A cell-based ribozyme reporter system employing a chromosomally-integrated 5\u2032 exonuclease gene

Aiyada Aroonsri,Philip James Shaw,Daniel Abidin Aubry,Jeremy David Gunawan,Jindaporn Kongsee

doi:10.1186/s12860-021-00357-7

Abstract

BackgroundBioinformatic genome surveys indicate that self-cleaving ribonucleic acids (ribozymes) appear to be widespread among all domains of life, although the functions of only a small number have been validated by biochemical methods. Alternatively, cell-based reporter gene assays can be used to validate ribozyme function. However, reporter activity can be confounded by phenomena unrelated to ribozyme-mediated cleavage of RNA.ResultsWe established a ribozyme reporter system in Escherichia coli in which a significant reduction of reporter activity is manifest when an active ribozyme sequence is fused to the reporter gene and the expression of a foreign Bacillus subtilis RNaseJ1 5′ exonuclease is induced from a chromosomally-integrated gene in the same cell.ConclusionsThe reporter system could be useful for validating ribozyme function in candidate sequences identified from bioinformatics.

Highlights

Bioinformatic genome surveys indicate that self-cleaving ribonucleic acids appear to be widespread among all domains of life, the functions of only a small number have been validated by biochemical methods
We constructed plasmids for expression of reporter protein and B. subtilis RNaseJ1 with compatible origins that can be co-transformed into E. coli similar to [23]. (Fig. 1)
An in vivo reporter system in E. coli was established in which ribozyme activity can be demonstrated by the specific attenuation of reporter under the condition in which RNaseJ1 expression is induced

Summary

Introduction

Bioinformatic genome surveys indicate that self-cleaving ribonucleic acids (ribozymes) appear to be widespread among all domains of life, the functions of only a small number have been validated by biochemical methods. Nucleolytic ribozymes (hereafter referred to as ribozymes for short) are small RNAs (less than 200 nt) that function independently of proteins, of which nine classes are known to exist (hammerhead, hairpin, Varkud satellite, hepatitis delta virus (HDV), glmS, twister, twister-sister, pistol and hatchet) each with distinctive folding patterns. They catalyze site-specific cleavage of RNA, and in some cases, the reverse ligation reaction via a concerted general acid–base mechanism [2]. Besides HDV and hammerhead, other classes of ribozyme may be widespread, including the twister ribozyme [10]

Methods

Results

Discussion

Conclusion