Finely tuned conformational dynamics regulate the protective function of the lncRNA MALAT1 triple helix

Abstract

Nucleic acid triplexes may regulate many important biological processes. Persistent accumulation of the oncogenic 7-kb long noncoding RNA MALAT1 is dependent on an unusually long intramolecular triple helix. This triplex structure is positioned within a conserved ENE (element for nuclear expression) motif at the lncRNA 3′ terminus and protects the entire transcript from degradation in a polyA-independent manner. A requisite 3′ maturation step leads to triplex formation though the precise mechanism of triplex folding remains unclear. Furthermore, the contributions of several peripheral structural elements to triplex formation and protective function have not been determined. We evaluated the stability, conformational fluctuations, and function of this MALAT1 ENE triple helix (M1TH) protective element using in vitro mutational analyses coupled with biochemical and biophysical characterizations. Using fluorescence and UV melts, FRET, and an exonucleolytic decay assay we define a concerted mechanism for triplex formation and uncover a metastable, dynamic triplex population under near-physiological conditions. Structural elements surrounding the triplex regulate the dynamicM1TH conformational variability, but increased triplex dynamics lead to M1TH degradation. Taken together, we suggest that finely tuned dynamics may be a general mechanism regulating triplex-mediated functions.