Deletion of exons 2 and 3 from Actb leads to widespread, beta-actin independent alterations in gene expression associated with cell cycle control

The cytoplasmic actin proteins, beta- and gamma-actin, are 99% identical but perform non-redundant functions. Genes encoding the cytoplasmic actins, Actb and Actg1, respectively, are less similar but still share 89% of their nucleotide sequences. Knockout (KO) of Actb by deletion of first coding exons 2 and 3 in mice is embryonic lethal while KO embryonic fibroblasts (MEFs) fail to proliferate. In contrast, KO of Actg1 is viable but mice lacking Actg1 present with increased perinatal lethality and Actg1 KO MEFs present with a much milder defect in cell proliferation. Recent studies have identified important protein-independent functions for both Actb and Actg1 and demonstrate that deletions within the Actb nucleotide sequence, and not loss of the beta-actin protein, cause the most severe phenotypes in KO mice and cells. Here, we use a multi-omics approach to better understand what drives the phenotypes of Actb KO MEFs. RNA-sequencing and mass spectrometry of Actb KO MEFs reveal largescale changes to the transcriptome, proteome, and phosphoproteome. Pathway analysis of genes and proteins differentially expressed upon Actb KO shows widespread dysregulation of genes involved in the cell cycle. Together, these data suggest novel, protein-independent roles for Actb in regulating gene expression associated with control of cell proliferation.

细胞质肌动蛋白β-肌动蛋白（β-actin）与γ-肌动蛋白（γ-actin）的序列同源性高达99%，但二者发挥的功能并不冗余。分别编码这两种细胞质肌动蛋白的基因Actb与Actg1，序列相似性虽稍低，但仍共享89%的核苷酸序列。在小鼠中通过敲除Actb的前两个编码外显子2与3实现基因敲除（Knockout, KO），会导致胚胎致死，且该基因敲除的小鼠胚胎成纤维细胞（Mouse Embryonic Fibroblasts, MEFs）无法正常增殖。与之相反，Actg1基因敲除小鼠可存活，但缺失Actg1的小鼠围产期致死率升高，且Actg1敲除的MEFs仅表现出程度更轻微的细胞增殖缺陷。近期研究证实，Actb与Actg1均存在重要的不依赖于其编码蛋白的功能，并且证明在基因敲除小鼠与细胞中引发最严重表型的是Actb核苷酸序列的缺失，而非β-肌动蛋白蛋白的丢失。本研究采用多组学（multi-omics）方法，旨在深入解析驱动Actb敲除MEFs出现异常表型的分子机制。对Actb敲除MEFs进行RNA测序（RNA-sequencing）与质谱分析（mass spectrometry），可揭示转录组、蛋白质组与磷酸化蛋白质组的大规模表达变化。对Actb敲除后差异表达的基因与蛋白质进行通路富集分析，结果显示细胞周期相关基因存在广泛的表达失调。综上，本研究数据提示Actb可通过不依赖于其编码蛋白的全新途径，调控与细胞增殖控制相关的基因表达。