Abstract
RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We useRNA:RNAbinding bySHAPE (RABS) to investigatemicroRNA-34a(miR-34a) binding its mRNA target, thesilent information regulator 1(mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the nakedmiR-34ais able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.
Funder
NIGMS
Cancerfonden
the Karolinska Institute and the Department of Medical Biochemistry and Biophysics
the Ragnar Söderberg Stiftelse
the Stiftelse för Strageskic Forskning
Knut och Alice Wallenberg foundation
Publisher
Cold Spring Harbor Laboratory
Cited by
1 articles.
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