cyPhyRNA-seq: a genome-scale RNA-seq method to detect active self-cleaving ribozymes by capturing RNAs with 2',3' cy clic p hosphates and 5' hy droxyl ends

RNA Biol. 2021 Nov 12;18(sup2):818-831. doi: 10.1080/15476286.2021.1999105. Epub 2021 Dec 14.

Abstract

Self-cleaving ribozymes are catalytically active RNAs that cleave themselves into a 5'-fragment with a 2',3'-cyclic phosphate and a 3'-fragment with a 5'-hydroxyl. They are widely applied for the construction of synthetic RNA devices and RNA-based therapeutics. However, the targeted discovery of self-cleaving ribozymes remains a major challenge. We developed a transcriptome-wide method, called cyPhyRNA-seq, to screen for ribozyme cleavage fragments in total RNA extract. This approach employs the specific ligation-based capture of ribozyme 5'-fragments using a variant of the Arabidopsis thaliana tRNA ligase we engineered. To capture ribozyme 3'-fragments, they are enriched from total RNA by enzymatic treatments. We optimized and enhanced the individual steps of cyPhyRNA-seq in vitro and in spike-in experiments. Then, we applied cyPhyRNA-seq to total RNA isolated from the bacterium Desulfovibrio vulgaris and detected self-cleavage of the three predicted type II hammerhead ribozymes, whose activity had not been examined to date. cyPhyRNA-seq can be used for the global analysis of active self-cleaving ribozymes with the advantage to capture both ribozyme cleavage fragments from total RNA. Especially in organisms harbouring many self-cleaving RNAs, cyPhyRNA-seq facilitates the investigation of cleavage activity. Moreover, this method has the potential to be used to discover novel self-cleaving ribozymes in different organisms. [Figure: see text].

Keywords: 5ʹ-3ʹ exonuclease Xrn1; RNA pyrophosphohydrolase (RppH); RtcB ligase; T4 RNA ligase truncated KQ; next-generation sequencing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics
  • Computational Biology
  • Escherichia coli / genetics
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Gene Expression Profiling* / methods
  • Gene Library
  • Genomics / methods
  • RNA, Catalytic / chemistry
  • RNA, Catalytic / genetics*
  • RNA-Seq / methods*

Substances

  • Fungal Proteins
  • RNA, Catalytic
  • hammerhead ribozyme