Metastasis-associated lung adenocarcinoma transcript 1 (Malat1/MALAT1, mouse/human), a highly conserved long noncoding (lnc) RNA, has been linked with several physiological processes, including the alternative splicing, nuclear organization, and epigenetic modulation of gene expression. MALAT1 has also been implicated in metastasis and tumor proliferation in multiple cancer types. The 3′ terminal stability element for nuclear expression (ENE) assumes a triple-helical configuration that promotes its nuclear accumulation and persistent function. Utilizing a novel small molecule microarray strategy, we identified multiple Malat1 ENE triplex-binding chemotypes, among which compounds 5 and 16 reduced Malat1 RNA levels and branching morphogenesis in a mammary tumor organoid model. Computational modeling and Förster resonance energy transfer experiments demonstrate distinct binding modes for each chemotype, conferring opposing structural changes to the triplex. Compound 5 modulates Malat1 downstream genes without affecting Neat1, a nuclear lncRNA encoded in the same chromosomal region as Malat1 with a structurally similar ENE triplex. Supporting this observation, the specificity of compound 5 for Malat1 over Neat1 and a virus-coded ENE was demonstrated by nuclear magnetic resonance spectroscopy. Small molecules specifically targeting the MALAT1 ENE triplex lay the foundation for new classes of anticancer therapeutics and molecular probes for the treatment and investigation of MALAT1-driven cancers.

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 dynamic M1TH 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.

Cellular and virus-coded long non-coding (lnc) RNAs support multiple roles related to biological and pathological processes. Several lncRNAs sequester their 3′ termini to evade cellular degradation machinery, thereby supporting disease progression. An intramolecular triplex involving the lncRNA 3′ terminus, the element for nuclear expression (ENE), stabilizes RNA transcripts and promotes persistent function. Therefore, such ENE triplexes, as presented here in Kaposi's sarcoma-associated herpesvirus (KSHV) polyadenylated nuclear (PAN) lncRNA, represent targets for therapeutic development. Towards identifying novel ligands targeting the PAN ENE triplex, we screened a library of immobilized small molecules and identified several triplex-binding chemotypes, the tightest of which exhibits micromolar binding affinity. Combined biophysical, biochemical, and computational strategies localized ligand binding to a platform created near a dinucleotide bulge at the base of the triplex. Crystal structures of apo (3.3 Å) and ligand-soaked (2.5 Å) ENE triplexes, which include a stabilizing basal duplex, indicate significant local structural rearrangements within this dinucleotide bulge. MD simulations and a modified nucleoside analog interference technique corroborate the role of the bulge and the base of the triplex in ligand binding. Together with recently discovered small molecules that reduce nuclear MALAT1 lncRNA levels by engaging its ENE triplex, our data supports the potential of targeting RNA triplexes with small molecules.

5 UUUUUUU, where U 11 and U 5 are loops of 11 and 5 uridines, respectively. We used a combination of temperature-dependent UV spectroscopy and differential scanning calorimetric techniques to determine the unfolding thermodynamics of each pseudoknot. Additional UV melting experiments were done as a function of oligomer, salt and osmolyte concentrations to determine the transition molecularity, ion and water binding, respectively. In the 16-116 mM NaCl range, U-PsKn has a higher T M s and is thermodynamically more stable (by 12.3 kcal/ mol) than dU-PsKn. This large differential free energy contribution is driven by a differential favorable enthalpy contribution of 34.3 kcal/mol. In addition, U-PsKn immobilizes more counterions (by 0.3 per mol,) and water molecules (by 16 per mol), and unfolds with a higher heat capacity contribution (by 1.0 kcal/ o C-mol) than its DNA analog. Furthermore, the overall enthalpy is higher than the corresponding enthalpy of its double helical stem (estimated from nearestneighbors contributions), suggesting the longer U 11 loop could potentially form local UAU base-triplets with the adjacent A 7 /U 7 stem. We estimate U-PsKn forms 7 base-triplet stacks while dU-PsKn forms 4 base-triplet stacks. The overall results imply U-PsKn forms a more compact structure.

No abstract