Modulating Heterologous Gene Expression with Portable mRNA-Stabilizing 5′-UTR Sequences

Abstract

RNA half-lives are frequently perceived as depending on too many variables, and transcript stability is generally missed as a checkpoint amenable to manipulation in synthetic designs. In this work, the contribution of mRNA stability to heterologous protein production levels in E. coli has been inspected. To this end, we capitalized on the wealth of information available on intrinsic mRNA stability determinants, four of which were formatted as portable modules consisting of 5'-untranslated regions (UTRs). The cognate DNA sequences were then assembled in a genetic frame in which mRNA stability endowed by the UTRs was the only variable to run expression of sfGFP. Reporter output and Northern blot-based measurements of absolute mRNA half-lives revealed that such UTRs were found to keep intact their ability to modulate transcript stability when excised from their natural context and placed as the upstream region of the reporter gene. By keeping transcription fixed and combining different UTRs with a constant ribosomal binding site, we showed that mRNA decay can be made the limiting constituent of the overall gene expression flow. Moreover, the data indicated that manipulating mRNA stability had little effect on expression noise in the corresponding population. Finally, augmented heterologous expression brought about by mRNA stability did not make cells more vulnerable to resource-consuming stresses. The tangible result of this work was a collection of well-characterized mRNA-stabilizing sequences that can be composed along with other expression signals in any construct following the assembly rules of the Standard European Vector Architecture (SEVA) format.

Pseudomonas mRNA 2.0: Boosting Gene Expression Through Enhanced mRNA Stability and Translational Efficiency

“…Lou et al integrated hairpins in both ribozymes used in this work, RiboJ and VtmoJ, to expose the RBS and confirmed their catalytic functionality through rapid amplification of cDNA 5 ′ -ends (Lou et al, 2012). The presence of such hairpins in the 5 ′ UTR region was reported to increase the respective mRNA half-life, which leads to higher protein production (Viegas et al, 2018). Further clarification of the stabilizing effect of the hairpin structure of RiboJ and its individual effect on protein expression has been published elsewhere (Clifton et al, 2018).…”

Predictable control of RNA lifetime using engineered degradation-tuning RNAs

“…Inspired by previous studies that demonstrated that naturally occurring stabilizers can be used to tune gene expression in synthetic gene circuits 31 , 33 , we inserted the 5’ UTR sequence from the E. coli ompA transcript between the promoter and RBS region to tune downstream GFP expression 20 , 34 , 35 ( Fig. 1a , right).…”

“…In E. coli , the endonucleases RNase E or RNase III target the underlying RNA molecule for primary cleavage followed by complete degradation via 3’ → 5’ exonucleases 27 – 29 . Previously discovered naturally occurring 5’ UTRs, termed RNA stabilizers, or rationally designed synthetic DNA cassettes can increase RNA half-life by forming 5’ secondary structures 30 – 33 . These 5’ hairpin structures have been shown to be able to control heterologous mRNA half-life and used to regulate recombinant protein expression without introducing stress to host cells 33 .…”

“…Previously discovered naturally occurring 5’ UTRs, termed RNA stabilizers, or rationally designed synthetic DNA cassettes can increase RNA half-life by forming 5’ secondary structures 30 – 33 . These 5’ hairpin structures have been shown to be able to control heterologous mRNA half-life and used to regulate recombinant protein expression without introducing stress to host cells 33 . However, most engineered 5’ stabilizing elements have been designed and tested on an ad-hoc basis.…”

Stories of the future: manipulating RNA and Intra/Interkingdom communication

“…The ribonucleases have a fundamental role in the turnover of RNAs since the accumulation of degradation products and aberrant transcripts is usually quite detrimental for the cell; furthermore, they actively participate in the recycling of valuable nucleotides. Engineering and modulating the lifespan of RNAs has great potential (Viegas et al, 2018) and will certainly be one of the highlights of future science. Synthetic Biology supported by all the recent RNA-derived technologies will lead to important Biotech applications.…”