Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75390, United States.
Department of Cardiology, Clinical Center for Human Gene Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei, P. R. China.
Biochemistry. 2020 Apr 28;59(16):1559-1564. doi: 10.1021/acs.biochem.0c00100. Epub 2020 Apr 17.
Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant and ubiquitous nuclear enzyme that catalyzes the transfer of ADP-ribose from donor NAD molecules to specific amino acids on substrate proteins. The catalytic activity of PARP-1 has long been known to be allosterically stimulated by the free ends of DNA, such as those found at double-strand breaks in the genome. A number of studies have also shown that the catalytic activity of PARP-1 can also be stimulated by various types of RNA. A recent study by Nakamoto et al., however, has contradicted these results, concluding that the apparent stimulatory activity of the RNAs was due to contaminating DNA in the RNA preparations used in the biochemical assays. Here we show using a carefully controlled set of biochemical assays that DNA-free, in vitro-transcribed, PARP-1-interacting snoRNAs can stimulate PARP-1 catalytic activity. We confirmed the activation of PARP-1 by snoRNAs using a chemically synthesized snoRNA, as well as CRISPR/Cas9-mediated knockout of snoRNAs in cells. Finally, we provide a set of considerations and experimental conditions for the careful evaluation of RNA-stimulated PARP-1 catalytic activity that will help researchers avoid artifacts.
聚（ADP-核糖）聚合酶-1（PARP-1）是一种丰富且普遍存在的核酶，能够催化从供体 NAD 分子向底物蛋白上特定氨基酸转移 ADP-核糖。PARP-1 的催化活性早已被证实可被 DNA 的游离末端（如基因组中双链断裂处）别构激活。许多研究还表明，PARP-1 的催化活性也可被各种类型的 RNA 刺激。然而，Nakamoto 等人的一项最新研究与这些结果相矛盾，他们得出结论，认为 RNA 中明显的刺激活性是由于生化测定中使用的 RNA 制剂中存在污染 DNA。在这里，我们使用一组精心控制的生化测定表明，无 DNA 的体外转录的与 PARP-1 相互作用的 snoRNA 可以刺激 PARP-1 的催化活性。我们通过化学合成的 snoRNA 以及通过 CRISPR/Cas9 介导的细胞 snoRNA 敲除证实了 snoRNA 对 PARP-1 的激活。最后，我们提供了一套考虑因素和实验条件，用于仔细评估 RNA 刺激的 PARP-1 催化活性，这将帮助研究人员避免假象。
