A viral ADP-ribosyltransferase attaches RNA chains to host proteins

The mechanisms by which viruses hijack their host’s genetic machinery are of current interest. When bacteriophage T4 infects Escherichia coli, three different ARTs (ADP-ribosyltransferases) reprogram the host’s transcriptional and translational apparatus through ADP-ribosylation using nicotinamide adenine dinucleotide (NAD) as substrate. Recently, NAD was identified as a 5’-modification of cellular RNAs. Here, we report that T4 ART ModB accepts not only NAD but also NAD-capped RNA (NAD-RNA) as substrate and attaches entire RNA chains to acceptor proteins in an “RNAylation” reaction. ModB specifically RNAylates ribosomal proteins rS1 and rL2 at defined arginine residues, and selected E. coli and T4 phage RNAs are linked to rS1 in vivo. T4 phages that express an inactive mutant of ModB show a decreased burst size and slowed lysis of E. coli. Our findings reveal a distinct biological role of NAD-RNA, namely activation of the RNA for enzymatic transfer to proteins. The attachment of specific RNAs to ribosomal proteins might provide a strategy for the phage to modulate the host’s translation machinery. This work exemplifies the first direct connection between RNA modification and post-translational protein modification. As ARTs play important roles far beyond viral infections, RNAylation may have far-reaching implications. Exponentially growing E. coli with endogenously His-tagged rS1 were infected with T4 phage WT or T4 phage ModB R73A,G74A or not infected for 8 minutes. His-tagged rS1 was captured and covalently bound RNA was reverse transcribed and sequenced using our RNAylomeSeq pipeline. His-tagged rS1 RNAylated with NAD-RNAI was used as a spike-in to monitor success of capture of RNAylated RNAs by RNAylomeSeq. Uninfected and ModB R73A,G74A infected samples serve as negative controls for the WT infected samples.

病毒劫持宿主遗传机器的相关机制是当前的研究热点。当T4噬菌体（bacteriophage T4）感染大肠杆菌（Escherichia coli）时，三种不同的ADP核糖基转移酶（ADP-ribosyltransferases，ART）会以烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide，NAD）作为底物，通过ADP核糖基化作用重编程宿主的转录与翻译系统。近期研究发现，NAD可作为细胞RNA的5'端修饰基团。本研究表明，T4噬菌体的ART ModB不仅可将NAD作为底物，还可识别带NAD帽的RNA（NAD-capped RNA，NAD-RNA）作为底物，并通过“RNA连接修饰（RNAylation）”反应将完整的RNA链连接至受体蛋白。ModB可在特定的精氨酸残基处，对核糖体蛋白rS1与rL2进行特异性RNA连接修饰，且在大肠杆菌体内，特定的大肠杆菌与T4噬菌体RNA可与rS1蛋白形成共价连接。表达ModB失活突变体的T4噬菌体，其子代噬菌体裂解量（burst size）下降、大肠杆菌的裂解进程延缓。本研究结果揭示了NAD-RNA的全新生物学功能：激活RNA使其可通过酶促反应连接至蛋白质。将特定RNA连接至核糖体蛋白，或许为噬菌体调控宿主翻译系统提供了一种可行策略。本研究首次建立了RNA修饰与蛋白质翻译后修饰之间的直接关联。鉴于ART的生物学功能远超病毒感染范畴，RNA连接修饰或具有深远的研究意义。将内源携带His标签的rS1的指数生长期大肠杆菌，分别以野生型T4噬菌体（WT）、T4噬菌体ModB R73A、G74A双突变株进行感染，或不进行感染处理，感染时长均为8分钟。通过亲和捕获获取His标签标记的rS1蛋白，对与其共价结合的RNA进行反转录，并通过我们建立的RNAylomeSeq流程完成测序。将经NAD-RNAI进行RNA连接修饰的His标签rS1作为加标内参（spike-in），以监测RNAylomeSeq流程对RNA连接修饰RNA的捕获效果。未感染组与ModB R73A、G74A双突变株感染组作为野生型T4噬菌体感染组的阴性对照。