ABSTRACT

Function of bacterial small non-coding RNAs (sRNAs) and overall RNA metabolism is largely shaped by a vast diversity of RNA-protein interactions. However, in non-model bacteria with defined non-coding transcriptomes the sRNA interactome remains almost unexplored. We used affinity chromatography to capture proteins associated in vivo with MS2-tagged trans-sRNAs that regulate nutrient uptake (AbcR2 and NfeR1) and cell cycle (EcpR1) mRNAs by antisense-based translational inhibition in the nitrogen-fixing α-rhizobia Sinorhizobium meliloti. The three proteomes were rather distinct, with that of EcpR1 particularly enriched in cell cycle-related enzymes, whilst sharing several transcription/translation-related proteins recurrently identified associated with sRNAs. Strikingly, MetK, the synthetase of the major methyl donor S-adenosylmethionine, was reliably recovered as a binding partner of the three sRNAs, which reciprocally co-immunoprecipitated with a FLAG-tagged MetK variant. Induced (over)expression of the trans-sRNAs and MetK depletion did not influence canonical riboregulatory traits, `for example, protein titration or sRNA stability, respectively. An in vitro filter assay confirmed binding of AbcR2, NfeR1 and EcpR1 to MetK and further revealed interaction of the protein with other non-coding and coding transcripts but not with the 5S rRNA. These findings uncover a broad specificity for RNA binding as an unprecedented feature of this housekeeping prokaryotic enzyme.

 抽象的

细菌小非编码 RNA (sRNA) 的功能和整体 RNA 代谢在很大程度上是由多种多样的 RNA-蛋白质相互作用决定的。然而，在具有明确非编码转录组的非模型细菌中，sRNA 相互作用组几乎尚未被探索。我们使用亲和层析捕获体内与 MS2 标记的反式sRNA 相关的蛋白质，这些蛋白质通过固氮 α-根瘤菌中华根瘤菌中的反义翻译抑制来调节营养吸收（AbcR2 和 NfeR1）和细胞周期（EcpR1）mRNA。这三个蛋白质组相当不同，其中 EcpR1 的蛋白质组特别富含细胞周期相关酶，同时共享一些经常被识别的与 sRNA 相关的转录/翻译相关蛋白质。引人注目的是，MetK（主要甲基供体 S-腺苷甲硫氨酸的合成酶）作为三种 sRNA 的结合配偶体被可靠地回收，这三种 sRNA 与 FLAG 标记的 MetK 变体相互共免疫沉淀。反式sRNA 的诱导（过度）表达和 MetK 消耗并不影响典型的核糖调节特性，例如蛋白质滴定或 sRNA 稳定性。体外过滤测定证实了 AbcR2、NfeR1 和 EcpR1 与 MetK 的结合，并进一步揭示了该蛋白质与其他非编码和编码转录本的相互作用，但不与 5S rRNA 相互作用。这些发现揭示了 RNA 结合的广泛特异性，这是这种管家原核酶的前所未有的特征。