Ligation of 2′, 3′‐cyclic phosphate RNAs for the identification of microRNA binding sites. Issue 2 (16th November 2020)

• Record Type: Journal Article
• Authors: Berk, Christian; Wang, Yuluan; Laski, Artur; Tsagkris, Stylianos; Hall, Jonathan
• Is Part Of: FEBS letters. Volume 595:Issue 2(2021)
• Page Start: 230
• Page End: 240
• Publication Date: 2020-11-16
• Subjects: 2′, 3′‐cyclic phosphate -- CLIP -- ligation -- microRNA -- RtcB; Biochemistry -- Periodicals; Biophysics -- Periodicals; Molecular biology -- Periodicals; Biochimie -- Périodiques; Biochemistry; Biophysics; Molecular biology; Periodicals; 572.05
• DOI: 10.1002/1873-3468.13976
• Languages: English
• ISSNs: 0014-5793

Abstract

Abstract : Identifying the targetome of a microRNA is key for understanding its functions. Cross‐linking and immunoprecipitation (CLIP) methods capture native miRNA‐mRNA interactions in cells. Some of these methods yield small amounts of chimeric miRNA‐mRNA sequences via ligation of 5′‐phosphorylated RNAs produced during the protocol. Here, we introduce chemically synthesized microRNAs (miRNAs) bearing 2′‐, 3′‐cyclic phosphate groups, as part of a new CLIP method that does not require 5′‐phosphorylation for ligation. We show in a system that models miRNAs bound to their targets, that addition of recombinant bacterial ligase RtcB increases ligation efficiency, and that the transformation proceeds via a 3′‐phosphate intermediate. By optimizing the chemistry underlying ligation, we provide the basis for an improved method to identify miRNA targetomes. Abstract :