Identification of metabolite caps including FAD on the 5′ end of RNA has uncovered a previously unforeseen intersection between cellular metabolism and gene expression. To understand the function of FAD caps in cellular physiology, we characterised the proteins interacting with FAD caps in budding yeast. Here we demonstrate that highly conserved 5′-3′ exoribonucleases, Xrn1 and Rat1, physically interact with the RNA 5′ FAD cap and both possess FAD cap decapping (deFADding) activity and subsequently degrade the resulting RNA. Xrn1 deFADding activity was also evident in human cells indicating its evolutionary conservation. Furthermore, we report that the recently identified bacterial 5′-3′ exoribonuclease RNase AM also possesses deFADding activity that can degrade FAD-capped RNAs in vitro and in Escherichia coli cells. To gain a molecular understanding of the deFADding reaction, an RNase AM crystal structure with three manganese ions coordinated by a sulfate molecule and the active site amino acids was generated that provided details underlying hydrolysis of the FAD cap. Our findings reveal a general propensity for 5′-3′ exoribonucleases to hydrolyse and degrade RNAs with 5′ end noncanonical caps in addition to their well characterized 5′ monophosphate RNA substrates indicating an intrinsic property of 5′-3′ exoribonucleases.

中文翻译：

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鉴定人、酵母和细菌 5' 至 3' 外切核糖核酸酶中的新脱FADding 活性

鉴定包括RNA 5'端FAD在内的代谢物帽揭示了细胞代谢和基因表达之间以前无法预料的交叉点。为了了解 FAD 帽在细胞生理学中的功能，我们表征了与出芽酵母中的 FAD 帽相互作用的蛋白质。在这里，我们证明高度保守的 5'-3' 外切核糖核酸酶 Xrn1 和 Rat1 与 RNA 5' FAD 帽发生物理相互作用，并且都具有 FAD 帽脱帽 (deFADding) 活性并随后降解产生的 RNA。Xrn1 deFADding 活性在人类细胞中也很明显，表明其进化保守。此外，我们报告最近发现的细菌 5'-3' 外切核糖核酸酶 RNase AM 还具有 deFADding 活性，可以在体外和大肠杆菌细胞中降解 FAD 加帽的 RNA。为了获得对去FADding反应的分子理解，产生了由硫酸盐分子和活性位点氨基酸配位的具有三个锰离子的RNase AM晶体结构，该结构提供了FAD帽水解的详细信息。我们的研究结果揭示了 5'-3' 外切核糖核酸酶具有水解和降解具有 5' 末端非规范帽的 RNA 的普遍倾向，此外它们的特征良好的 5' 单磷酸 RNA 底物表明 5'-3' 外切核糖核酸酶的内在特性。