Identification and targeting of G-quadruplex structures in MALAT1 long non-coding RNA

Abstract

RNA G-quadruplexes (rG4s) have functional roles in many cellular processes in diverse organisms. While a number of rG4 examples have been reported in coding messenger RNAs (mRNA), so far only limited works have studied rG4s in non-coding RNAs (ncRNAs), especially in long non-coding RNAs (lncRNAs) that are of emerging interest and significance in biology. Herein, we report that MALAT1 lncRNA contains conserved rG4 motifs, forming thermostable rG4 structures with parallel topology. We also show that rG4s in MALAT1 lncRNA can interact with NONO protein with high specificity and affinity in vitro and in nuclear cell lysate, and we provide cellular data to support that NONO protein recognizes MALAT1 lncRNA via rG4 motifs. Notably, we demonstrate that rG4s in MALAT1 lncRNA can be targeted by the rG4-specific small molecule, peptide, and L-aptamer, leading to the dissociation of MALAT1 rG4-NONO protein interaction. Altogether, this study uncovers new and important rG4s in MALAT1 lncRNAs, reveals their specific interactions with NONO protein, offers multiple strategies for targeting MALAT1 and its RNA–protein complex via its rG4 structure and illustrates the prevalence and significance of rG4s in ncRNAs.

Essential Roles and Risks of G-Quadruplex Regulation: Recognition Targets of ALS-Linked TDP-43 and FUS

“…Due to incomplete identification of in the high-throughput screening, it is necessary to confirm the prediction by biochemical analysis of direct interactions between the proteins and the target RNA candidates using such as the gel shift assay. However, the sharply bent DNA/RNA-protein complexes do not migrate normally in native acrylamide gel electrophoresis and G4-protein complex often retained in the top of gel, if the complex is correctly formed ( Carey et al, 2010 ; Al-Furoukh et al, 2013 ; Ariyo et al, 2015 ; Huang et al, 2017 ; Mou et al, 2022 ). Furthermore, it is difficult to increase the concentration of monocations in the gel during electrophoretic fractionation, which is necessary for the stabilization of G4 tetrads.…”

“…thus often leading to incorrect identification ( Yakhnin et al, 2012 ; Kim et al, 2015 ). To avoid these problems, use in combination with other methods such as the filter-binding assay, thermophoresis assay or surface plasmon resonance analysis is effective to determine the specific interaction between G4-RNA and G4-RNA binding protein ( von Hacht et al, 2014 ; Ishiguro et al, 2016 ; Mou et al, 2022 ).…”

“…Surprisingly, these RNA binding-proteins recognize and bind to mRNA containing G4 motifs ( Ishiguro et al, 2021 ). For instance, ALS-linked intronic expansion of the human C9orf72 gene and potentially causative two long noncoding RNAs, NEAT1 (nuclear enriched abundant transcript 1) and MALAT1 (metastasis associated in lung adenocarcinoma transcript 1/NEAT2) contain abundant G4 motifs ( Grigg et al, 2014 ; Haeusler et al, 2014 ; Simko et al, 2020 ; Mou et al, 2022 ). More interestingly, these ALS-related proteins/RNAs constitute RNP granules or regulate the formation of RNP granules ( Tourrière et al, 2003 ; Fujii et al, 2005 ; Guil et al, 2006 ; Bosco et al, 2010 ; Molliex et al, 2015 ; Mensch et al, 2018 ; Ishiguro et al, 2021 ).…”

Novel L-RNA Aptamer Controls APP Gene Expression in Cells by Targeting RNA G-Quadruplex Structure

“…G4 prediction analysis which consisted of cGcC (consecutive G over consecutive C ratio), G4H (G4Hunter) and G4NN (G4 neural network) was conducted by G4screener v0.2 () with default settings of cGcC > 4.5, G4H > 0.9, G4NN > 0.5 to define a G4 threshold …”

The Non-Coding RNA Journal Club: Highlights on Recent Papers—10

“…The N6-methyladenosine (m6A) writer METTL3 for example methylates MALAT1 lncRNA leading to its stabilization, while the m6A reader YTHDC1 binds to m6A-modified MALAT1 and regulates the composition/function of nuclear speckles, strongly contributing to the metastatic ability of cancer cells. Mou and colleagues have now reported that MALAT1 activity is not only controlled by epitranscriptomic modifications but also by complex RNA structures termed RNA G-quadruplexes (rG4) [ 2 ]. rG4 take part in cancer development through numerous biological functions, such as the control of RNA localization and translation.…”