Investigating the Structure:Dynamics:Function Relationship of the MALAT1 Triple Helix

Abstract

The “noncodingRNA (ncRNA) revolution” in the late 20th and early 21st century triggered the transition from scientists viewing ncRNA as cellular junk to realizing that ncRNA plays a variety of roles in biological functions in both healthy and disease related processes. With this discovery came the desire to drug the transcriptome and develop therapies to ameliorate diseases that had previously been thought of as undruggable. Among the potential RNA targets discovered was the ncRNA MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) a long non-coding RNA that is expressed at relatively high levels in cells. MALAT1 was first identified as a marker for lung cancer, then as an oncogenic transcript shown to be over accumulated in several different cancer phenotypes. Previous work has shown the therapeutic potential of knocking down this transcript, making it an attractive potential target. In addition, the 3′-end of the mature MALAT1 transcript forms a U•A-U rich triple helix that evades normal cellular degradation pathways through the sequestration of an A-rich tail between two U-rich regions. Targeting this region with small molecules was shown to decrease metastasis in an organoid model, indicating the promise of the region as a drug target. However, the most effective targeting of an RNA must begin with understanding the underlying structure:dynamics:function relationship. Towards that end, this work aims to increase the understanding of the structure:dynamics:function relationship of the 3′-end of MALAT1 by: 1) probing the conformational landscape of the triple helix in different sequence contexts, 2) examining the protective function of the triple helix in different sequence contexts, and 3) investigating binding-competent structures found in the MALAT1 triple helix ensemble. In the first aim, we show that the 3′-end of MALAT1 is predicted to form modular, independently folding secondary structures. In addition, we report evidence of non-triplex contacts forming within the triple helix, supporting the presence of alternate, non-triple helix structures in the ensemble of the MALAT1 3′-end. In the second aim, we probe the change in protective function of the triple helix within different native sequence contexts and report the development of an enzymatic assay that we believe will be of use in probing the protective function of RNA triple helices and other RNA motifs in general. In the third aim, we investigate binding-competent structures within the triple helix ensemble through use of a mutation construct.