In a recent paper in Nucleic Acid Research, Yao et al (2015) report on their efforts to develop a universal molecular tool for translation enhancement. Although the work was carried out in mammalian cells there may be possible applications to insect studies.
Exploring biological pathways using a classical genetic approach usually involves the identification of mutations which cause phenotypes of interest. Phenotypes caused by overexpression of wild-type gene products can provide researchers with an alternative tool to identify pathway components that may remain undetectable using traditional loss-of-function analysis.
Moderation is a fatal thing. Nothing succeeds like excess.
Unlike previous approaches such as the Gal/UAS system, that work at the transcriptional level to enhance protein expression, the authors here report on a system they have coined RNAe which they state is the only technology for gene expression enhancement at the post-transcriptional level.
Building on previous work by Carrieri et al (2012), the authors engineered long non-coding RNAs to increase the translation efficiency of selected mRNAs. They determined that sufficient activity of these RNAe’s only required a 72 nucleotide (nt) 5’ pairing sequence which determines specificity, a 167-nt short non-pairing interspersed nuclear element (SINE)B2 sequence which enhances ribosome recruitment to the target mRNA and a poly (A) tail.
Using a fluorescence reporter system, the authors showed that co-transfection of an EGFP containing plasmid with an RNAe specific to EGFP resulted in a dose dependent increase in EGFP protein expression compared to the control. They also observed that when the expression of an EGFP-BBCK fusion protein was enhanced using RNAe, endogenous BBCK in the HEK293T host cells was not enhanced, showing the specificity of the RNAe system.
Yao et al (2015) also explored the feasibility of enhancing endogenous proteins. Here the authors used the rat cell line C5 to show that SOX9, a key transcription factor could be enhanced using RNAe while maintaining functionality.
They also demonstrated the flexibility of the RNAe system by testing it in multiple eukaryotic cell lines. The authors state that though the level of enhancement differed in each cell type, there was significant enhancement of their protein target.
Finally the authors also show possible novel applications for RNAe. RNAe could be used in a models for high throughput screening; for use as an RNAe vectors for enhanced expression of heterologous eukaryotic proteins; and eukaryotic antibody production.
In summary, RNAe may be a powerful new tool for protein expression in biological research.
Yao, Y., Jin, S., Long, H., Yu, Y., Zhang, Z. et al., 2015 RNAe: an effective method for targeted protein translation enhancement by artificial non-coding RNA with SINEB2 repeat. Nucleic Acids Res 43: e58doi: 10.1093/nar/gkv125.