Figure 3. Worm
mrp-5 Encodes a Putative Intestinal Heme Exporter(A) GFP expression in IQ5051 [Pmrp-5::GFP::
unc-54 30 UTR;
unc-119(
ed3);
unc-119 rescue fragment] as determined using confocal microscopy.
mrp-5 isexpressed in the hypodermis and some neurons and at higher levels in the pharynx and intestine. P, pharynx; I, intestine; H, hypodermis; E, embryo. Scalebars, 20 μM.(B) Transgenic IQ5351 worms [Pvha-6::MRP-5:GFP::
unc-54 30 UTR;
unc-119(
ed3);
unc-119 rescue fragment] expressing an
mrp-5 translational reporter were imaged by confocal microscopy. Dotted lines indicate apical membrane, dashed lines indicate basolateral membrane, and arrowheads indicate lateral membranes between adjacent intestinal cells. Scale bar, 50 μM.(C) The MRP-5::GFP fusion gene can rescue the embryonic lethality of
mrp-5 RNAi. RNAi targeting the
mrp-5 ORF causes embryonic lethality in both wild-type N2 and transgenic worms. RNAi against the
mrp-5 30 UTR results in a less severe embryonic lethal phenotype in N2 worms, but this lethality is significantly rescued by expression of the MRP-5::GFP transgene from either the
mrp-5 or the intestinal
vha-6 promoter. ***p < 0.001 when compared to wild-type N2 worms under identical conditions, n = 3 (two-way ANOVA, Bonferroni post test). Error bars represent mean ± SEM.(D) Intestinal RNAi of
mrp-5 recapitulates the embryonic lethality of whole animal
mrp-5 RNAi. Wild-type N2 worms and the tissue-specific RNAi strains were grown on RNAi plates with no added heme. Int, intestinal RNAi; Mus, muscle RNAi; Hyp, hypodermal RNAi; n = 2. See Results and Figure S2 for further strain information. Error bars represent mean ± SEM.(E) Ectopic expression of
hrg-3 does not rescue the embryonic lethality of whole-animal
mrp-5 RNAi. The experiment was performed as in Figure 1C using wildtype N2 worms and worms ectopically expressing HRG-3 and GFP separated by the SL2 intercistronic sequence [
hrg-3(
tm2468); Pvha-6::HRG-3::ICS::GFP,
unc119(ed3);
unc-119 rescue fragment] grown on RNAi plates with no added heme. Error bars represent mean ± SEM.(F) Loss of
mrp-5 activates an extraintestinal heme depletion signal. Left: yellow fluorescent protein fluorescence (60-100 worms per treatment) quantified using COPAS BioSort in the
hrg-2 translational fusion line IQ8122 [Phrg-2::HRG-2:YFP::
hrg-2 30 UTR,
unc-119(
ed3);
unc-119 rescue fragment] exposed to vector,
hrg-4, or
mrp-5 RNAi at varying heme concentrations. ***p < 0.001, **p < 0.01 when compared to vector control worms (two-way ANOVA, Bonferroni post test). Right: representative images of worms grown at 1 mM heme from the left panel. Left: error bars represent mean ± SEM.