The proteomic response to mutants of the Escherichia coli RNA degradosome

Abstract

The Escherichia coli RNA degradosome recognizes and degrades RNA through the coordination of four main protein components, the endonuclease RNase E, the exonuclease PNPase, the RhlB helicase and the metabolic enzyme enolase. To help our understanding of the functions of the RNA degradosome, we quantified expression changes of >2,300 proteins by mass spectrometry based shotgun proteomics in E. coli strains deficient in rhlB, eno, pnp (which displays temperature sensitive growth), or rne(1-602) which encodes a C-terminal truncation mutant of RNaseE and is deficient in degradosome assembly. Global protein expression changes are most similar between the pnp and rhlB mutants, confirming the functional relationship between the genes. We observe down-regulation of protein chaperones including GroEL and DnaK (which associate with the degradosome), a decrease in translation related proteins in Δpnp, ΔrhlB and rne(1-602) cells, and a significant increase in the abundance of aminoacyl-tRNA synthetases. Analysis of the observed proteomic changes point to a shared motif, CGCTGG, that may be associated with RNA degradosome targets. Further, our data provide information on the expression modulation of known degradosome-associated proteins, such as DeaD and RNase G, as well as other RNA helicases and RNases – suggesting or confirming functional complementarity in some cases. Taken together, our results emphasize the role of the RNA degradosome in the modulation of the bacterial proteome and provide the first large-scale proteomic description of the response to perturbation of this major pathway of RNA degradation.

TheEscherichia colimajor exoribonuclease RNase II is a component of the RNA degradosome

“…Gel affinity (far Western) blotting experiments provided evidence that the failure of co- IP of RNase II and RNaseE (1–417) was not due to other changes associated with loss of RNaseE C in the RNaseE 1–417 cells [ 1 , 29 , 33 ]. The ability of purified RNaseE–HA to interact with RNase II–Flag in far Western blots was tested using PEs of cells that coexpressed RNase II–Flag together with either truncated RNaseE (1–417) ( Figure 2 B left lane) or full-length RNaseE ( Figure 2 B right lane).…”

“…Loss of degradosome assembly was associated with global changes in normal mRNA turnover [ 2 ] and protein synthesis [ 1 ] in cells that lack the RNaseE C-terminal scaffold domain, RhlB, or PNPase, which are required for formation of the RNase II–degradosome complex described here. RNase II, a hydrolytic exoribonuclease, accounts for 90% of exoribonuclease activity of cell extracts, suggesting that E. coli RNA degradation is a predominantly hydrolytic [ 6 ].…”

“…RNA degradation plays an important role in controlling the synthesis of proteins by directly affecting the half-life of mRNAs. In Escherichia coli , normal degradation of mRNAs requires a four-protein complex, the RNA degradosome, whose components include the essential RNaseE (endoribonuclease E), RhlB (RNA helicase B), the exoribonuclease PNPase (polynucleotide phosphorylase) and the glycolytic enzyme Eno (enolase) [ 1 , 2 ]. RNaseE, the 1061 amino-acid product of the rne gene, is the core degradosomal component.…”

Systems-Level Response to Point Mutations in a Core Metabolic Enzyme Modulates Genotype-Phenotype Relationship

“…They have been widely used to analyze systems-level changes associated with disease phenotypes in mammalian cells (Vogel and Marcotte, 2012). Other applications include the study of the systems-level response to major perturbations such as whole genome duplication (de Godoy et al, 2008), osmolarity and oxidative stresses (Maier et al, 2011; Vogel and Marcotte, 2012), and loss of function mutations in the RNA degradosome in E. coli , which affect global RNA turnover and regulation (Bernstein et al, 2004; Zhou et al, 2013). Also, quantitative proteomics was used to explore the general relationship between cells proteome and growth rates (Brauer et al, 2008; Geiler-Samerotte et al, 2013; Slavov and Botstein, 2011).…”

Bridging the physical scales in evolutionary biology: from protein sequence space to fitness of organisms and populations

“…The map between the biophysical to systems to organismal scales can also be assessed quantitatively by global transcriptomics and proteomics techniques [2,77,78]. Bershtein et al .…”