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

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.

A Structural View of miRNA Biogenesis and Function

“…Structural biology information combined with biochemical assays also allowed to dissect specific molecular details involved in the dicing activity and additional modulators of the pre-miRNA maturation [58]. For instance, the flexibility of a sliding bulge located within the catalytic RNAse III region of Dicer has been involved in the alternative processing mechanism observed in humans and higher eukaryotes for the generation of miRNA isoforms from the same pre-miRNA precursor [59].…”

HT-SELEX-based identification of binding pre-miRNA hairpin-motif for small molecules

“…Secondary structures in a pre-miRNA and the local structural environment of dicer binding sites have a profound impact on dicer-mediated cleavage and its efficiency. 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 To evaluate the effect of CMBL3aL on dicer cleavage of these aptamers, we performed an in vitro dicer cleavage reaction in the absence and presence of CMBL3aL using R-Seq1, 2, and 4 as the substrates. Formation of cleaved products (I + P) was observed for R-Seq1, 2, and 4, with a fraction of 15.8%, 16.0%, and 39.3%, respectively, in the absence of CMBL3aL ( Figures 4 G, 4H, and S6 ).…”

“…R-Seq4 form preferable hairpin loop structures, which are favorable binding substrates for dicers ( Figures 4 G and 4H). 67 Change in the conformation of R-Seq2 upon CMBL3aL binding might lead to a more favorable structure for dicer recognition, resulting in an increase in cleaved products.…”

The Non-Coding RNA Journal Club: Highlights on Recent Papers—10

“…The characteristic structural shape of Dicer confers the property of acting as a “molecular ruler” that measures and cleave double-stranded RNAs (dsRNA) generating products with specific sizes. The manuscript by Luo and co-workers, published in Nature Communications , describes a very interesting approach to study the substrate specificity of Dicer by combining a new RNA structure-probing method (icSHAPE-MaP, an approach combining in vivo selective 2′-hydroxyl acylation with 2-methylnicotinic acid imidazolide-azide, NAI-N3, and mutational profiling to probe intact RNA structures), together with an antibody pull-down of the enzyme [ 9 ]. The authors proposed a structural landscape for Dicer targets that depends on the size of the terminal loop of the dsRNA substrate.…”