Figure 3.

Improvements on transcriptional labelling. (A) Neurons and pharynx have less effective RNAi. After exposure to dsRNA against GFP, YFP::NRDE-3 is nuclear localized in most tissues (white arrowheads: muscle nuclei), but localizes to the cytoplasm in pharynx and neurons. (B) Restoring expression of SID-1 in all tissues improves nuclear localization. After exposure to dsRNA against GFP, YFP::NRDE-3 is nuclear localized in neurons and, partially, in the posterior pharynx. (C) Improved transcriptional labelling in neurons. An embryo expressing SID-1 in all tissues, after maternal exposure to dsRNA against ttx-3, shows nuclear localization and transcriptional foci in AIY neurons (compare with Figure 2). (D) Principle of trimolecular fluorescence complementation. N-terminal and C-terminal fragments of the fluorescent protein Venus are fused to NRDE-3. In the cytoplasm or nucleus, local concentration of NRDE-3 molecules does not allow fluorescence complementation, thus reducing background fluorescence; once bound on the target transcript, VenusN::NRDE-3 and VenusC::NRDE-3 are in sufficient proximity to allow for fluorescence complementation. (E) Labelling of transcription with trimolecular fluorescence complementation. After exposure to dsRNA against hlh-1, bright transcription foci are visible in multiple nuclei (red arrows); additionally, some cytoplasmic foci are visible in the cytoplasm of muscle cells (white arrowheads), but not in other tissues. (F) Trimolecular fluorescence complementation labels cytoplasmic transcripts. Cytoplasmic spots (white arrowheads), due to fluorescence complementation between VenusN::NRDE-3 and VenusC::NRDE-3, as well as nuclear active transcription sites (red arrows), colocalize with smiFISH probes labelling hlh-1 transcripts.