title: Hoogsteen-position pyrimidines promote the stability and function of the MALAT1 RNA triple helix.
abstract: 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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Brown
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
authors.forename: Charles G
authors.initials: CG
authors.lastname: Kinzig
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
authors.forename: Suzanne J
authors.initials: SJ
authors.lastname: DeGregorio
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
authors.forename: Joan A
authors.initials: JA
authors.lastname: Steitz
mesh_terms: D011743:Pyrimidines / Q000737:chemistry*; D062085:RNA, Long Noncoding / Q000235:genetics*; D013816:Thermodynamics
chemical_list: D011743:Pyrimidines; D062085:RNA, Long Noncoding
keywords: MALAT1; RNA base triples; RNA stability; RNA triple helix; intronless β-globin reporter assay