Figure 1.
Nuclear localization of trans-acting siRNA pathway members.
Double immunolocalization in interphase nuclei of RDR6, SGS3, DCL4 and AGO7 proteins indicate that, with the exception of RDR6, all the proteins are colocalized within a round-shaped signal in the nucleolar periphery (in yellow in the merged panels). In the top nuclei, RDR6 was detected with a mouse anti-Flag antibody, while SGS3 was detected with an antibody raised in rabbit against the native protein. In the middle and bottom panels, SGS3 was detected using a mouse anti-Flag antibody, whereas DCL4 and AGO7 were visualized by making use of specific native antibodies raised in rabbit. The arrow denotes colocalization foci between RDR6 and SGS3. n = number of nuclei analyzed and % indicates the percentage of nuclei with the representative immunolocalization pattern. Nuclear DNA was counterstained by DAPI (in blue). Scale bar denotes 5 µm.
Figure 2.
Nuclear localization of Dicer proteins.
Immunofluorescence analysis of DCL1, DCL2, DCL3 and DCL4 shows that these functionally redundant proteins are colocalized within a round-shaped signal in the nuclear periphery, as suggested by the strong yellow signal displayed after merging the green and red channels. DCL proteins were detected using a specific antibody combination originated from different hosted species to avoid cross reactivity issues. n = number of nuclei analyzed and % indicates the percentage of nuclei with the representative immunolocalization pattern. Nuclear DNA was counterstained by DAPI (in blue). Scale bar denotes 5 µm.
Figure 3.
Relative localization of HEN1 and Dicer proteins.
Simultaneous immunofluorescence of HEN1 and DCL1, DCL3 and DCL4 indicates these proteins colocalize within the nucleolar periphery (in yellow in the merged panels). HEN1 was visualized by immunolocalization using an anti-Flag antibody. DCL1, DCL3 and DCL4 were detected within the nucleus by using specific native antibodies. n = number of nuclei analyzed and % indicates the percentage of nuclei with the representative immunolocalization pattern. Nuclear DNA was counterstained by DAPI (in blue). Scale bar denotes 5 µm.
Figure 4.
Nuclear localization of Argonaute proteins.
The analysis of sub-nuclear localization of Argonaute proteins was performed by immunofluorescence. The overlay of green and red channels (resulting in the displayed yellow signal) indicates that, in a fraction of the nuclei, the proteins were colocalized within the nucleolar periphery. AGO4 was detected using an anti-cMyc antibody, whereas AGO1 and AGO7 were visualized by making use of specific native antibodies raised in chicken and rabbit, respectively. n = number of nuclei analyzed and % indicates the percentage of nuclei with the representative immunolocalization pattern. Nuclear DNA was counterstained by DAPI (in blue). Scale bar denotes 5 µm.
Figure 5.
Nuclear colocalization of Cajal body components.
Immunoflourescence of specific Cajal body components, U2B”, smD3 and coilin, indicates that those are colocalized in a large fraction of nuclei (in yellow in the merged panels). SmD3 was detected using an anti-Flag antibody and both coilin and U2B” were visualized by making use of specific native antibodies. Nuclear DNA was counterstained by DAPI (in blue). Scale bar denotes 5 µm.
Figure 6.
Colocalization analysis of Cajal body markers and components of miRNA and siRNA pathways.
Coilin- and smD3-containing Cajal bodies overlapped with siRNA and miRNA pathway proteins in a fraction of nuclei (in yellow in the merged panels). n = number of nuclei analyzed and % indicates the percentage of nuclei with the representative immunolocalization pattern. Proteins were localized using a combination of antibodies specific to the epitope tag in the respective transgenic lines and native antibodies. Nuclear DNA was counterstained by DAPI (in blue). Scale bar denotes 5 µm.
Figure 7.
Effect of Cajal body mutants in the localization of miRNA and siRNA pathway components.
Immunofluorescence of several proteins functioning in siRNA and miRNA pathways using specific native antibodies showed that the loss of function of coilin and smD3 interfered with their nuclear localization. Representative nuclei for each class are displayed. In the table, n = number of nuclei analyzed and % indicates the percentage of nuclei with the representative immunolocalization pattern. Nuclear DNA was counterstained by DAPI (in blue). Scale bar denotes 5 µm.
Figure 8.
Multiple endogenous small RNA-directed silencing pathways in A. thaliana plants potentially intersect in a specific nuclear domain corresponding to a CB.
The model depicts the scenario where pathways possibly converge in the same CB. A. RNA polymerase IV (Pol IV) transcription provides a single-stranded RNA template to RDR2. RDR2 is localized around the nucleolus and in the CB and acts exclusively in the hc-siRNA pathway. RDR2 activity produces a dsRNA precursor, processed by DCL3 into 24nt siRNA duplexes. B. SGS3 stabilization of a RDR6-derived dsRNA precursor can occur in the nucleoplasm and/or in the Cajal body. C. Dicer activity hypothetically occurs within the CB nuclear domain, with different DCLs directed to its respective substrates by DRB proteins, like HYL1 and DRB4. Upon dicing, smRNAs are methylated by HEN1 and loaded onto AGO proteins according to their size and 5′- end nucleotide. D. Loaded AGO proteins will exit the CB and be assigned to the nucleus or cytoplasm to carry out its function. Traffic to the cytoplasm might involve HASTY and other proteins not yet identified. E. In the cytoplasm, AGO7 has been found associated with RDR6 and SGS3 within a specific compartment related to post-transcriptional gene silencing [50], [51]. F. AGO4 localization to the cytoplasm as part of RISC complex maturation has been reported [57] followed by its return to the nucleus to target epigenetic modifications.