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

Abstract

Background Bioinformatic 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. Results We 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. Conclusions The reporter system could be useful for validating ribozyme function in candidate sequences identified from bioinformatics.

Prevention of ribozyme catalysis through cDNA synthesis enables accurate RT-qPCR measurements of context-dependent ribozyme activity

“…Many methods for measuring self-cleaving ribozyme activity exist, such as electrophoretic separation (26)(27)(28), Förster resonance energy transfer (FRET) (29)(30)(31), connecting ribozyme activity to the expression of a reporter gene (32)(33)(34)(35)(36)(37), RNA sequencing (RNA-seq) (32,(38)(39)(40)(41)(42)(43), and reverse-transcription-polymerase chain reaction (RT-PCR)-based techniques (5,(44)(45)(46). Gel electrophoresis and FRET are well suited for IVT studies, but these techniques can be difficult for RNA produced in cells.…”

“…Particularly for RNAs with low expression, for which signal may be below the detection limit (47). Connecting ribozyme activity to the expression of a reporter gene is ideal for in situ measurements in cells but can be difficult to generalize across cell lines, typically requiring different implementations for prokaryotic (33,36,37) and eukaryotic systems (32,34,35). These methods are also not applicable outside of cells as they rely on cellular machinery not usually present in many cell-free systems.…”