Moores Alexandra, Riesco Ana B, Schwenk Stefan, Arnvig Kristine B
Institute for Structural and Molecular Biology, University College London, London, United Kingdom.
PLoS One. 2017 Mar 21;12(3):e0174079. doi: 10.1371/journal.pone.0174079. eCollection 2017.
Mycobacterium tuberculosis depends on the ability to adjust to stresses encountered in a range of host environments, adjustments that require significant changes in gene expression. Small RNAs (sRNAs) play an important role as post-transcriptional regulators of prokaryotic gene expression, where they are associated with stress responses and, in the case of pathogens, adaptation to the host environment. In spite of this, the understanding of M. tuberculosis RNA biology remains limited. Here we have used a DosR-associated sRNA as an example to investigate multiple aspects of mycobacterial RNA biology that are likely to apply to other M. tuberculosis sRNAs and mRNAs. We have found that accumulation of this particular sRNA is slow but robust as cells enter stationary phase. Using reporter gene assays, we find that the sRNA core promoter is activated by DosR, and we have renamed the sRNA DrrS for DosR Regulated sRNA. Moreover, we show that DrrS is transcribed as a longer precursor, DrrS+, which is rapidly processed to the mature and highly stable DrrS. We characterise, for the first time in mycobacteria, an RNA structural determinant involved in this extraordinary stability and we show how the addition of a few nucleotides can lead to acute destabilisation. Finally, we show how this RNA element can enhance expression of a heterologous gene. Thus, the element, as well as its destabilising derivatives may be employed to post-transcriptionally regulate gene expression in mycobacteria in combination with different promoter variants. Moreover, our findings will facilitate further investigations into the severely understudied topic of mycobacterial RNA biology and into the role that regulatory RNA plays in M. tuberculosis pathogenesis.
结核分枝杆菌依赖于其在一系列宿主环境中应对压力的能力，而这种应对需要基因表达发生显著变化。小RNA（sRNA）作为原核基因表达的转录后调节因子发挥着重要作用，它们与应激反应相关，在病原体的情况下，还与适应宿主环境有关。尽管如此，对结核分枝杆菌RNA生物学的了解仍然有限。在这里，我们以一种与DosR相关的sRNA为例，研究了分枝杆菌RNA生物学的多个方面，这些方面可能适用于其他结核分枝杆菌的sRNA和mRNA。我们发现，随着细胞进入稳定期，这种特定sRNA的积累缓慢但稳定。通过报告基因分析，我们发现sRNA核心启动子被DosR激活，我们将该sRNA重新命名为DrrS，即DosR调控的sRNA。此外，我们表明DrrS转录为一个更长的前体DrrS+，它会迅速加工成成熟且高度稳定的DrrS。我们首次在分枝杆菌中鉴定出一个参与这种非凡稳定性的RNA结构决定因素，并展示了添加几个核苷酸如何导致急剧的不稳定。最后，我们展示了这个RNA元件如何增强异源基因的表达。因此，该元件及其不稳定衍生物可与不同的启动子变体结合，用于在转录后调节分枝杆菌中的基因表达。此外，我们的发现将有助于进一步研究分枝杆菌RNA生物学这一严重未被充分研究的课题，以及调节性RNA在结核分枝杆菌发病机制中的作用。
