Engineering a Circular Riboregulator in Escherichia coli.

William Rostain,Alfonso Jaramillo,Guillermo Rodrigo,Teresa Cordero,Shensi Shen

doi:10.34133/2020/1916789

Abstract

RNAs of different shapes and sizes, natural or synthetic, can regulate gene expression in prokaryotes and eukaryotes. Circular RNAs have recently appeared to be more widespread than previously thought, but their role in prokaryotes remains elusive. Here, by inserting a riboregulatory sequence within a group I permuted intron-exon ribozyme, we created a small noncoding RNA that self-splices to produce a circular riboregulator in Escherichia coli. We showed that the resulting riboregulator can trans-activate gene expression by interacting with a cis-repressed messenger RNA. We characterized the system with a fluorescent reporter and with an antibiotic resistance marker, and we modeled this novel posttranscriptional mechanism. This first reported example of a circular RNA regulating gene expression in E. coli adds to an increasing repertoire of RNA synthetic biology parts, and it highlights that topological molecules can play a role in the case of prokaryotic regulation.

Highlights

RNA molecules can regulate gene expression in trans through many different mechanisms in bacteria, either by themselves such as with positive/negative control of protein translation [1,2,3] and transcription termination [4, 5] or in complex with proteins such as in CRISPR interference [6]
We focus on riboregulators, transregulating small RNAs (sRNAs) that interact with cis-repressed messenger RNAs to activate protein translation [1], which are programmable as their interactions are mostly governed by simple base pairing rules
The gel revealed the linear products that are unspliced, partly spliced, and fully spliced. This was again performed with purified RNA from E. coli cells expressing the sRNA circRAJ31 [23]

Summary

Introduction

RNA molecules can regulate gene expression in trans through many different mechanisms in bacteria, either by themselves such as with positive/negative control of protein translation [1,2,3] and transcription termination [4, 5] or in complex with proteins such as in CRISPR interference [6]. These developments have allowed the application of small RNAs (sRNAs) to engineer synthetic circuits that can be used for sensing, computation, and control. Our aim is not to show that circular riboregulators could perform better than linear ones, but to demonstrate that topological molecules can regulate gene expression even in simple organisms like bacteria by engineering one instance

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Conclusion