A highly ordered, nonprotective MALAT1 ENE structure is adopted prior to triplex formation.

The 3' end of the ∼7 kb lncRNA MALAT1 contains an evolutionarily and structurally conserved element for nuclear expression (ENE) which confers protection from cellular degradation pathways. Formation of an ENE triple helix is required to support transcript accumulation, leading to persistent oncogenic activity of MALAT1 in multiple cancer types. Though the specific mechanism of triplex-mediated protection remains unknown, the MALAT1 ENE triplex has been identified as a promising target for therapeutic intervention. Interestingly, a maturation step of the nascent lncRNA 3' end is required prior to triplex formation. We hypothesize that disruption of the maturation or folding process may be a viable mechanism of inhibition. To assess putative cotranscriptional ENE conformations prior to triplex formation, we perform microsecond MD simulations of a partially folded ENE conformation and the ENE triplex. We identify a highly ordered ENE structure prior to triplex formation. Extensive formation of U•U base pairs within the large U-rich internal loops produces a global rod-like architecture. We present a three-dimensional structure of the isolated ENE motif, the global features of which are consistent with small angle X-ray scattering (SAXS) experiments. Our structural model represents a nonprotective conformation of the MALAT1 ENE, providing a molecular description useful for future mechanistic and inhibition studies. We anticipate that targeting stretches of U•U pairs within the ENE motif will prove advantageous for the design of therapeutics targeting this oncogenic lncRNA.