Hfq is a conserved RNA-binding protein with multiple regulatory roles within the prokaryotic cell. One of the key roles is to promote stable duplex formation between small RNAs and mRNAs and thus Hfq deletion mutants have pleiotropic phenotypes. This study represents first attempt to characterize Hfq regulon in bacterial pathogen using an integrative omics approach by comparing RNA Seq and microarray technique.
Hfq protein in action. Figure shows binding mechanism by forming duplex formation.
This recent study by Dienstbier et al (1) performs a comparison across next generation sequencing using RNA Seq analysis and DNA microarray to establish regulatory action for Hfq protein in B. pertussis.
Total RNA isolated from biological triplicates of B. pertussis Tohama I strain and its isogenic hfq strain cultures were analyzed by RNA-seq. Differential gene expression analysis showed significant modulation of B. pertussis genes including non-coding RNAs. A parallel DNA microarray analysis was performed and results were compared to determine sensitivity between the two approaches. Compared to microarray profiling, RNA seq analysis showed significant sensitivity in retrieving novel genes. RNA Seq analysis pipeline was used for clustering and principle component analysis.
Heatmap showing clustering of deferentially expressed genes. Differential gene expression analysis identified 653 significantly modulated B. pertussis genes including 40 non-coding RNAs and 11 transfer RNA genes.
Gene Ontology (GO) analysis provided insights about the functional profiles of Hfq-dependent genes. As displayed in figure below, genes related to Translation, Regulation of transcription, and Transmembrane transport terms were up regulated while genes belonging to Transmembrane transport, Iron–sulfur cluster assembly, Oxido-reduction process, Pathogenesis, and Protein secretion by the type III secretion system terms were enriched among the transcripts which were significantly down regulated in the hfq mutant.
Gene Ontology (GO) analysis for functional profiling
Above study clearly highlights advantages of next generation sequencing – Transcriptome sequencing or RNA seq and bioinformatics RNA Seq analysis pipelines to establish differential gene expression profiles.
(1) Comparative Integrated Omics Analysis of the Hfq Regulon in Bordetella pertussis. Int J Mol Sci. 2019 Jun 24;20(12). pii: E3073. doi: 10.3390/ijms20123073.