Recently, the Blue Carbon Team of the Institute of Marine Science and Technology (IMST) published a research paper titled "Selective RNA processing and stabilization are multi-layer and stoichiometric regulators of gene expression in Escherichia coli" in the journal Advanced Science. Associate Professor Huang Ranran from the IMST is the corresponding author. Assistant Professor Liu Daixi from the School of Pharmaceutical Sciences, and Master students Lv Haibo and Wang Yafei from the IMST are the co-first authors.

Selective RNA processing and stabilization (SRPS) is an important post-transcriptional regulatory mechanism for regulating the stoichiometry of protein complex subunits in microorganisms, which can regulate the differential expression of multiple genes in polycistronic operons. However, how SRPS-related enzymes coordinate to achieve stoichiometric regulation is still unclear.

In this study, by using RNA SEnd-seq and comparative transcriptome analysis, the authors first revealed the genome-wide targetomes of SRPS-related enzymes in Escherichia coli at a single-nucleotide resolution, revealing the synergistic effects of pyrophosphohydrolase (RppH), RNA endonuclease (RNase E) and RNA exonuclease in determining gene transcription abundance. The authors quantitatively characterized SRPS processes and depicted a quantitative regulation model of SRPS in E. coli. At the post-transcriptional layer, the process efficiency (pyrophosphate removal ratio) of RppH is regulated by the first three nucleotides at the 5′ end of the primary transcript, and thus determines the cleavage efficiency of RNase E by the cascade; the stability of the stem-loops formed by the 3′ nucleotide sequences of the processed mRNA fragments positively affects the termination of the 3′-to-5′ exoRNase activities. These synergistic actions collectively modulate the transcript abundance in E. coli. At the translational layer, different endoRNase cleavage sites in the 5′-UTR lead to differential stem-loop formation in the 5′-UTR, which affects ribosome-binding and thus modulates translational initiation efficiency. The effectiveness of these SRPS elements was validated using a dual-fluorescence reporter system. To further explore and utilize SRPS elements, we constructed the first database of prokaryotic SRPS sites (ProSRPSite,http://prosrpsite.sdu.edu.cn/).

In summary, this study revealed the molecular mechanism of SRPS and presents a multi-layer, quantitative, and ubiquitous post-transcriptional regulatory mechanism as well as elements to accurately optimize gene expression stoichiometry. In addition, it provides a solution for generating “polycistronic operon plus SRPS elements”, to overcome the classic challenge faced by synthetic biology researchers, that is, “balanced expression of multiple genes in chassis cells”. The related research work was supported by scientific research projects such as the National Natural Science Foundation, National Key R&D Program, Key R&D Program of Shandong Province, etc.

Associate Professor Huang Ranran has been engaged in microbial bioinformatics analysis based on next-generation sequencing data for a long time. On the one hand, he uses (meta)genomics, (meta)transcriptomics sequencing and other means to identify microbial metabolic processes and regulatory modes, and analyze microbial community structure changes and driving mechanisms; on the other hand, he uses single-cell and other technologies to carry out quantitative synthetic biology application research based on the regulatory mechanisms he analyzed. His research results have been published as the first or corresponding author in academic journals such as Nature Communications, Advanced Science, Chemical Engineering Journal, Microbiology Spectrum, Biotechnology for Biofuels, etc.

Link to the paper: http://doi.org/10.1002/advs.202301459