Biochem. J. (2014) 458, 11–22 (Printed in Great Britain) doi:10.1042/BJ20131287 11 The Escherichia coli RNA processing and degradation machinery is compartmentalized within an organized cellular network Aziz TAGHBALOUT* 1 , Qingfen YANG* and V´ eronique ARLUISON† *Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06032, U.S.A. †Universit´ e Paris Diderot-Paris 7, Sorbonne Paris Cit´ e & Laboratoire L´ eon Brillouin, Commissariat ` a l’Energie Atomique, CNRS-UMR 12, CEA-Saclay, 91191 Gif-sur-Yvette Cedex, France Bacterial RNA processing and degradation involves the co- ordinated action of a large number of RNases, RNA helicases and other proteins. It is not known how this functional network is organized within the cell nor how it is co-ordinated or regulated. In the present study, we show that multiple components of the RNA degradation and processing network of Escherichia coli are localized within extended cellular structures that appear to coil around the periphery of the cell. These include Orn, Hfq, PAP I, RNase III, RppH, RraA and RraB in addition to the previously reported proteins RNase II and RNaseE. Double-label localization studies of several of the proteins showed co-localization of the proteins within the observed structures. Assembly of the proteins into the structures was independent of the MreBCD or MinCDE cytoskeletal systems, RNA synthesis, or nucleoid positioning within the cell. Our results indicate that the components of the RNA processing and degradation network are compartmentalized within the cell rather than diffusely distributed in the cytoplasm. This sequestration provides the cell with a possible mechanism to control access to RNA substrates and to functionally co-ordinate the multiple players of the RNA processing and degradation pathways. Key words: cellular compartmentalization, cellular organization, macromolecular assembly, membrane association, RNA degrado- some, RNA processing and degradation. INTRODUCTION A fundamental problem for the bacterial cell is how to organize components that comprise a functionally coupled pathway or that catalyse conflicting cellular events taking place within its cytoplasm. This is well illustrated by the RNA processing and degradation pathways which deploy a large number of proteins to carry out essential roles in the life of the organism. In bacterial cells, the RNA processing and degradation proteins include endoribonucleases and exoribonucleases, RNA helicases, RNA polyadenylation and pyrophosphohydrolase enzymes, RNA- binding proteins, and regulator proteins (Figure 1A). The RNA degradation and processing proteins function as an interconnected system that leads either to the formation of short oligoribonucleotides and monoribonucleotides or to the formation of mature tRNA and rRNA (reviewed in [1–4]). Despite the importance of the RNA degradation and processing pathways, the cellular location and organization of most of these proteins and the way in which their individual functions are co-ordinated within the cell are not known. It has been shown previously that two components of the RNA degradation and processing machinery of Escherichia coli, i.e. RNase II and the proteins of the RNA degradosome, appear to be present in extended structures that seem to coil around the cell periphery [5–7]. In the present paper, we describe the cellular organization patterns of other major proteins of the pathway: the essential oligoribonuclease Orn, the endoribonuclease RNase III, the RNA pyrophosphohydrolase RppH, the poly(A) polymerase PAP I, the RNA chaperone Hfq, and two known regulators of RNaseE activity RraA and RraB. We have shown that these proteins are not diffusely distributed throughout the cytoplasm, but are localized in extended structures indistinguishable from those of RNase II and the RNA degradosome proteins. Double-label localization studies of several of the proteins showed co-localization of the proteins within the observed structures. The results suggest that most, and perhaps all, of the elements of the RNA degradation and processing machinery are compartmentalized within composite cellular elements. The sequestration of the machinery could provide the cell with a mechanism to control access to RNA substrates and to co-ordinate the coupled or overlapping biochemical functions of the different components of the network. EXPERIMENTAL E. coli strains, plasmids and growth conditions Strains and plasmids are listed in Supplementary Table S1 (at http://www.biochemj.org/bj/458/bj4580011add.htm) and the details of their construction are available from A.T. on request. Strains were grown at 37 ◦ C to mid-exponential phase in LB medium [8] to which 100 μg/ml ampicillin, 50 μg/ml kanamycin, 30 μg/ml chloramphenicol or 0.4 % glucose was added when indicated. Cell growth was monitored by measuring the attenuance at 600 nm (D 600 ). To inhibit transcription, 200 μg/ml rifampicin was added to the medium for 2 h when indicated. dnaA ts mutants were initially grown at 30 ◦ C then shifted to 42 ◦ C for 2 h. Cells containing plasmids coding for YFP-labelled proteins were grown in the presence of 10 μM IPTG essentially as described previously [9]. Gene knockouts were constructed by linear DNA recombination [10]. HA (haemagglutinin) and FLAG epitope tagging were carried out as described previously [11] and the as- sociated antibiotic cassettes were eliminated, when indicated, by the use of the FLP-expressing plasmid pCP20 [12]. P1-mediated transduction was used to move mutations to different strains [13]. Abbreviations: GlpD, glycerol-3-phosphate dehydrogenase; HA, haemagglutinin; PNPase, polynucleotide phosphorylase. 1 To whom correspondence should be addressed (email taghbalout@neuron.uchc.edu). c  The Authors Journal compilation c  2014 Biochemical Society Biochemical Journal www.biochemj.org