Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a cancer-promoting long noncoding RNA, accumulates in cells by using a 3'-triple-helical RNA stability element for nuclear expression (ENE). The ENE, a stem-loop structure containing a U-rich internal loop, interacts with a downstream A-rich tract (ENE+A) to form a blunt-ended triple helix composed of nine U•A-U triples interrupted by a C•G-C triple and C-G doublet. This unique structure prompted us to explore the possibility of protein binding. Native gel-shift assays revealed a shift in radiolabeled MALAT1 ENE+A RNA upon addition of HEK293T cell lysate. Competitive gel-shift assays suggested that protein binding depends not only on the triple-helical structure but also its nucleotide composition. Selection from the lysate using a biotinylated-RNA probe followed by mass spectrometry identified methyltransferase-like protein 16 (METTL16), a putative RNA methyltransferase, as an interacting protein of the MALAT1 ENE+A. Gel-shift assays confirmed the METTL16-MALAT1 ENE+A interaction in vitro: Binding was observed with recombinant METTL16, but diminished in lysate depleted of METTL16, and a supershift was detected after adding anti-METTL16 antibody. Importantly, RNA immunoprecipitation after in vivo UV cross-linking and an in situ proximity ligation assay for RNA-protein interactions confirmed an association between METTL16 and MALAT1 in cells. METTL16 is an abundant (∼5 × 105 molecules per cell) nuclear protein in HeLa cells. Its identification as a triple-stranded RNA binding protein supports the formation of RNA triple helices inside cells and suggests the existence of a class of triple-stranded RNA binding proteins, which may enable the discovery of additional cellular RNA triple helices.

Triple-stranded RNA was first deduced to form in vitro more than 50 years ago and has since been implicated in RNA catalysis, stability, and small molecule binding. Despite the emerging biological significance of RNA triple helices, it remains unclear how their nucleotide composition contributes to their thermodynamic stability and cellular function. To investigate these properties, we used in vitro RNA electrophoretic mobility shift assays (EMSAs) and in vivo intronless β-globin reporter assays to measure the relative contribution of 20 RNA base triples (N•A–U, N•G–C, N•C–G, N•U–A, and N•G–U) to triple-helical stability. These triples replaced a single internal U•A–U within the known structure of the triple-helical RNA stability element of human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), which contains 10 major-groove base triples. In addition to the canonical C•G–C triple, the noncanonical base triples U•G–C, U•G–U, C•C–G, and U•C–G exhibited at least 30% stability relative to the wild-type U•A–U base triple in both assays. Of these triples, only U•A–U, C•G–C, and U•G–C, when tested as four successive triples, formed stabilizing structures that allowed accumulation of the intronless β-globin reporter. Overall, we find that Hoogsteen-position pyrimidines support triple helix stability and function and that thermodynamic stability, based on EMSA results, is necessary but not sufficient for stabilization activity of the MALAT1 triple helix in cells. These results suggest that additional RNA triple helices containing noncanonical triples likely exist in nature.