Reproducible and efficient new method of RNA 3'-end labelling by CutA nucleotidyltransferase-mediated CC-tailing. (12th November 2021)

• Record Type: Journal Article
• Authors: Tomecki, Rafal; Kobylecki, Kamil; Drazkowska, Karolina; Hyjek-Skladanowska, Malwina; Dziembowski, Andrzej
• Is Part Of: RNA biology. Volume 18(2021)Supplement 2
• Page Start: 623
• Page End: 639
• Publication Date: 2021-11-12
• Subjects: CutA -- nucleotidyltransferase -- 3ʹ-end RNA labelling -- T4 RNA ligase I -- radioactive nucleotides -- ssRNA -- dsRNA -- Xrn1 -- Dis3 -- exoribonuclease directionality; RNA -- Periodicals; Molecular biology -- Periodicals; Molecular biology; RNA; Periodicals; 572.8805
• DOI: 10.1080/15476286.2021.1999104
• Languages: English
• ISSNs: 1547-6286

Abstract

ABSTRACT: Despite the development of non-radioactive DNA/RNA labelling methods, radiolabelled nucleic acids are commonly used in studies focused on the determination of RNA fate. Nucleic acid fragments with radioactive nucleotide analoguesincorporated into the body or at the 5ʹ or 3ʹ terminus of the molecule can serve as probes in hybridization-based analyses of in vivo degradation and processing of transcripts. Radiolabelled oligoribonucleotides are utilized as substrates in biochemical assays of various RNA metabolic enzymes, such as exo- and endoribonucleases, nucleotidyltransferases or helicases. In some applications, the placement of the label is not a concern, while in other cases it is required that the radioactive mark is located at the 5ʹ- or 3ʹ-end of the molecule. An unsurpassed method for 5ʹ-end RNA labelling employs T4 polynucleotide kinase (PNK) and [γ- 32 P]ATP. In the case of 3ʹ-end labelling, several different possibilities exist. However, they require the use of costly radionucleotide analogues. Previously, we characterized an untypical nucleotidyltransferase named CutA, which preferentially incorporates cytidines at the 3ʹ-end of RNA substrates. Here, we demonstrate that this unusual feature can be used for the development of a novel, efficient, reproducible and economical method of RNA 3ʹ-end labelling by CutA-mediated cytidine tailing. The labelling efficiency is comparable to that achieved with the most common method applied to date, i.e . [5ʹ- 32 P]pCp ligation to the RNA 3ʹ-terminus catalysed by T4 RNA ligase I. We show the utility of RNA substrates labelled using our new method in exemplary biochemical assays assessing directionality of two well-known eukaryotic exoribonucleases, namely Dis3 and Xrn1.