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

Splice modulating small molecules have been developed to promote the U1 snRNP to engage with pre-mRNAs with strong and altered sequence preference. Transcriptomic profiling of bulk RNA from compound treated cells enables detection of RNAs impacted; however, it is difficult to delineate whether transcriptional changes are a consequence of direct compound treatment or trans-acting effects. To identify RNA targets that bind directly with splice modulating compounds, we deployed a photoaffinity labeling (PAL)-based Chem-CLIP approach. Through this workflow, we identify the telomerase lncRNA (TERC) as a previously unknown target of this class of clinically relevant small molecules. Using SHAPE-MaP, we orthogonally validate and further define the compound binding site as likely to be the conserved CR4/5 domain. Additionally, a thorough analysis of the PAL-based Chem-CLIP data reveals that considering competed RNAs, irrespective of magnitude of enrichment, adds a rich dimension of hit calling. We multiplexed PAL-based Chem-CLIP samples with identically and contemporaneously treated samples for bulk transcriptomic profiling. We anticipated that this additional layer of multiplexing would enable correlations of enrichment for PAL-labeled RNAs with changes in expression or splicing. These datasets include: 1) parallel processed chem-CLIP and bulk RNAseq samples of PAL-treated SH-SY5Y cells and 2) in-cellulo SHAPE-MaP (TERC lncRNA target) in SH-SY5Y cells treated with DMSO or NV1.