RNA Infrastructure Profiling Illuminates Transcriptome Structure in Crowded Spaces

The cellular function of RNA is intimately linked to its structure. The 3D structure of RNA is intricate and compact, and is often complexed with other macromolecules for regulatory interaction. These interactions frequently lead to occluded environments that block structure probing by current reagents. Our RNA infrastructure profiling method (RISP) quantitatively compares standard acylation probes to new small-sized probes, and reveals ca. 80% more structural data for intracellular RNAs underlying protein contacts. Comparative analysis also reveals information about close contacts in ribonucleoprotein complexes such as small nuclear RNAs in the spliceosome. In addition, RISP analysis with small agent AcIm reveals pronounced signals for m6A methylation sites of RNAs in their native cellular setting, even in crowded environments. The sequencing experiments were performed in two biological replicates. Comparative analysis of deep sequencing data using our RBRP popeline with certain adjustment identifies structure information in crowded space.