Title | Hoogsteen-position pyrimidines promote the stability and function of the MALAT1 RNA triple helix. |
Publication Type | Journal Article |
Year of Publication | 2016 |
Authors | Brown JA, Kinzig CG, DeGregorio SJ, Steitz JA |
Journal | RNA |
Volume | 22 |
Issue | 5 |
Pagination | 743-9 |
Date Published | 2016 May |
ISSN | 1469-9001 |
Keywords | Pyrimidines, RNA, Long Noncoding, Thermodynamics |
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. |
DOI | 10.1261/rna.055707.115 |
Alternate Journal | RNA |
PubMed ID | 26952103 |
PubMed Central ID | PMC4836648 |
Grant List | K99GM111430 / GM / NIGMS NIH HHS / United States GM026154 / GM / NIGMS NIH HHS / United States CA193300 / CA / NCI NIH HHS / United States / / Howard Hughes Medical Institute / United States P01 CA016038 / CA / NCI NIH HHS / United States R01 GM026154 / GM / NIGMS NIH HHS / United States R01 CA193300 / CA / NCI NIH HHS / United States K99 GM111430 / GM / NIGMS NIH HHS / United States |
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