Peterman Emma L, Ploessl Deon S, Love Kasey S, Sanabria Valeria, Daniels Rachel F, Johnstone Christopher P, Godavarti Diya R, Kabaria Sneha R, Oakes Conrad G, Pai Athma A, Galloway Kate E
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
Nucleic Acids Res. 2025 Jun 6;53(11). doi: 10.1093/nar/gkaf528.
Concentrations of RNAs and proteins provide important determinants of cell fate. Robust gene circuit design requires an understanding of how the combined actions of individual genetic components influence both messenger RNA (mRNA) and protein levels. Here, we simultaneously measure mRNA and protein levels in single cells using hybridization chain reaction Flow-FISH (HCR Flow-FISH) for a set of commonly used synthetic promoters. We find that promoters generate differences in both the mRNA abundance and the effective translation rate of these transcripts. Stronger promoters not only transcribe more RNA but also show higher effective translation rates. While the strength of the promoter is largely preserved upon genome integration with identical elements, the choice of polyadenylation signal and coding sequence can generate large differences in the profiles of the mRNAs and proteins. We used long-read direct RNA sequencing to define the transcription start and splice sites of common synthetic promoters and independently vary the defined promoter and 5' UTR sequences in HCR Flow-FISH. Together, our high-resolution profiling of transgenic mRNAs and proteins offers insight into the impact of common synthetic genetic components on transcriptional and translational mechanisms. By developing a novel framework for quantifying expression profiles of transgenes, we have established a system for building more robust transgenic systems.
RNA和蛋白质的浓度是细胞命运的重要决定因素。强大的基因回路设计需要了解单个遗传元件的联合作用如何影响信使RNA（mRNA）和蛋白质水平。在这里，我们使用杂交链式反应流式荧光原位杂交技术（HCR Flow-FISH）同时测量一组常用合成启动子在单细胞中的mRNA和蛋白质水平。我们发现启动子会导致这些转录本在mRNA丰度和有效翻译速率上产生差异。更强的启动子不仅转录更多的RNA，而且显示出更高的有效翻译速率。虽然启动子的强度在与相同元件进行基因组整合时基本保持不变，但聚腺苷酸化信号和编码序列的选择会在mRNA和蛋白质的表达谱上产生很大差异。我们使用长读长直接RNA测序来确定常见合成启动子的转录起始位点和剪接位点，并在HCR Flow-FISH中独立改变定义的启动子和5'非翻译区（UTR）序列。总之，我们对转基因mRNA和蛋白质的高分辨率分析深入了解了常见合成遗传元件对转录和翻译机制的影响。通过开发一种用于量化转基因表达谱的新框架，我们建立了一个用于构建更强大转基因系统的体系。
