In Vivo Mapping of Eukaryotic RNA Interactomes Reveals Principles of Higher-Order Organization and Regulation

Identifying pairwise RNA-RNA interactions is key to understanding how RNAs fold and interact with other RNAs inside the cell. We present a high-throughput approach, sequencing of psoralen crosslinked, /igated, and selected hybrids (SPLASH), that maps pairwise RNA interactions in vivo with high sensitivity and specificity, genome-wide. Applying SPLASH to human and yeast transcriptomes revealed the diversity and dynamics of thousands of long-range intra-and intermolecular RNA-RNA interactions. Our analysis highlighted key structural features of RNA classes, including the modular organization of mRNAs, its impact on translation and decay, and the enrichment of long-range interactions in noncoding RNAs. Additionally, intermolecular mRNA interactions were organized into network clusters and were remodeled during cellular differentiation. We also identified hundreds of known and new snoRNA-rRNA binding sites, expanding our knowledge of rRNA biogenesis. These results highlight the under-explored complexity of RNA interactomes and pave the way to better understanding how RNA organization impacts biology.