期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：49
页码：14013-14018
DOI：10.1073/pnas.1614759113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceRNA triple helices were deduced to form in vitro almost 60 years ago, yet only three examples from eukaryotic cellular RNAs have been structurally validated. The longest triple helix, the MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) ENE+A (element for nuclear expression with a downstream A-rich tract), presents an opportunity to investigate the biological roles of these enigmatic structures. We have discovered that the MALAT1 triple helix is specifically recognized and bound by METTL16 (methyltransferase-like protein 16). There are two important implications: (i) the MALAT1 triple helix is a bona fide structure in the cellular environment and (ii) there may exist an undiscovered class of triple-stranded RNA binding proteins. METTL16s antiproliferative role in Caenorhabditis elegans suggests that the METTL16-MALAT1 complex may contribute to the oncogenic activity of MALAT1. 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[bullet]A-U triples interrupted by a C[bullet]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 x 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.
关键词：RNA binding protein ; RNA triple helix ; noncoding RNA
