Reproducible and efficient new method of RNA 3'-end labelling by CutA nucleotidyltransferase-mediated CC-tailing

Area of Science:

• Molecular Biology
• Biochemistry
• Nucleic Acid Chemistry

Background:

• Radiolabeled nucleic acids are crucial for studying RNA fate, degradation, and processing in vivo.
• Current methods for 3'-end RNA labeling often rely on expensive radionucleotide analogues.
• T4 polynucleotide kinase (PNK) with [γ-32P]ATP is a standard for 5'-end RNA labeling.

Purpose of the Study:

• To develop a novel, efficient, and economical method for 3'-end RNA labeling.
• To utilize the unique cytidine incorporation activity of the CutA enzyme for RNA labeling.
• To demonstrate the applicability of CutA-mediated labeling in biochemical assays.

Main Methods:

• Characterization of the nucleotidyltransferase CutA for its RNA 3'-end modification activity.
• Development of a CutA-mediated cytidine tailing protocol for RNA 3'-end labeling.
• Comparative analysis of labeling efficiency against established methods like [5'-32P]pCp ligation.

Main Results:

• CutA enzyme efficiently and reproducibly labels the 3'-terminus of RNA substrates with cytidine.
• The labeling efficiency of the CutA method is comparable to T4 RNA ligase I-mediated [5'-32P]pCp ligation.
• RNA substrates labeled via CutA-mediated tailing were successfully used in assays for exoribonuclease directionality.

Conclusions:

• CutA-mediated cytidine tailing provides a valuable and economical alternative for 3'-end RNA labeling.
• This method simplifies RNA labeling for various biochemical applications, including enzyme activity studies.
• The developed method enhances the toolkit for investigating RNA metabolism and function.