Mechanistic insights into RNA ENE triplex function and inhibition by small molecules

Abstract

The emerging role of RNA triple helices (TH) in many biological functions has attracted many research studies. Recently, this structural element was found at the 3’ end of two long noncoding RNAs (lncRNAs) involved in human disease: metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) RNA and polyadenylated nuclear (PAN) RNA. MALAT1 is an oncogenic lncRNA that is overexpressed in many cancer cells. PAN RNA is a viral lncRNA that is encoded by the Kaposi sarcoma-associated herpesvirus (KSHV). The TH in both RNAs prevents their degradation by normal exonucleolytic degradation pathways. Because of the role of these RNAs in regulating human disease progression, they are considered excellent candidates for therapeutic intervention. Therefore, we aimed to target these RNA triplexes with small molecules. Toward this goal, we thoroughly characterized the folding and stability of MALAT 1 triple helix (M1TH), revealing that finely tuned conformational dynamics of the triplex controls its function. Additional extensive characterizations of the linker region of M1TH showed that it adapts a double helix that is extended beneath the P1 stem. Targeting this RNA with small molecules reduced the RNA level as well as the branching morphology in mammary tumor organoid. Even though M1TH and PAN triple helix (PANTH) are similar, they exhibit different folding and stability properties in vitro. Our initial studies of PANTH indicated that a truncated construct, PANTHA9-CGG, demonstrated promising results with in vitro triplex formation and protection against exonucleolytic activity. We used this construct to characterize the folding and stability of PANTH and investigate the mechanisms of interaction between PANTH and small molecule inhibitors. Taken together, these data demonstrated the applicability of targeting RNA triplexes with small molecules.