Background

We describe here a protocol to image transcription at transcription sites in C. elegans using an Argonaute protein NRDE-3, based on a method we recently published (Toudji-Zouaz et al., 2021). In C. elegans, double-stranded RNA targeting the gene of interest is uptaken by the systemic RNAi mechanisms and distributed to all cells by the dsRNA transporter SID-1.

The exoRNAi pathway will process dsRNA and use them for recognition of target mRNAs for degradation. This recruits the RNA dependent RNA polymerase RRF-1 to the transcript, which will synthesise triphosphorylated, antisense, secondary small RNAs that are loaded into secondary Argonautes. The only somatic secondary Argonaute, NRDE-3, when unladen, is cytoplasmic. Once loaded with small RNA, NRDE-3 moves to the nucleus and binds to the nascent transcript, where it recruits other proteins to silence transcription.

In a wild-type background, the endogenous RNAi pathway will induce synthesis of secondary small RNAs, and constitutive nuclear localisation of NRDE-3 (Figure 1A, B). To prevent this, we introduce a mutation in eri-1. NRDE-3 is therefore unladen in most tissues (except for the germline, intestine, and early embryo), localises to the cytoplasm, and will move into the nucleus only if the gene of interest is expressed in the cell (Guang et al., 2008) (Figure 1C, D). To prevent downstream transcriptional silencing, we also introduce a mutation in nrde-2 (Guang et al., 2010).

Figure 1. NRDE-3 nuclear localisation is dependent on small RNAs. Nuclear localisation of fluorescently labelled NRDE-3 in the embryo (A) and adult somatic tissues (head) (B) in a eri-1(+) genetic background. Cytoplasmic localisation of NRDE-3 in a eri-1(-) background, in the embryo (C) and adult (D). Note the nuclear localisation in the germline primordium in the embryo (C).