The functions of long noncoding (lnc)RNAs, such as MEG3, are defined by their interactions with other RNAs and proteins. These interactions, in turn, are shaped by their subcellular localization and temporal context. Therefore, it is important to be able to analyze the relationships of lncRNAs while preserving cellular architecture. The ability of MEG3 to suppress cell proliferation led to its recognition as a tumor suppressor. MEG3 has been proposed to activate p53 by disrupting the interaction of p53 with mouse double minute 2 homolog (Mdm2). To test this mechanism in the native cellular context, we employed two-color direct stochastic optical reconstruction microscopy, a single-molecule localization microsco... More
The functions of long noncoding (lnc)RNAs, such as MEG3, are defined by their interactions with other RNAs and proteins. These interactions, in turn, are shaped by their subcellular localization and temporal context. Therefore, it is important to be able to analyze the relationships of lncRNAs while preserving cellular architecture. The ability of MEG3 to suppress cell proliferation led to its recognition as a tumor suppressor. MEG3 has been proposed to activate p53 by disrupting the interaction of p53 with mouse double minute 2 homolog (Mdm2). To test this mechanism in the native cellular context, we employed two-color direct stochastic optical reconstruction microscopy, a single-molecule localization microscopy technique, to detect and quantify the localizations of p53, Mdm2, and MEG3 in U2OS cells. We developed a new cross-nearest neighbor/Monte Carlo algorithm to quantify the association of these molecules. Proof of concept for our method was obtained by examining the association between FKBP1A and mTOR, MEG3 and p53, and Mdm2 and p53. In contrast to previous models, our data support a model in which MEG3 modulates p53 independently of the interaction with Mdm2.
