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

Area of Science:

• Molecular Biology
• Epigenetics
• RNA Biology

Background:

• Long noncoding RNAs (lncRNAs) function as scaffolds for chromatin complexes, but their structural dynamics and protein interactions are not well understood.
• The RNA helicase MLE (Male-Less) is crucial for X-chromosome hyperactivation, incorporating roX RNAs into the MSL complex to increase X-linked gene expression.

Purpose of the Study:

• To elucidate the nucleotide-level interactions between MLE and roX2 RNA.
• To investigate how ATP hydrolysis influences MLE-mediated RNA remodeling.

Main Methods:

• SHAPE chemical probing
• Hydroxyl radical footprinting
• Electrophoretic Mobility Shift Assay (EMSA)
• Fluorescence polarization

Main Results:

• MLE binds specific 5'-helices of roX2 RNA in an ATP-independent manner.
• ATP hydrolysis induces localized unfolding in a 3'-domain helix, exposing the roX-box motif.
• MLE interacts with specific nucleotides in R2H1-R2H3, followed by ATP-dependent rearrangement at the 3' end of roX2 RNA.

Conclusions:

• Provides the first direct evidence of domain-specific MLE-roX2 interaction.
• Demonstrates ATP-driven lncRNA rearrangement by a helicase.
• Establishes a framework for understanding helicase-guided lncRNA structural changes in chromatin regulation.