Enhanced neuronal RNAi in C. elegans using SID-1

Abstract

We expressed SID-1, a transmembrane protein from Caenorhabditis elegans that is required for systemic RNA interference (RNAi), in C. elegans neurons. This expression increased the response of neurons to double-stranded (ds)RNA delivered by feeding. Mutations in the lin-15b and lin-35 genes enhanced this effect. Worms expressing neuronal SID-1 showed RNAi phenotypes when fed with bacteria expressing dsRNA for known neuronal genes and for uncharacterized genes with no previously known neuronal phenotypes. Neuronal expression of sid-1 decreased nonneuronal RNAi, suggesting that neurons expressing transgenic sid-1(+) served as a sink for dsRNA. This effect, or a sid-1(-) background, can be used to uncover neuronal defects for lethal genes. Expression of sid-1(+) from cell-specific promoters in sid-1 mutants results in cell-specific feeding RNAi. We used these strains to identify a role for integrin signaling genes in mechanosensation.

Figure 1. Expression of sid-1 in neurons enhances neuronal RNAi

(a) Neuronal YFP fluorescence in different areas of P_unc-119sid-1 (TU3270) and control worms (TU3310) fed with bacteria producing dsRNA for gfp. Both strains contain P_unc-119yfp. Worms fed mec-4 dsRNA were used as controls. Similar results were obtained with the TU3311 strain. Arrows indicate PLML neurons. Scale bars = 25 µm, except ALML (10 µm). (b) Touch sensitivity of worms of the indicated genotypes or expressing the indicated transgenes, fed with either mec-4 dsRNA (gray bars) and gfp dsRNA (white bars). Similar results were obtained when worms expressing P_mec-18sid-1 were fed bacteria expressing dsRNA for mec-12. The same effect as for P_unc-119sid-1 worms (TU3270) was obtained with TU3311. Values represent the mean ± S.E.M. of four experiments, each with 30 adults, except for the mec-18 data, which was from nine experiments with 20 adults.

Figure 2. Mutations in lin-35 and lin-15b enhance RNAi in neurons expressing sid-1

(a) YFP expression in the nerve ring and ventral cord of worms with the indicated genotypes after feeding with bacteria making dsRNA for gfp or for mec-4 (compare with worms in Fig. 1). Both strains contain P_unc-119yfp. Scale bars = 25 µm. (b) YFP fluorescence in the nerve ring of the indicated strains after feeding with bacteria making gfp dsRNA. The control strain is TU3310, expressing P_unc-119yfp alone. Strains with P_unc-119sid-1 were derived from TU3270 strain and have the mec-6(+) transgene. Results are presented as the mean percentage of fluorescence (± S.E.M.) measured in the same strain fed bacteria making mec-4 dsRNA (three experiments, each with 30 adult worms). (c) Anterior touch response in worms of the indicated strains fed bacteria making dsRNA for gfp (white) or mec-4 (gray). Values represent the mean ± S.E.M. of four experiments, each with 30 adults. The asterisk represents significance at p < 0.05.

Figure 3. Enhanced RNAi for genes needed for touch sensitivity

The plots show the anterior touch response (out of five touches) in worms of the indicated genotypes fed bacteria making dsRNA for known mec genes (which give a touch insensitive phenotype when mutated). mec mutants were examined for comparison. Strains with P_unc-119sid-1 were derived from TU3270 strain and have the mec-6(+) transgene. Except for the mec mutants, each value is the mean response (± SEM) of worms on 9 RNAi plates with 20 adult worms each. The values for the mec mutants represent the mean response (± SEM) of 20 adult worms.

Figure 4. Expression of sid-1 in neurons decreases RNAi responses in non-neuronal tissues

The fraction of worms of the indicated genotypes that resisted treatment with dsRNA for various genes (rpl-3, unc-52, elt-2 and nhx-2) is plotted. For rpl-3 we counted the number of worms that reached adulthood and became fertile; for unc-52 we counted the number of paralyzed worms, and for elt-2 and nhx-2 we counted the number of fertile F1 worms. We used wild type (N2) as controls that do not express P_unc-119sid-1(+) and strain TU3270 as controls that do. sid-1 mutants were included as negative controls. Values represent the mean ± S.E.M. of nine plates, 50 worms scored per plate.

Figure 5. Eliminating integrin signaling proteins by RNAi in neurons

(a, b) The plots show the touch response (out of five touches) of P_unc-119sid-1-expressing worms (a), P_mec-18sid-1; sid-1 worms (b, grey bars) or P_mec-18sid-1; sid-1; lin-5b worms (b, white bars) fed bacteria making dsRNA for the indicated genes. In (a), combined results for three strains (TU3270, TU3311, and TU3401, see Methods for details) are shown, because all gave similar results. Values are mean response ± S.E.M, 20 adult animals/plate; numbers indicate the number of plates examined.