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Proc Natl Acad Sci U S A. 2016 Dec 6;113(49):14013-14018. Epub 2016 Nov 21.

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

Author information

1
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.
2
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06536.

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 × 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.

KEYWORDS:

RNA binding protein; RNA triple helix; noncoding RNA

PMID:
27872311
PMCID:
PMC5150381
[Available on 2017-06-06]
DOI:
10.1073/pnas.1614759113
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