Primary Characterization of Small RNAs in Symbiotic Nitrogen-Fixing Bacteria

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1734; p. 277
• Main Authors: Robledo, Marta, García-Tomsig, Natalia I, Jiménez-Zurdo, José I
• Format: Journal Article
• Language: English
• Published: United States 2018
• Subjects: Gene Expression; Gene Expression Regulation, Bacterial; Genes, Reporter; Nitrogen-Fixing Bacteria - genetics; Nitrogen-Fixing Bacteria - metabolism; Reproducibility of Results; RNA, Bacterial; RNA, Messenger - genetics; RNA, Small Untranslated - genetics; Symbiosis; GFP; Genetic reporter assay; mRNA target; Northern hybridization; RACE; Riboregulation
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-7604-1_22

High-throughput transcriptome profiling (RNAseq) has uncovered large and heterogeneous populations of small noncoding RNA species (sRNAs) with potential regulatory roles in bacteria. A large fraction of sRNAs are differentially regulated and rely on protein-assisted antisense interactions to trans-encoded target mRNAs to fine-tune posttranscriptional reprogramming of gene expression in response to external cues. However, annotation and function of sRNAs are still largely overlooked in nonmodel bacteria with complex lifestyles. Here, we describe experimental protocols successfully applied for the accurate annotation, expression profiling and target mRNA identification of trans-acting sRNAs in the nitrogen-fixing α-rhizobium Sinorhizobium meliloti. The protocols presented here can be similarly applied for the characterization of trans-sRNAs in genetically tractable α-proteobacteria of agronomical or clinical relevance interacting with eukaryotic hosts.