Herzog Roman, Papenfort Kai
Faculty of Biology I, Department of Microbiology, Ludwig-Maximilians-University of Munich, Munich, Germany.
Faculty of Biology I, Department of Microbiology, Ludwig-Maximilians-University of Munich, Munich, Germany; Munich Center for Integrated Protein Science (CIPSM), Munich, Germany.
Methods Enzymol. 2018;612:303-342. doi: 10.1016/bs.mie.2018.09.008. Epub 2018 Oct 24.
Transcriptome analysis using RNA-sequencing (RNA-seq) has now become the standard approach to determine the transcriptional output of an organism. Various modifications to this technology have been developed over the years, usually aiming to improve the annotation of transcript borders, or to identify novel classes of RNAs, such as small regulatory RNAs (sRNAs) and antisense transcripts. RNA-seq has also led to the identification of dozens of new sRNAs in the major human pathogen, Vibrio cholerae. Several of these sRNAs function in the context of a cell-to-cell communication process, called quorum sensing (QS). QS is key for pathogenicity and biofilm formation of V. cholerae and the sRNAs involved typically act by base pairing with multiple target mRNAs to control gene expression at the posttranscriptional level. In this chapter, we describe the use of RNA-seq technologies for the discovery and characterization of regulatory RNAs in V. cholerae and discuss their relevance to QS and collective functions, such as biofilm formation. We further outline possible methods for the identification and validation of sRNA target genes, which can provide crucial information as to the physiological roles of an sRNA.
使用RNA测序（RNA-seq）进行转录组分析现已成为确定生物体转录输出的标准方法。多年来，人们对这项技术进行了各种改进，通常旨在改善转录本边界的注释，或识别新型RNA类别，如小调控RNA（sRNA）和反义转录本。RNA-seq还促使人们在主要人类病原体霍乱弧菌中鉴定出数十种新的sRNA。其中一些sRNA在一种称为群体感应（QS）的细胞间通信过程中发挥作用。群体感应对于霍乱弧菌的致病性和生物膜形成至关重要，所涉及的sRNA通常通过与多个靶标mRNA碱基配对，在转录后水平控制基因表达。在本章中，我们描述了使用RNA-seq技术发现和表征霍乱弧菌中的调控RNA，并讨论它们与群体感应和生物膜形成等集体功能的相关性。我们还概述了识别和验证sRNA靶基因的可能方法，这些方法可以提供有关sRNA生理作用的关键信息。
