title: Methyltransferase-like protein 16 binds the 3'-terminal triple helix of MALAT1 long noncoding RNA.
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 × 105molecules 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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06536 jbrown33@nd.edu joan.steitz@yale.edu.
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, CT 06536.
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, CT 06536.
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, CT 06536 jbrown33@nd.edu joan.steitz@yale.edu.
authors.forename: Joan A
authors.initials: JA
authors.lastname: Steitz
mesh_terms: D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D059370:RNA Folding / Q000502:physiology; D020871:RNA Stability; D062085:RNA, Long Noncoding / Q000235:genetics / Q000378:metabolism*
chemical_list: C557041:MALAT1 long non-coding RNA, human; D062085:RNA, Long Noncoding; C000621191:METTL16 protein, human; D008780:Methyltransferases
keywords: RNA binding protein; RNA triple helix; noncoding RNA