression through genomic imprinting (Vu et al, 2013) and paramutation (reviewed in Hollick, 2012).…”

RNA-Directed DNA Methylation and Transcriptional Silencing in Arabidopsis

“…Immunolocalization experiments with isolated nuclei have shown that RDR2, DCL3, and AGO4 localize in the Cajal body [62,63]. The Cajal body is involved in a variety of functions including pre-mRNA splicing, rRNA processing, and telomere maintenance [64].…”