SEMPER:真核核糖体对 mRNA 多单元的定量表达,实现紧凑、比例可调的多基因表达。

Cell systems Pub Date : 2024-07-17 Epub Date: 2024-07-05 DOI:10.1016/j.cels.2024.06.001
Mengtong Duan, Ishaan Dev, Andrew Lu, Goar Ayrapetyan, Mei Yi You, Mikhail G Shapiro
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摘要

在这里,我们介绍了一种利用翻译起始的漏扫描模式从单个转录本表达多个开放阅读框(ORF)的方法。在这种被称为 "真核核糖体对 mRNA 多单链体的定量表达"(SEMPER)的方法中,相邻的 ORF 以可调节的比例从单个 mRNA 翻译出来,这种比例由它们在序列中的顺序和翻译起始位点的强度决定。我们通过在两种不同的细胞系中用一个质粒表达多达三种荧光蛋白来验证这种方法。然后,我们用它来编码一个按比例调整的多聚组蛋白构建体,该构建体编码气泡声学报告基因,能有效地形成多蛋白复合物,同时将细胞毒性降到最低。我们还证明,SEMPER 可使质粒 DNA 中的重组单克隆抗体和体外转录的单个 mRNA 中的两种荧光蛋白实现多聚体表达。最后,我们提供了一个概率模型来阐明 SEMPER 的内在机制。本文的同行评审过程透明,其记录见补充信息。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes for compact, ratio-tunable multi-gene expression.

Here, we present a method for expressing multiple open reading frames (ORFs) from single transcripts using the leaky scanning model of translation initiation. In this approach termed "stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes" (SEMPER), adjacent ORFs are translated from a single mRNA at tunable ratios determined by their order in the sequence and the strength of their translation initiation sites. We validate this approach by expressing up to three fluorescent proteins from one plasmid in two different cell lines. We then use it to encode a stoichiometrically tuned polycistronic construct encoding gas vesicle acoustic reporter genes that enables efficient formation of the multi-protein complex while minimizing cellular toxicity. We also demonstrate that SEMPER enables polycistronic expression of recombinant monoclonal antibodies from plasmid DNA and of two fluorescent proteins from single mRNAs made through in vitro transcription. Finally, we provide a probabilistic model to elucidate the mechanisms underlying SEMPER. A record of this paper's transparent peer review process is included in the supplemental information.

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