Abstract
The ability of the long noncoding RNA MEG3 to suppress cell proliferation led to its recognition as a tumor suppressor. MEG3 has previously been shown to bind to p53 in vitro, which led us to hypothesize that MEG3 functions by disrupting the interaction of p53 and its E3 ubiquitin ligase Mdm2. To test this hypothesis in vivo, we built a cross‐nearest neighbor/Monte Carlo analytical method based on two color direct stochastic optical reconstruction microscopy (dSTORM), a single‐molecule localization microscopy (SMLM) technique. Our data support the interaction of MEG3 and p53. Surprisingly, this association had no effect on the binding of p53 and Mdm2, distinct from the most commonly proposed model for the mechanism of MEG3 action. Additionally, our mathematical approach to analyzing SMLM data has general applicability to assessing molecular interactions in a native cellular context.Support or Funding InformationRJS: NIH R01CA193520, NIH R01DK062472, NIH S10RR027931; NCB: NIH T32DK007540; AK and YZ: NIH R01CA193520; and The Jarislowsky Foundation
Highlights
Long noncoding RNAs function in cell-type and subcellular localization–dependent contexts; how they do so is poorly understood
To fully understand the interactions of MEG3 with p53 and to test the hypothesis that MEG3 disrupts p53–Mdm2 binding, we developed a new cross–nearest neighbor/Monte Carlo algorithm to quantify the association between molecules from direct stochastic optical reconstruction microscopy data
Using our cross–nearest neighbor/Monte Carlo method, we found a stark difference between MEG3 and GAPDH mRNA in terms of fraction bound (Figure 6A)
Summary
Long noncoding RNAs (lncRNAs) function in cell-type and subcellular localization–dependent contexts; how they do so is poorly understood. MEG3 expression is significantly reduced or absent in meningiomas [8], epithelial ovarian cancer [9], and squamous cell carcinoma of the tongue [10]; supporting its role as a tumor suppressor. These values were accumulated across the whole cell (plotted in G), and the fraction of pairs with a probability of chance association < 0.1 and within a physically possible binding distance (< 200 nm), the fraction bound, was calculated (H) To fully understand the interactions of MEG3 with p53 and to test the hypothesis that MEG3 disrupts p53–Mdm binding, we developed a new cross–nearest neighbor/Monte Carlo algorithm to quantify the association between molecules from direct stochastic optical reconstruction microscopy (dSTORM) data. In contrast to previous models, our data support a model in which MEG3 modulates p53 independently of Mdm
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