Title | Methyltransferase-like protein 16 binds the 3'-terminal triple helix of MALAT1 long noncoding RNA. |
Publication Type | Journal Article |
Year of Publication | 2016 |
Authors | Brown JA, Kinzig CG, DeGregorio SJ, Steitz JA |
Journal | Proc Natl Acad Sci U S A |
Volume | 113 |
Issue | 49 |
Pagination | 14013-14018 |
Date Published | 2016 12 06 |
ISSN | 1091-6490 |
Keywords | HEK293 Cells, HeLa Cells, Humans, Methyltransferases, Nucleic Acid Conformation, RNA Folding, RNA Stability, RNA, Long Noncoding |
Abstract | 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 × 10 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. |
DOI | 10.1073/pnas.1614759113 |
Alternate Journal | Proc. Natl. Acad. Sci. U.S.A. |
PubMed ID | 27872311 |
PubMed Central ID | PMC5150381 |
Grant List | R00 GM111430 / GM / NIGMS NIH HHS / United States UL1 TR001863 / TR / NCATS NIH HHS / United States P01 CA016038 / CA / NCI NIH HHS / United States R01 GM026154 / GM / NIGMS NIH HHS / United States K99 GM111430 / GM / NIGMS NIH HHS / United States |
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