An RNA degradosome assembly in Caulobacter crescentus

Abstract

In many bacterial species, the multi-enzyme RNA degradosome assembly makes key contributions to RNA metabolism. Powering the turnover of RNA and the processing of structural precursors, the RNA degradosome has differential activities on a spectrum of transcripts and contributes to gene regulation at a global level. Here, we report the isolation and characterization of an RNA degradosome assembly from the α-proteobacterium Caulobacter crescentus, which is a model organism for studying morphological development and cell-cycle progression. The principal components of the C. crescentus degradosome are the endoribonuclease RNase E, the exoribonuclease polynucleotide phosphorylase (PNPase), a DEAD-box RNA helicase and the Krebs cycle enzyme aconitase. PNPase and aconitase associate with specific segments in the C-terminal domain of RNase E that are predicted to have structural propensity. These recognition 'microdomains' punctuate structurally an extensive region that is otherwise predicted to be natively disordered. Finally, we observe that the abundance of RNase E varies through the cell cycle, with maxima at morphological differentiation and cell division. This variation may contribute to the program of gene expression during cell division.

Figure 1.

RNase E of α-proteobacteria have a distinguishing S1 domain insert not found in RNase E of other bacterial classes. (A) Structure based sequence alignment of the E. coli and C. crescentus RNase E catalytic domains. The secondary structural elements of E. coli RNase E are shown on the lines above the sequence alignment using the PDB file 2BX2. The arrows indicate β-sheet, the coils indicate α-helices, TT indicates β turns and η indicates 3₁₀ helices. Red letters indicate homology and blue boxes show similarity. The red highlights indicate identity across the sequences. The green stars represent the antigenic peptide used in this study. Structural sub-domains of RNase E are coloured (RNase H: light blue; S1: purple; 5′ sensor: yellow; DNase I: dark grey; zinc link: red; small domain: dark blue). Alignments were prepared using CLUSTALW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html) and ESPript (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). (B) E. coli RNase E NTD tetramer in complex with 13-mer RNA (pale orange). Structural sub-domains are highlighted for one protomer, coloured as in Figure 1A. The position of the S1 insert absent in E. coli RNase E catalytic domain is represented by the dashed loops.

Figure 2.

Identification of an RNA degradosome complex in C. crescentus. (A) SDS-PAGE analysis of immunoprecipitate from cell lysate of C. crescentus using RNase E antibody (lane 2). Cell lysates were applied to protein–A sepharose with bound antibody raised against C. crescentus RNase E and eluted with the peptide epitope. Degradosome components identified by MALDI fingerprinting are marked with an asterisk and correspond to RNase E, aconitase, polynucleotide phosphorylase and a DEAD-box RNA helicase. Lane 1 shows molecular weight standard (kDa). Lane 3 is a control in which C. crescentus lysates were eluted from protein–A sepharose in the absence of the anti-RNase E antibody. The gel is Coomassie stained. (B) Identification of PNPase partners by pull down. Cell lysates were applied to a glutathione matrix with bound fusion protein of PNPase and glutathionine-S-transferase (GST) and eluted with glutathione. Lane 1, molecular weight marker. Lane 2, GST–PNPase input. Lane 3, material eluted from glutathione-sepharose pre-incubated with GST–PNPase and C. crescentus cell lysate. Lane 4, GST alone input. Lane 5, control: elution from glutathione sepharose following incubation with GST and C. crescentus cell lysate. The asterisk indicates the position of GST-PNPase, and RNase E is marked with an arrow. The gel is Coomassie stained. (C) Western blot of same gel, probed with antibody against C. crescentus RNase E.

Figure 3.

A PNPase interaction motif in C. crescentus RNase E. (A) Sequence alignment of multiple α-proteobacterial RNase E sequences (C-terminal end only). The absolutely conserved GWW motif is highlighted with asterisks. (B) Pull-down experiment with GST-GWW peptide. The GWW bait sequence is from the C-terminus of C. crescentus RNase E (tappekprrGWWrr). Lane 1, molecular weight marker. Lane 2, GST–GWW input. Lane 3, GST only input. Lane 4, elution from glutathione sepharose following incubation with GST-GWW and C. crescentus cell lysate. Lane 5, elution from glutathione sepharose following incubation with GST and C. crescentus cell lysate. Co-purifying PNPase is marked with an asterisk. (C) Overlay of the 20 best NMR structural models of the 10-mer peptide (EKPRRGWWRR), showing the sidechains of residues WWRR only. A single peptide backbone trace is shown in black with WWRR coloured orange, red, magenta and blue, respectively. (D) NOE residue interaction matrix, highlighting inter-residue contacts clustering about the GWW motif.

Figure 4.

Identification of an aconitase binding region in C. crescentus RNase E. (A) Disorder prediction based on RNase E protein sequence from E. coli (left) and C. crescentus (right). N-terminal catalytic domains and C-terminal scaffold domains are separated by a vertical dashed line and extended regions of disorder are represented with a solid horizontal black line. C-terminal microdomains and the C. crescentus S1 insert are indicated with arrows. (B) Pull downs from cell extracts using immobilised C. crescentus micro-domain (RNase E_675–725). Lane 1, molecular weight marker. Lane 2, input GST-RNase E_675–725. Lane 3, the material eluted from GST–microdomain fusion, the asterisk indicates aconitase. Lane 4, input GST. Lane 5, material eluted from pulldown with GST alone. The gel is Coomassie stained.

Figure 5.

9S ribosomal RNA precursor processing by C. crescentus and E. coli RNase E catalytic domain. Digestion time points (0, 15 min) were analysed by denaturing polyacrylamide gel and detected with SYBR Gold stain. The p5S product, as seen to be released by both RNase E enzymes, is a precursor of the mature 120 nt 5S rRNA. The length of RNA species were determined by comparing to RiboRuler^™ low-range RNA marker (Fermentas), in the leftmost lane.

Figure 6.

Cell-cycle variation of RNase E. (Top) Representative western blots of RNase E, PNPase, aconitase and FliF protein abundance during the cell division cycle. The two maxima of RNase E abundance are marked at the top with +. (Bottom) Cartoon representation of the stages of C. crescentus cell division.