Small regulatory RNAs and antisense RNAs play important roles in the regulation of gene expression in bacteria but are underexplored, especially in natural populations. While environmentally relevant microbes often are not amenable to genetic manipulation or cannot be cultivated in the laboratory, extensive metagenomic and metatranscriptomic datasets for these organisms might be available. Hence, dedicated workflows for specific analyses are needed to fully benefit from this information. Here, we identified abundant sRNAs from oceanic environmental populations of the ecologically important primary producer Prochlorococcus starting from a metatranscriptomic differential RNA-Seq (mdRNA-Seq) dataset. We tracked their homologs in laboratory isolates, and we provide a framework for their further detailed characterization. Several of the experimentally validated sRNAs responded to ecologically relevant changes in cultivation conditions. The expression of the here newly discovered sRNA Yfr28 was highly stimulated in low-nitrogen conditions. Its predicted top targets include mRNAs encoding cell division proteins, a sigma factor, and several enzymes and transporters, suggesting a pivotal role of Yfr28 in the coordination of primary metabolism and cell division. A cis-encoded antisense RNA was identified as a possible positive regulator of atpF encoding subunit b' of the ATP synthase complex. The presented workflow will also be useful for other environmentally relevant microorganisms for which experimental validation abilities are frequently limiting although there is wealth of sequence information available.

中文翻译：

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微生物环境群体中非编码 RNA 的计算预测和分析及其实验验证的框架。

小调节 RNA 和反义 RNA 在细菌基因表达的调节中发挥着重要作用，但尚未得到充分研究，特别是在自然群体中。虽然与环境相关的微生物通常不适合基因操作或无法在实验室中培养，但可能可以获得这些生物体的广泛宏基因组和宏转录组数据集。因此，需要针对特定​​分析的专用工作流程才能充分受益于这些信息。在这里，我们从宏转录组差异 RNA-Seq (mdRNA-Seq) 数据集出发，从生态重要的初级生产者原绿球藻的海洋环境种群中鉴定出了丰富的 sRNA。我们在实验室分离株中追踪了它们的同源物，并为它们的进一步详细表征提供了一个框架。一些经过实验验证的 sRNA 对培养条件的生态相关变化做出了反应。这里新发现的 sRNA Yfr28 的表达在低氮条件下受到高度刺激。其预测的首要靶标包括编码细胞分裂蛋白、西格玛因子以及多种酶和转运蛋白的 mRNA，这表明 Yfr28 在协调初级代谢和细胞分裂中发挥着关键作用。顺式编码的反义 RNA 被鉴定为 ATP 合酶复合物的 atpF 编码亚基 b' 的可能正调节因子。所提出的工作流程对于其他环境相关微生物也很有用，尽管有大量可用的序列信息，但实验验证能力经常受到限制。