Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix

Abstract

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly-abundant nuclear long noncoding RNA that promotes malignancy. A 3′-stem-loop structure is predicted to confer stability by engaging a downstream A-rich tract in a triple helix, similar to the expression and nuclear retention element (ENE) from the KSHV polyadenylated nuclear RNA. The 3.1-Å resolution crystal structure of the human MALAT1 ENE and A-rich tract reveals a bipartite triple helix containing stacks of five and four U•A-U triples separated by a C+•G-C triplet and C-G doublet, extended by two A-minor interactions. In vivo decay assays indicate that this blunt-ended triple helix, with the 3′ nucleotide in a U•A-U triple, inhibits rapid nuclear RNA decay. Interruption of the triple helix by the C-G doublet induces a “helical reset” that explains why triple-helical stacks longer than six do not occur in nature.

Hoogsteen-position pyrimidines promote the stability and function of the MALAT1 RNA triple helix

“…The crystal structure of the MALAT1 ENE + A confirmed that the U-rich ENE coalesces with a downstream A-rich tract to form a bipartite triple helix, allowing MALAT1 to accumulate in cells (Brown et al 2014). Preceded by two A-minor triples, the first triplex segment consists of five U•A-U triples followed by a C•G-C triple, and the second triplex contains four U•A-U triples.…”

“…Most RNA triple helices identified to date rely heavily on canonical U•A-U and C•G-C triples (Brown et al 2014). However, the discovery of noncanonical triples in bacterial riboswitches, the group II intron, and the spliceosome suggests that other nucleotide combinations are possible (Gilbert et al 2008;Toor et al 2008;Hang et al 2015).…”

“…Separating these two triple-helical regions is a C-G base pair that resets the helical axis and allows the adjacent formation of these two independent stacks of RNA base triples (see triple helix in Fig. 1A; Brown et al 2014). The MENβ ENE + A has been proposed to adopt a similar bipartite triple-helical structure based on sequence similarity (Brown et al 2012).…”

“…To date, structural validation of natural RNA triple helices, which we define as three or more stacked major-groove base triples, has been reported for telomerase (Theimer et al 2005;Cash et al 2013), bacterial S-adenosylmethionine-II (SAM-II) and class II prequeuosine 1 (preQ 1 ) riboswitches (Gilbert et al 2008;Liberman et al 2013), group II introns and spliceosomal U2/U6 snRNAs (Toor et al 2008;Hang et al 2015), Kaposi's sarcoma-associated herpesvirus (KSHV) polyadenylated nuclear (PAN) RNA (Mitton-Fry et al 2010), and human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) (Brown et al 2014). These RNA triple helices are predominantly composed of canonical U•A-U base triples and typically contain 3-5 consecutive triples.…”

“…To probe the effects of altering nucleotide composition within an RNA triple helix, we selected the MALAT1 ENE + A as a model. With nine U•A-U triples and one C•G-C triple, it is the largest of the known naturally occurring majorgroove RNA triple helices (Brown et al 2014). We altered a U•A-U triple at a single position within the MALAT1 ENE + A triple helix and performed electrophoretic mobility shift assays (EMSAs) to determine the relative thermodynamic stability of 20 distinct major-groove RNA base triples (N•A-U, N•G-C, N•C-G, N•U-A, and N•G-U).…”