Institute of Biophysics and Physical Biochemistry, Regensburg Center for Biochemistry, University of Regensburg, D-93040 Regensburg, Germany.
Institute of Biochemistry, Faculty of Biology and Chemistry, University of Giessen, D-35392 Giessen, Germany.
Int J Mol Sci. 2023 Jul 16;24(14):11536. doi: 10.3390/ijms241411536.
An open research field in cellular regulation is the assumed crosstalk between RNAs, metabolic enzymes, and metabolites, also known as the REM hypothesis. High-throughput assays have produced extensive interactome data with metabolic enzymes frequently found as hits, but only a few examples have been biochemically validated, with deficits especially in prokaryotes. Therefore, we rationally selected nineteen enzymes from such datasets and examined their ability to bind RNAs using two complementary methods, iCLIP and SELEX. Found interactions were validated by EMSA and other methods. For most of the candidates, we observed no RNA binding (12/19) or a rather unspecific binding (5/19). Two of the candidates, namely glutamate-5-kinase (ProB) and quinone oxidoreductase (QorA), displayed specific and previously unknown binding to distinct RNAs. We concentrated on the interaction of QorA to the mRNA of , a grounded prophage gene, which could be validated by EMSA and MST. Because the physiological function of both partners is not known, the biological relevance of this interaction remains elusive. Furthermore, we found novel RNA targets for the MS2 phage coat protein that served us as control. Our results indicate that RNA binding of metabolic enzymes in procaryotes is less frequent than suggested by the results of high-throughput studies, but does occur.
细胞调节中一个开放的研究领域是假定的 RNA、代谢酶和代谢物之间的串扰，也称为 REM 假说。高通量测定法产生了广泛的互作组数据，其中代谢酶经常被发现是靶点，但只有少数例子得到了生化验证，尤其是在原核生物中。因此，我们从这些数据集有理性地选择了 19 种酶，并使用两种互补的方法 iCLIP 和 SELEX 来检查它们结合 RNA 的能力。发现的相互作用通过 EMSA 和其他方法进行了验证。对于大多数候选物，我们观察到没有 RNA 结合（12/19）或结合相当非特异性（5/19）。两个候选物，即谷氨酸-5-激酶（ProB）和醌氧化还原酶（QorA），显示出与特定 RNA 的特异性和先前未知的结合。我们集中研究了 QorA 与地面噬菌体基因 mRNA 的相互作用，可以通过 EMSA 和 MST 进行验证。由于两个伙伴的生理功能未知，因此这种相互作用的生物学相关性仍然难以捉摸。此外，我们发现了 MS2 噬菌体外壳蛋白的新 RNA 靶标，作为对照。我们的结果表明，原核生物代谢酶的 RNA 结合比高通量研究结果所暗示的要少，但确实存在。
