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

Qing-Jun Luo,Jinsong Zhang,Jianpeng Xu,Biswajoy Roy-Chaudhuri,Qiangfeng Cliff Zhang,Mark A Kay,Pan Li,Qing Wang,Yue Zhang

doi:10.1038/s41467-021-23607-w

Abstract

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.

Highlights

It is known that an RNA’s structure determines its biological function, yet current RNA structure probing methods only capture partial structure information
We developed an RNA structure probing method, which we call icSHAPE-Mutational Profiling, that uses the icSHAPE reagent NAI-N3 to modify RNA, and subsequently maps mis-incorporation events generated by the reverse transcriptase Superscript II at the nucleotides with NAI-N3-induced RNA modifications (Fig. 1a, see “Methods”)
Superscript II reverse transcriptase usually adds a random number of non-template nucleotides at the 3′ end of cDNA18, which confounds accurate reverse transcription stops (RT-stop) identification

Summary

Results

Development of icSHAPE-MaP to probe intact RNA structures. We developed an RNA structure probing method, which we call icSHAPE-Mutational Profiling (icSHAPE-MaP), that uses the icSHAPE reagent NAI-N3 to modify RNA, and subsequently maps mis-incorporation events generated by the reverse transcriptase Superscript II (i.e., reverse transcription mutations) at the nucleotides with NAI-N3-induced RNA modifications (Fig. 1a, see “Methods”). To evaluate the ability of icSHAPE-MaP to capture structural information from intact RNAs, we examined sRNA species (

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Discussion

Methods

Code availability