Powell Geralle, Pavlovic Djuranovic Slavica, Djuranovic Sergej
Department of Cell Biology and Physiology, Washington University School of Medicine, 600 South Euclid Avenue, Campus Box 8228, St. Louis, MO 63110, USA.
Mol Ther Nucleic Acids. 2021 Oct 8;26:865-878. doi: 10.1016/j.omtn.2021.10.005. eCollection 2021 Dec 3.
Manipulation of gene activity through creation of hypomorphic mutants has been a long-standing tool in examining gene function. Our previous studies have indicated that hypomorphic mutants could be created by inserting -regulatory sequences composed of consecutive adenosine nucleotides called poly(A) tracks. Here we use poly(A) tracks to create hypomorphic mutants and functional characterization of membrane, secretory, and endogenous proteins. Insertion of poly(A) tracks into the sequences of interleukin-2 and membrane protein CD20 results in a programmable reduction of mRNA stability and attenuation of protein expression regardless of the presence of a signaling sequence. Likewise, CRISPR-Cas9 targeted insertion of poly(A) tracks into the coding sequence of the endogenous human genes and results in a programmable reduction of targeted protein and mRNA levels. Functional analyses of AUF1-engineered hypomorphs indicate a direct correlation between gene levels and the stability of AUF1-regulated mRNAs. Hypomorphs of TP53 affect expression of the target genes differentially depending on the severity of the hypomorphic mutation. Finally, decreases in TP53 protein affect the same cellular pathways in poly(A) track-engineered cells as in cancer cells, indicating these variants' biological relevance. These results highlight this technology's power to create predictable, stable hypomorphs in recombinant or endogenous genes in combination with CRISPR-Cas9 engineering tools.
通过创建亚效突变体来操纵基因活性一直是研究基因功能的长期工具。我们之前的研究表明，可以通过插入由连续腺苷核苷酸组成的调控序列（称为聚腺苷酸序列）来创建亚效突变体。在这里，我们使用聚腺苷酸序列来创建亚效突变体，并对膜蛋白、分泌蛋白和内源性蛋白进行功能表征。将聚腺苷酸序列插入白细胞介素-2和膜蛋白CD20的序列中，无论是否存在信号序列，都会导致mRNA稳定性的可编程降低和蛋白质表达的减弱。同样，CRISPR-Cas9介导的将聚腺苷酸序列靶向插入内源性人类基因和的编码序列中，会导致靶向蛋白和mRNA水平的可编程降低。对AUF1工程化亚效突变体的功能分析表明，基因水平与AUF1调节的mRNA稳定性之间存在直接相关性。TP53的亚效突变体根据亚效突变的严重程度差异地影响靶基因的表达。最后，TP53蛋白的减少在聚腺苷酸序列工程化细胞中影响的细胞途径与在癌细胞中相同，表明这些变体的生物学相关性。这些结果突出了这项技术与CRISPR-Cas9工程工具相结合，在重组或内源性基因中创建可预测、稳定的亚效突变体的能力。
