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

• Published in: Nature communications 2021-06, Vol.12 (1), p.3397-12, Article 3397
• Main Authors: Luo, Qing-Jun, Zhang, Jinsong, Li, Pan, Wang, Qing, Zhang, Yue, Roy-Chaudhuri, Biswajoy, Xu, Jianpeng, Kay, Mark A., Zhang, Qiangfeng Cliff
• Format: Article
• Language: English
• Subjects: 13/89; 38; 38/91; 631/114; 631/1647/2258; 631/45/500; Acylation; Binding; Binding sites; Cleavage; Computational Biology; DEAD-box RNA Helicases - genetics; DEAD-box RNA Helicases - metabolism; Gene expression; Gene Knockdown Techniques; Health care; HEK293 Cells; Humanities and Social Sciences; Humans; Immunoprecipitation; Laboratories; Life sciences; multidisciplinary; Mutagenesis, Site-Directed; Mutation; Nucleic Acid Conformation; Protein Binding - genetics; Reagents; Ribonuclease III - genetics; Ribonuclease III - metabolism; Ribonucleic acid; RNA; RNA - chemistry; RNA - genetics; RNA - metabolism; RNA Probes; RNA-protein interactions; RNA-Seq; Science; Science (multidisciplinary); Site selection; Substrate Specificity - genetics; Substrates; Transcriptomes; Transfer RNA
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-23607-w

It is known that an RNA’s structure determines its biological function, yet current RNA structure probing methods only capture partial structure information. The ability to measure intact (i.e., full length) RNA structures will facilitate investigations of the functions and regulation mechanisms of small RNAs and identify short fragments of functional sites. Here, we present icSHAPE-MaP, an approach combining in vivo 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 based on the combination of RNA immunoprecipitation pull-down and icSHAPE-MaP small RNA structural profiling. We discover distinct structural categories of Dicer substrates in correlation to both their binding affinity and cleavage efficiency. And by tertiary structural modeling constrained by icSHAPE-MaP RNA structural data, we find the spatial distance measuring as an influential parameter for Dicer cleavage-site selection. Sequencing methods such as icSHAPE were developed to probe RNA structures transcriptome-wide in cells. To probe intact RNA structures, the authors develop icSHAPE-MaP and apply to Dicer-bound substrates showing that distance measuring is important for Dicer cleavage of pre-miRNAs.