Methyltransferase-like protein 16 binds the 3′-terminal triple helix of MALAT1 long noncoding RNA

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 113; no. 49; pp. 14013 - 14018
• Main Authors: Brown, Jessica A., Kinzig, Charles G., DeGregorio, Suzanne J., Steitz, Joan A.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 06.12.2016
• Subjects: Biological Sciences; Cancer; Cells; Mass spectrometry; Metastasis; Proteins; Ribonucleic acid; RNA; RNA triple helix; noncoding RNA; RNA binding protein
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1614759113

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.