Figure 1.
Characterization of Small RNA Changes following Rescue of RDE-4 in Neurons
(A) Nervous system-specific rescue of RDE-4. A typical image demonstrating the neuronal expression pattern of the rescued RDE-4 (Psng-1::rde-4::SL2::yfp), as monitored by examination of a trans-spliced YFP fluorescent reporter. Bar, 20 μm.
(B and C) smFISH staining of yfp transcripts (magenta) and DAPI nuclei staining (blue) in one typical worm expressing the integrated single-copy Psng-1::rde-4::SL2::yfp pan-neuronal rescue transgene. Shown are focal plains focusing on the neuronal ventral chord (B, yellow dashed lines), and the germline (C, white dashed lines). Bar, 20 μm.
(D) Expression of STGs in rescued rde-4(n299);Psng-1::rde-4 worms (y axis) compared to rde-4(ne299) mutants (x axis). Shown are the averaged expression values (log2 of RPM) of STGs (see also Table S2). Each dot represents an STG. Red dots, STGs that display differential expression between groups (analyzed with Deseq2, adjusted p value < 0.1).
(E) x-fold enrichment and depletion values of upregulated STGs and downregulated STGs following RDE-4 rescue in neurons. We tested the enrichment of the RDE-4-dependent STGs against lists of STGs that are known to require DCR-1 for their biogenesis, to bind the Argonaute ERGO-1, and to depend on somatic mut-16 activity (Welker et al., 2010, Zhang et al., 2011, Vasale et al., 2010). p values for enrichment were calculated using 10,000 random gene sets identical in size to the tested group (see STAR Methods for details). Enrichments were considered significant if p < 0.05. Not significant [ns], p > 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 10−4.
(F) STGs distributions. Shown are STGs normalized read counts (y axis) as function of genomic location (x axis) of small RNAs targeting the genes ser-5 and C46G7.5 STGs (in red) in N2 wild-type worms, rde-4 mutants, and Psng-1::rde-4 rescue worms. Exons appear on a gray background. Blue arrow points to the direction of transcription.
See also Figure S1.