Phase-separated bacterial ribonucleoprotein bodies organize mRNA decay

Abstract

In bacteria, mRNA decay is controlled by megadalton scale macromolecular assemblies called, "RNA degradosomes," composed of nucleases and other RNA decay associated proteins. Recent advances in bacterial cell biology have shown that RNA degradosomes can assemble into phase-separated structures, termed bacterial ribonucleoprotein bodies (BR-bodies), with many analogous properties to eukaryotic processing bodies and stress granules. This review will highlight the functional role that BR-bodies play in the mRNA decay process through its organization into a membraneless organelle in the bacterial cytoplasm. This review will also highlight the phylogenetic distribution of BR-bodies across bacterial species, which suggests that these phase-separated structures are broadly distributed across bacteria, and in evolutionarily related mitochondria and chloroplasts. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Export and Localization > RNA Localization RNA Turnover and Surveillance > Regulation of RNA Stability.

Keywords: RNA degradosome; bacterial ribonucleoprotein bodies; mRNA decay; phase separation.

Figure 1.

Overview of biomolecular condensates. A.) Cartoon highlighting the organization of biomolecules via phase separation. On the left, a well-mixed solution of macromolecules A and B. On the right, demixing of molecules A and B by phase separation into a biomolecular condensate. B.) Cartoon of material states of biomolecular condensates. Left, Liquid-liquid phase separation can generate biomolecular condensates with rather dynamic internal diffusion as represented as the particle trajectory by the red arrow. Middle, phase separation to form a gel or hydrogel can lead to biomolecular condensates with slower internal diffusion. Right, liquid-solid phase separation leading to very slow internal motion.

Figure 2.

C. crescentus BR-bodies organize mRNA decay through biomolecular condensation. A.) Domain organization of the Ccr-RNase E. Structured N-terminal domain (NTD) containing the catalytic E/G domain and “small domain” for multimerization are shown in neon green. In red, the intrinsically disordered CTD with patches of negative (red) and positive (blue) amino acids that facilitate self-assembly. Below, cartoons of the RNase E monomer which assembles into a tetramer. The RNA degradosome which assembles with a suite of RNA decay related proteins which associated predominantly with the CTD is also shown with red ovals as degradosome proteins. B.) Ccr-RNase E phase separates in vitro (N. Al-Husini et al., 2018), forming a biomolecular condensate. C.) Subcellular localization of BR-bodies in C. crescentus and E. coli. RNase E subcellular localization is highlighted in green and the cell envelope is colored black. D.) Evidence of liquid like properties for C. crescentus and E. coli BR-bodies. Left, RNase E-YFP foci were observed to fuse in vivo, suggestive of a liquid-like state. Images were taken every 10s for C. crescentus (N. Al-Husini et al., 2018) and 200ms for E. coli (Strahl et al., 2015). Right, single molecule particle trajectories showed confined diffusion in C. crescentus cells in the presence of mRNA. E.) BR-bodies compete with ribosomes for mRNA substrates. Free mRNA (red) can either be directed down a decay path by the RNA degradosome or be translated by ribosomes. Both Eco- and Ccr-RNase E foci are rifampicin sensitive, suggesting they require mRNA. Translation inhibitor treatments and translation initiation factor depletion experiments in C. crescentus suggest that the untranslated mRNA pool directs BR-body assembly. In addition, C. crescentus BR-bodies were shown to exclude ribosomes. While eukaryotic P-bodies and stress granules tend to lead to mRNA storage upon stress, its unclear whether BR-bodies can provide a similar storage function. In conditions of logarithmic growth, BR-bodies accelerate the mRNA decay rate.

Figure 3.

α-proteobacterial RNase E IDR sequences contain charge-patterning. Phylogenetic tree containing several α-proteobacterial RNase E sequences (black) and two γ proteobacterial RNase E sequences (orange). Sequences are aligned to the catalytic NTD which is represented as a light-grey box. To the right, the IDR is colored corresponding to a 10 amino-acid sliding window of the electrostatic charge (scale below).

Figure 4.

RNase E IDRs show charge patterning across bacterial phyla. A representative RNase E from each major bacterial class or from plant chloroplasts is shown to highlight the charge-patterning of RNase E IDRs. Sequences are aligned to the catalytic domain which is represented as a light-grey box. Below, the IDR is colored corresponding to a 11 amino-acid sliding window of the electrostatic charge (scale below). For Mxa-RNase E, the individual amino acids are displayed based on their charge to reveal micro-charge patterning (right).

Figure 5.

The B. subtilis RNA degradosome scaffold RNase Y likely forms BR-bodies. A.) Charge-patterning of the mRNA decay nuclease Bsu-RNase Y. The sequence is colored corresponding to a 11 amino-acid window of electrostatic charge (scale below). RNase Y domain architecture is shown below, with its IDR shown in a black box (Lehnik-Habrink et al., 2011). TM=transmembrane, CC=coiled-coil, KH=RNA binding domain, HD=nuclease active site. B.) TIRF microscopy time-lapse of and Bsu-RNase Y-msfGFP fusion (Hamouche et al., 2020). Red arrows mark two foci that ultimately fuse together, suggesting liquid-like properties. Images were taken every 100ms (Hamouche et al., 2020).

Figure 6.

BR-bodies facilitate the mRNA life cycle. BR-bodies are stimulated by untranslated mRNAs causing condensation of RNA degradosomes with mRNA. mRNA acts as a scaffold to which RNA degradosomes can self-assemble. The mRNA decay pathway is shown on the right, with both the monophosphate stimulated decay pathway and the direct entry pathways highlighted. The localization of RppH, the enzyme which catalyzes the conversion to a 5′-monophosphate containing RNA which enhances the RNase E cleavage rate. Within the condensate, mRNA decay is stimulated by high concentrations of the RNA degradosome and poorly translated mRNA (top middle). The initial step of mRNA decay is controlled by the endoribonuclase RNase E and is stimulated within the BR-body (right). The subsequent steps of mRNA performed by RNA degradosomes associated exoribonucleases that are also stimulated within the BR-body (right). Upon cleavage of the mRNA down into small oligonucleotides by both the endoribonuclase RNase E and RNA degradosome-associated exoribonucleases, the multivalent bridging functionality of the mRNA is lost, causing a dissolution of the BR-body, releasing short oligo RNA products and RNA degradosomes that can reassemble on new mRNAs.