Evaluation of synthetic post-transcription regulatory sequences reveals design principle to enhance mRNA stability and translation efficiency

Le résumé fourni par la source

The Shine-Dalgarno (SD) sequence and its adjacent flanking regions, including the translation standby site (TSS) and the N-terminal coding sequence (NCS), play critical roles in regulating ribosome recruitment, translation efficiency, and mRNA stability against RNase degradation. However, structure-activity relationships governing these regions remain poorly characterized, and their functional interplay introduces substantial complexity. In this study, we employed one-pot technology to build a post-transcriptional regulatory component (PTRC) library of 576 variants to clarify the relationship between sequence variants and both protein expression and mRNA levels via high-throughput sequencing. Our results show that although unstructured TSSs do not enhance mRNA stability, they markedly increase translation efficiency, causing a 16 %–100 % rise in protein expression. In contrast, structured TSSs increase mRNA levels by 43 %–90 %. Additionally, highly conserved SD sequences boost translation efficiency by up to 10 % and mRNA abundance by up to 12 %. Moreover, it was found that optimized linear N-terminal coding sequence (NCS) positively affects protein expression and mRNA levels. The effects of these optimized regulatory components were verified in the expression control of the nrk and sam2 genes, resulting in enhanced production. These findings underscore the crucial role of structural optimization, guiding the rational design of synthetic post-transcriptional regulatory elements.

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