A tetracycline-dependent ribozyme switch allows conditional induction of gene expression in Caenorhabditis elegans

Abstract

The nematode Caenorhabditis elegans represents an important research model. Convenient methods for conditional induction of gene expression in this organism are not available. Here we describe tetracycline-dependent ribozymes as versatile RNA-based genetic switches in C. elegans. Ribozyme insertion into the 3'-UTR converts any gene of interest into a tetracycline-inducible gene allowing temporal and, by using tissue-selective promoters, spatial control of expression in all developmental stages of the worm. Using the ribozyme switches we established inducible C. elegans polyglutamine Huntington's disease models exhibiting ligand-controlled polyQ-huntingtin expression, inclusion body formation, and toxicity. Our approach circumvents the complicated expression of regulatory proteins. Moreover, only little coding space is necessary and natural promoters can be utilized. With these advantages tetracycline-dependent ribozymes significantly expand the genetic toolbox for C. elegans.

Fig. 1

Impact of self-cleaving ribozymes on gene expression in C. elegans. a Schematic showing how ribozymes in the 3’-UTR affect mRNA stability. Blue arrow indicates self-cleavage site. (A)_N indicates poly-A tail. goi, gene of interest. b Sequence and secondary structure of type 3 hammerhead ribozyme with characteristic stems I–III. Blue arrow indicates cleavage site. Box indicates inactivating A-to-G mutation. c Schematic showing constructs injected into worms. Active and inactive ribozyme variants were inserted into the myo-2p::mCherry construct. P promoter. Rz ribozyme. d Microscope images of young adult worms injected with plasmids shown in c. BF, bright field. Scale bar, 300 µm. e RT-qPCR analysis showing the relative mCherry mRNA levels in young adult animals as shown in d. GFP was used as a reference gene. Error bars, s.d. ***p < 0.001 (two-tailed t-test); n = 3. Source data are provided as a Source Data file

Fig. 2

Tetracycline-dependent ribozymes enable conditional gene expression in C. elegans. a Schematic of the tetracycline-induced ribozyme expression ON-switch. A communication module (gray) connects the ribozyme to a tetracycline-binding aptamer (red). Self-cleavage (blue arrow) in the absence of the ligand leads to mRNA decay. Binding of tetracycline induces a conformational change in the ribozyme and its inactivation leading to mRNA stabilization and gene expression. (A)_N indicates poly-A tail. goi, gene of interest. b Microscope images of stable transgenic worms carrying aptazyme-regulated icd-1p::mCherry reporter. Worms were treated with indicated tetracycline concentrations from hatch until adulthood (3 days, 20 °C). BF, bright field. Scale bar, 300 µm. c Immunoblot analysis of mCherry protein levels in animals as shown in b. Actin served as loading control. Tet, tetracycline. d Flow cytometry analysis of worms carrying aptazyme-regulated icd-1p::mCherry reporter. Synchronized L1 larvae were treated with 5 µM tetracycline for 48 h at 20 °C. Histogram plots mCherry fluorescence intensity against the number of worms. n > 300. e Worm flow cytometry analysis as in d. Animals were treated at indicated developmental stages with 10 µM tetracycline for 24 h at 20 °C. Bar diagram shows the fold-induction of mCherry fluorescence compared to control. Error bars, s.d. ***p < 0.001 (two-tailed t-test); n > 300. L1-4 larval stage 1-4. YA young adult. D1 day 1 adult. Source data are provided as a Source Data file

Fig. 3

Aptazymes allow tissue-selective induction of gene expression in C. elegans. a Microscope images of worms carrying 3’-UTR aptazyme-regulated mCherry reporters driven by tissue-specific promoters for the pharynx (myo-2p), body wall muscles (myo-3p) and neurons (rab-3p). Worms were treated with 10 µM tetracycline from hatch until adulthood (3 days at 20 °C). Boxed sections in images of worms carrying the pan-neuronal reporter are shown enlarged with adjusted contrast to visualize the ventral nerve cord (VNC), dorsal nerve cord (DNC) and tail ganglion (TG). NR nerve ring, BF bright field. Scale bar, 300 µm. b Confocal microscope images of animals as in a. Images show the head region of worms carrying aptazyme-regulated mCherry reporters expressed in body wall muscles and neurons. BF bright field. Scale bar, 30 µm

Fig. 4

Aptazyme-inducible proteotoxic C. elegans disease models. a, b Schematics showing constructs for inducible expression of human Huntingtin exon 1 (Htt) containing a polyglutamine stretch of either 109 (Htt109Q) or 25 glutamines (Htt25Q) fused to mCherry driven either by the ubiquitous promoter icd-1p (a) or the pan-neuronal promoter rab-3p (b). Rz, ribozyme. c Microscope images of worms carrying icd-1p::Htt109Q::mCherry construct. Animals were fed 10 µM tetracycline from hatch until adulthood (3 days, 20 °C). Upper row shows overlay of bright field images with mCherry fluorescence. Fluorescence signal in boxed sections is shown enlarged to visualize Htt109Q::mCherry aggregates (bottom row). Scale bar, 300 µm. d Same analysis as in c but with rab-3p::Htt109Q::mCherry strain. White arrows indicate Htt109Q::mCherry inclusion bodies. e, b Same analysis as in b and c but with Htt25Q::mCherry worms, respectively. g Strains carrying the constructs shown in a were treated with 10 µM tetracycline from hatch. N2 worms served as wildtype control. Diagram shows the percentage of paralyzed worms at indicated days of adulthood. Error bars, s.d. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Two-Way ANOVA, Bonferroni post-hoc test; n = 3. h Strains carrying the constructs shown in b were treated with 10 µM tetracycline from hatch until day 2 of adulthood (4 days at RT). Box plot shows the number of body bends made by worms in a drop of liquid in 20 s. ***p < 0.001 (two-tailed t-test); ns not significant; n = 40. Center line = median; box length = upper + lower quartile; whiskers = minimum/maximum quartile. Source data are provided as a Source Data file

Fig. 5

Aptazymes enable rescue control of mutant phenotypes in C. elegans. a Schematics showing constructs injected into unc-119(ed3) strain. A constitutive unc-119p::Cbr-unc-119 rescue plasmid (unc-119::WT) and a tetracycline-inducible unc-119p::Cbr-unc-119 rescue construct with the aptazyme in the 3’-UTR (unc-119::Rz) were used to rescue the unc phenotype of unc-119(ed3) worms. Rz ribozyme. b Parental unc-119(ed3) worms (−) and transgenic strains carrying the constructs shown in a were treated with 10 µM tetracycline from hatch. Diagram shows the percentage of unc worms at day 1 of adulthood. Twenty-four animals per group were used and experiments were carried out in biological triplicates. Error bars, s.d. ***p < 0.001 (two-tailed t-test); n = 3. Source data are provided as a Source Data file