Nucleotide-Resolution Mapping Reveals Specific MLE Binding Site on roX2 lncRNA

Long noncoding RNAs (lncRNAs) often serve as dynamic scaffolds for chromatin-related complexes, but the structural details that control their interaction with proteins remain poorly understood. The core of the X-chromosome hyperactivation process is the RNA helicase MLE (Male-Less), which helps incorporate roX RNAs into the Male-Specific Lethal (MSL) complex and causes a 2-fold increase in X-linked gene expression in males. In this study, we map MLE-roX2 interactions at the nucleotide level to show how (1) the helicase (MLE) binds the 5' RNA helices and (2) ATP hydrolysis drives region-specific RNA remodeling. Using a combination of biochemical techniques, including SHAPE chemical probing, hydroxyl radical footprinting, Electrophoretic Mobility Shift Assay (EMSA), and fluorescence polarization, we demonstrate that MLE binds two of the 5'-helices of roX2 RNA without ATP, while ATP specifically induces localized unfolding in one of the helices at the 3'-domain. This rearrangement specifically exposes the AU-rich roX-box motif, which in turn remodels the roX2 RNA. We show that MLE specifically interacts with particular nucleotides in R2H1-R2H3, followed by ATP-dependent local structural rearrangement at the 3' end of the roX2 RNA. Our findings provide the first direct evidence of domain-specific, roX2-MLE interaction and ATP-driven lncRNA rearrangement, advancing our understanding of helicase-guided lncRNA structural changes and establishing a framework linking lncRNA rearrangements to the regulation of chromatin-associated complexes.