Abstract
The in vitro formation of stable G-quadruplexes (G4s) in human rRNA was recently reported. However, their formation in cells and their cellular roles were not resolved. Here, by taking a chemical biology approach that integrates results from immunofluorescence, G4 ligands, heme-affinity reagents, and a genetically encoded fluorescent heme sensor, we report that human ribosomes can form G4s in vivo that regulate heme bioavailability. Immunofluorescence experiments indicate that the vast majority of extra-nuclear G4s are associated with rRNA. Moreover, titrating human cells with a G4 ligand alters the ability of ribosomes to bind heme and disrupts cellular heme bioavailability as measured by a genetically encoded fluorescent heme sensor. Overall, these results suggest that ribosomes play a role in regulating heme homeostasis.
Highlights
HEK293 cells were cultured in DMEM containing 4.5 g/liter glucose without sodium pyruvate and L-glutamine (Corning) supplemented with 10% FBS (Corning) and 2% penicillin-streptomycin solution (Gibco) in a humidified incubator kept at 37 °C with a 5% carbon dioxide atmosphere
After solutions were incubated for 30 min at room temperature, PhenDC3 or carrier DMSO was added to final concentrations consisting of 1.5 mM, 3 mM, and 6 mM
After RNA-heme solutions were incubated for 30 min at room temperature, PhenDC3 was added to a final concentration range consisting of 1.33–133 nM and allowed to mix for 15 min
Summary
Confocal microscopy and G4-pulldowns were used to determine whether human ribosomes form G4s in vivo. Unlike the in vitro experiments with K1, it seems probable that most rRNA G-tracts are unfolded in cells This inference is based on our observation that only 5% of ribosomes bind to the BG4 antibody in vivo until the addition of PhenDC3, upon which BG4 binding increases to 24% of ribosomes (Fig. 2, C and D). Weaker signal is seen for the SSU, in agreement with the higher abundance of G4 regions in the LSU than in the SSU (Fig. 1A) These data are consistent with our observations that PhenDC3 promotes rRNA G4 formation in cells (Fig. 2, C–D), providing additional heme-binding sites that can interact with heminagarose. Our data indicate that rRNA G4s bind heme and regulate intracellular heme bioavailability
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