Photoaffinity enabled transcriptome-wide identification of splice modulating small molecule-RNA binding events in...

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Chemical Biology

Background:

• Splice-modulating small molecules are designed to alter U1 snRNP engagement with pre-mRNAs.
• Transcriptomic profiling can detect compound-affected RNAs but struggles to distinguish direct from indirect effects.

Purpose of the Study:

• To identify direct RNA targets of splice-modulating small molecules.
• To investigate the binding site of these molecules on identified RNA targets.

Main Methods:

• Photoaffinity labeling (PAL)-based Chem-CLIP was employed to capture direct RNA-compound interactions.
• Cellular ΔSHAPE-MaP was used for orthogonal validation and precise binding site determination.
• Analysis of competed RNAs within the Chem-CLIP data was performed to refine hit identification.

Main Results:

• The telomerase RNA component (TERC) was identified as a novel direct target of splice-modulating small molecules.
• The conserved CR4/5 domain of TERC was validated as the likely small molecule binding site.
• Considering competed RNAs improved the identification of direct RNA targets, even those with low enrichment.

Conclusions:

• TERC is a previously unrecognized direct target of clinically relevant splice-modulating small molecules.
• The CR4/5 domain of TERC is a key interaction site for these compounds.
• PAL-based Chem-CLIP, with analysis of competed RNAs, is a powerful method for identifying direct RNA-small molecule interactions.