Abstract
As matchmaker between mRNA and sRNA interactions, the RNA chaperone Hfq plays a key role in riboregulation of many bacteria. Often, the global influence of Hfq on the transcriptome is reflected by substantially altered proteomes and pleiotropic phenotypes in hfq mutants. Using quantitative proteomics and co-immunoprecipitation combined with RNA-sequencing (RIP-seq) of Hfq-bound RNAs, we demonstrate the pervasive role of Hfq in nutrient acquisition, metabolism and motility of the plant pathogen Agrobacterium tumefaciens. 136 of 2544 proteins identified by iTRAQ (isobaric tags for relative and absolute quantitation) were affected in the absence of Hfq. Most of them were associated with ABC transporters, general metabolism and motility. RIP-seq of chromosomally encoded Hfq3xFlag revealed 1697 mRNAs and 209 non-coding RNAs (ncRNAs) associated with Hfq. 56 ncRNAs were previously undescribed. Interestingly, 55% of the Hfq-bound ncRNAs were encoded antisense (as) to a protein-coding sequence suggesting that A. tumefaciens Hfq plays an important role in asRNA-target interactions. The exclusive enrichment of 296 mRNAs and 31 ncRNAs under virulence conditions further indicates a role for post-transcriptional regulation in A. tumefaciens-mediated plant infection. On the basis of the iTRAQ and RIP-seq data, we assembled a comprehensive model of the Hfq core regulon in A. tumefaciens.
Highlights
Post-transcriptional gene regulation is a key strategy in the dynamic adaption to changing environmental conditions
Given the calculated upper (FC 2.3) and lower (FC 0.45) bounds we found a total of 136 proteins, encoded from all four replicons, that were differentially abundant in the hfq mutant compared to the WT (Fig. 1B). 100 proteins were upregulated, whereas 36 proteins were down-regulated (Table S4)
We combined two global approaches targeted at the identification of Hfq-affected proteins and Hfqassociated RNAs (RIP-seq) to further our understanding of the fundamental role of the RNA chaperone in Agrobacterium physiology
Summary
Post-transcriptional gene regulation is a key strategy in the dynamic adaption to changing environmental conditions. Small regulatory RNAs (sRNAs, non-coding RNAs, ncRNAs) rapidly adjust gene expression to the physiological needs and have been implicated in virulence control [1,2]. Bacterial ncRNAs usually influence translation or stability of their cognate mRNA target. Translational regulation occurs via blockage or release of the Shine-Dalgarno (SD) sequence on the mRNA [3,4]. The double-stranded ncRNA-mRNA duplex is often recognized and degraded by RNase III [5,6,7,8]. Rapid and dynamic adjustment of the cellular RNA pool involves other RNases (e.g. RNaseE, PNPase) that interact with single-stranded RNAs and modulate processing, degradation and general quality control of ncRNAs and mRNAs
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