Single molecule imaging of the central dogma reveals myosin-2A gene expression is regulated by contextual translational buffering.
Single molecule imaging of the central dogma reveals myosin-2A gene expression is regulated by contextual translational buffering.
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中心法则的单分子成像揭示肌球蛋白-2A 基因表达受上下文翻译缓冲调节。
DOI：
10.1101/2024.02.11.579797
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Stasevich,TimothyJ
中科院分区：
文献类型：
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作者：
Wiggan,O'Neil;Stasevich,TimothyJ
虽然蛋白质稳态是基因调控的标志，但使用传统方法很难解开维持稳态的隐藏调控机制。为了解决这个问题，我们CRISPR工程化了一种在内源性MYH 9基因中具有多个标签的人类细胞系，该基因编码必需的和普遍存在的肌球蛋白-2A细胞骨架马达。使用这些细胞，我们以不同的颜色对MYH 9的转录、翻译和成熟mRNA和蛋白质进行成像，从而能够全面剖析中心法则。我们的数据表明，MYH 9转录上调的SRF依赖性的方式响应于细胞骨架线索和MYH 9翻译可以缓冲或匹配的转录反应取决于上下文。在敲低肌动蛋白解聚蛋白如cofilin后，翻译效率下降了两倍，以缓冲强烈的转录上调，可能有助于防止过度的肌球蛋白活性。相比之下，血清刺激后，翻译匹配转录反应，容易重新建立稳定状态。我们的研究结果确定上下文翻译缓冲作为一个重要的调控机制驱动稳定MYH 9的表达。它们还证明了我们的细胞系的能力和广泛的适用性，现在可以用来准确地量化中心法则动态响应不同形式的细胞扰动。
While protein homeostasis is a hallmark of gene regulation, unraveling the hidden regulatory mechanisms that maintain homeostasis is difficult using traditional methods. To confront this problem, we CRISPR engineered a human cell line with multiple tags in the endogenous MYH9 gene, which encodes the essential and ubiquitous myosin-2A cytoskeletal motor. Using these cells, we imaged MYH9 transcription, translation, and mature mRNA and protein in distinct colors, enabling a full dissection of the central dogma. Our data show that MYH9 transcription is upregulated in an SRF-dependent manner in response to cytoskeletal cues and that MYH9 translation can either buffer or match the transcriptional response depending on context. Upon knockdown of actin-depolymerizing proteins like cofilin, translation efficiency drops by a factor of two to buffer strong transcriptional upregulation, likely to help prevent excessive myosin activity. In contrast, following serum stimulation, translation matches the transcriptional response to readily reestablish steady state. Our results identify contextual translational buffering as an important regulatory mechanism driving stable MYH9 expression. They also demonstrate the power and broad applicability of our cell line, which can now be used to accurately quantify central dogma dynamics in response to diverse forms of cellular perturbations.