Institute of Biomedical Chemistry, Pogodinskaya St 10/8, 119121, Moscow, Russia; Peoples Friendship University of Russia (RUDN University), Miklukho-Maklaya 6, 117198, Moscow, Russia.
Institute of Biomedical Chemistry, Pogodinskaya St 10/8, 119121, Moscow, Russia.
Biochimie. 2019 Feb;157:158-176. doi: 10.1016/j.biochi.2018.11.020. Epub 2018 Dec 3.
Apoptotic endonucleases act cooperatively to fragment DNA and ensure the irreversibility of apoptosis. However, very little is known regarding the potential regulatory links between endonucleases. Deoxyribonuclease 1 (DNase I) inactivation is caused by alternative splicing (AS) of DNase I pre-mRNA skipping exon 4, which occurs in response to EndoG overexpression in cells. The current study aimed to determine the role of EndoG in the regulation of DNase I mRNA AS and the modulation of its enzymatic activity. A strong correlation was identified between the EndoG expression levels and DNase I splice variants in human lymphocytes. EndoG overexpression in CD4 T cells down-regulated the mRNA levels of the active full-length DNase I variant and up-regulated the levels of the non-active spliced variant, which acts in a dominant-negative fashion. DNase I AS was induced by the translocation of EndoG from mitochondria into nuclei during the development of apoptosis. The DNase I spliced variant was induced by recombinant EndoG or by incubation with EndoG-digested cellular RNA in an in vitro system with isolated cell nuclei. Using antisense DNA oligonucleotides, we identified a 72-base segment that spans the adjacent segments of exon 4 and intron 4 and appears to be responsible for the AS. DNase I-positive CD4 T cells overexpressing EndoG demonstrated decreased progression towards bleomycin-induced apoptosis. Therefore, EndoG is an endonuclease with the unique ability to inactivate another endonuclease, DNase I, and to modulate the development of apoptosis.
凋亡内切核酸酶协同作用以片段化 DNA，并确保细胞凋亡的不可逆性。然而，对于内切核酸酶之间潜在的调控联系，人们知之甚少。脱氧核糖核酸酶 1（DNase I）的失活是由 DNase I 前体 mRNA 跳过外显子 4 的选择性剪接（AS）引起的，这种现象发生在细胞中 EndoG 过表达时。本研究旨在确定 EndoG 在调控 DNase I mRNA AS 及其酶活性中的作用。在人类淋巴细胞中，鉴定到 EndoG 表达水平与 DNase I 剪接变体之间存在很强的相关性。CD4 T 细胞中 EndoG 的过表达下调了活性全长 DNase I 变体的 mRNA 水平，并上调了非活性剪接变体的水平，后者以显性负性方式起作用。凋亡过程中，EndoG 从线粒体易位到细胞核，诱导了 DNase I 的 AS。在体外系统中，用重组 EndoG 或用经 EndoG 消化的细胞 RNA 孵育分离的细胞核，可诱导 DNase I 剪接变体。通过使用反义 DNA 寡核苷酸，我们鉴定出一个跨越外显子 4 和内含子 4 相邻片段的 72 个碱基片段，该片段似乎负责 AS。过表达 EndoG 的 DNase I 阳性 CD4 T 细胞显示出向博来霉素诱导的凋亡进展减少。因此，EndoG 是一种具有独特能力的内切核酸酶，可使另一种内切核酸酶 DNase I 失活，并调节细胞凋亡的发生。
