snoCLASH Reveals Extensive snoRNA-mRNA Interaction Networks.

Small nucleolar RNAs (snoRNAs) are classically defined as guides for ribosomal RNA (rRNA) modification, yet increasing evidence suggests that box C/D snoRNAs also interact with non-rRNA transcripts. Systematic discovery of such interactions has been hindered by overwhelming rRNA abundance and technical limitations in RNA-RNA capture. Here, we present snoCLASH, an optimized snoRNA RNA binding protein (RBP)-based crosslinking, ligation, and sequencing framework that integrates phenol-toluol extraction, polyA enrichment, nuclear fractionation, rRNA depletion, and dual-reference chimeric read analysis to enable transcriptome-scale identification of snoRNA-non-rRNA interactions. Applying this approach reveals thousands of snoRNA-associated mRNA regions spanning coding and regulatory elements and enriched for RBPs linked to epitranscriptomic regulation. Using this framework, we identify high-confidence snoRNA-mRNA interactions and functionally validate one candidate, demonstrating that a snoCLASH-discovered target undergoes snoRNA-dependent 2'-O-methylation with downstream effects on protein expression. Together, this work establishes snoCLASH as a scalable platform for discovering and validating non-canonical snoRNA targets beyond the ribosome.