Transcriptome-wide RNA structure probing reveals the structural basis of Dicer binding and cleavage

RNA structure is vital for its function. Current transcriptome-wide RNA structure probing methods only capture partial structure information. Measuring RNA structure in full length is critical to the function and regulation study of small RNAs and short fragments of functional sites. Here, we present icSHAPE-MaP, an approach combining in vivo click selective 2'-hydroxyl acylation and mutational profiling to probe intact RNA structures. We further showcase the RNA structural landscape of substrates bound by human Dicer, by combining RNA immunoprecipitation pull-down and small RNA structure profiling through icSHAPE-MaP. Structural categories of Dicer substrates were unveiled with distinct patterns in correlation to their binding affinity and cleavage efficiency. And by tertiary structural modeling for pre-miRNAs, one of the major binding and cleavage substrates for Dicer, we find the spatial distance measuring as an important parameter for Dicer cleavage-site selection. Overall design: icSHAPE-MaP of six samples for small RNAs (sRNAs, <200 nt) from HEK293T cells, including two treated with DMSO as control, two treated with NAI-N3 in the culture medium, and two treated with NAI-N3 after RNA extraction and refolding in vitro; Dicer-RIP-icSHAPE-MaP of eight samples from Dicer deficient 293T cells, including two RIP samples treated with DMSO as control, three RIP samples treated with NAI-N3, and three input samples as the control for RIP; sRNA-seq of eight samples (40-200 nt in size) from Dicer deficient 293T cells, including four with Dicer wildtype (WT) reconstitution and four with catalytic-dead Dicer reconstitution; miRNA-seq (~20 nt in size ) of 12 samples from HEK293T cells, including two replicates for the overexpression of each miR-217 variant (total five variants plus WT).