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

Proc Natl Acad Sci U S A. 2016 Dec 6;113(49):14013-14018. doi: 10.1073/pnas.1614759113. Epub 2016 Nov 21.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Methyltransferases / genetics
  • Methyltransferases / metabolism*
  • Nucleic Acid Conformation
  • RNA Folding / physiology
  • RNA Stability
  • RNA, Long Noncoding / genetics
  • RNA, Long Noncoding / metabolism*


  • MALAT1 long non-coding RNA, human
  • RNA, Long Noncoding
  • METTL16 protein, human
  • Methyltransferases