A novel strategy for cell-autonomous gene knockdown in Caenorhabditis elegans defines a cell-specific function for the G-protein subunit GOA-1

Abstract

We developed a novel knockdown strategy to examine cell-specific gene function in Caenorhabditis elegans. In this strategy a null mutation in any gene is replaced with a genetically stable transgene that contains a wild-type copy of the gene fused to a 3' tag that targets the mRNA transcript for degradation by the host nonsense-mediated decay (NMD) machinery. In NMD-defective animals, tagged transgene mRNA is expressed at levels similar to the endogenous gene it replaced and is translated into wild-type protein that fully rescues gene function. Cell-specific activation of NMD cell autonomously knocks down transgene expression in specific cell types without affecting its expression or function in other cells of the organism. To demonstrate the utility of this system, we replaced the goa-1 gene, encoding the pan-neuronally expressed G-protein subunit GOA-1, with a degradation-tagged transgene. We then knocked down expression of the transgene from only two neurons, the hermaphrodite-specific neurons (HSNs), and showed that GOA-1 acts cell autonomously in the HSNs to inhibit egg-laying behavior.

Keywords: RNA interference; gene expression; gene knockdown; nonsense-mediated decay.

Figure 1

Expression of degradation-tagged mCherry transgene is NMD dependent. (A) Schematic representation of degradation-tagged mCherry transgene. Boxes represent exons and lines between boxes are introns. Positions of stop codons are indicated. Bar, 1 kb. (B) Expression of tagged (rab-3p::mCherry::let-858) and untagged (rab-3p::GFP) transgenes in smg-5 mutants (top panels) and wild-type animals (bottom panels). The rab-3 promoter drives expression in all neurons. mCherry protein is detected in all neurons of NMD-defective (smg-5) mutants but is not detected in wild-type animals while GFP is easily detected in all neurons of both mutant and wild-type animals. Bars, 50 µm. (C) Expression of tagged (rab-3p::mCherry::let-858) and untagged (unc-17::GFP) transgenes in GABAergic and cholinergic ventral cord motor neurons of smg-5 mutants that either lack (left panels) or express (right panels) the unc-17::SMG-5 NMD-rescuing transgene. The unc-17 promoter drives expression in all cholinergic cells, and thus all green cells are cholinergic. mCherry protein in detected in both cholinergic and GABAergic neurons in smg-5 mutants lacking the rescue transgene (left panels) while mCherry protein is detected only in GABA neurons in smg-5 mutants that express the rescue transgene (right panels). Note the presence of yellow cells in the bottom left panel, indicating expression of the tagged mCherry transgene in cholinergic cells, and the lack of yellow cells in the bottom right panel, indicating degradation of tagged transgene in NMD-rescued cholinergic cells. GABA neuron cell bodies are indicated by arrows. Bars, 20 µm.

Figure 2

Expression of a degradation-tagged transgene rescues endogenous gene function in NMD-defective but not NMD-competent animals. (A–C) Quantification of (A) locomotion rate, (B) spontaneous reversal frequency, and (C) egg-lying behavior. Genotypes and transgenes expressed for each strain are indicated at the bottom of each panel. The behavior of each of 30 animals was recorded. The mean and standard deviation are shown.

Figure 3

NMD-dependent knockdown of tagged transgenes is robust and can be restricted to individual cell types. (A–C) Quantification of (A) locomotion rate, (B) spontaneous reversal frequency, and (C) egg-laying behavior. Genotypes and transgenes expressed for each strain are indicated at the bottom of each panel. Rescue of each behavior by expression of the goa-1p::GOA-1 tagged transgene is dependent on NMD. NMD-dependent knockdown of the tagged transgene is cell autonomous as expression of SMG-5 in HSNs by the tph-1p::SMG-5 transgene caused egg-laying defects similar to those observed in goa-1 null animals but did not cause defects in either locomotion rate or reversal frequency. The behavior of each of 30 animals was recorded. The mean and standard deviation are shown.

Figure 4

Levels of goa-1 expression from tagged transgenes are similar to those of wild-type animals and are dramatically reduced by NMD. (A) Quantification of goa-1 mRNA levels by qRT-PCR in wild-type and transgenic animals. All values represent the average from three independent RNA preparations for each strain and were normalized to cdc-42 expression. Error bars represent SEM. Comparisons shown are to goa-1 mRNA levels in the wild type. (B) Analysis of GOA-1 protein expression by Western blot. Worm lysates were probed with GOA-1 (top) and control UNC-64 (bottom) antibodies. Genotypes and transgenes expressed for each strain are indicated at the bottom of each panel.