Liquid-Liquid Phase Separation of the DEAD-Box Cyanobacterial RNA Helicase Redox (CrhR) into Dynamic Membraneless Organelles in Synechocystis sp. Strain PCC 6803.

Compartmentalization of macromolecules into discrete non-lipid-bound bodies by liquid-liquid phase separation (LLPS) is a well-characterized regulatory mechanism frequently associated with the cellular stress response in eukaryotes. In contrast, the formation and importance of similar complexes is just becoming evident in bacteria. Here, we identify LLPS as the mechanism by which the DEAD-box RNA helicase, cyanobacterial RNA helicase redox (CrhR), compartmentalizes into dynamic membraneless organelles in a temporal and spatial manner in response to abiotic stress in the cyanobacterium Synechocystis sp. strain PCC 6803. Stress conditions induced CrhR to form a single crescent localized exterior to the thylakoid membrane, indicating that this region is a crucial domain in the cyanobacterial stress response. These crescents rapidly dissipate upon alleviation of the stress conditions. Furthermore, CrhR aggregation was mediated by LLPS in an RNA-dependent reaction. We propose that dynamic CrhR condensation performs crucial roles in RNA metabolism, enabling rapid adaptation of the photosynthetic apparatus to environmental stresses. These results expand our understanding of the role that functional compartmentalization of RNA helicases and thus RNA processing in membraneless organelles by LLPS-mediated protein condensation performs in the bacterial response to environmental stress. IMPORTANCE Oxygen-evolving photosynthetic cyanobacteria evolved ~3 billion years ago, performing fundamental roles in the biogeochemical evolution of the early Earth and continue to perform fundamental roles in nutrient cycling and primary productivity today. The phylum consists of diverse species that flourish in heterogeneous environments. A prime driver for survival is the ability to alter photosynthetic performance in response to the shifting environmental conditions these organisms continuously encounter. This study demonstrated that diverse abiotic stresses elicit dramatic changes in localization and structural organization of the RNA helicase CrhR associated with the photosynthetic thylakoid membrane. These dynamic changes, mediated by a liquid-liquid phase separation (LLPS)-mediated mechanism, reveal a novel mechanism by which cyanobacteria can compartmentalize the activity of ribonucleoprotein complexes in membraneless organelles. The results have significant consequences for understanding bacterial adaptation and survival in response to changing environmental conditions.

Degron-mediated proteolysis of CrhR-like DEAD-box RNA helicases in cyanobacteria.

Conditional proteolytic degradation is an irreversible and highly regulated process that fulfills crucial regulatory functions in all organisms. As proteolytic targets tend to be critical metabolic or regulatory proteins, substrates are targeted for degradation only under appropriate conditions through the recognition of an amino acid sequence referred to as a "degron". DEAD-box RNA helicases mediate all aspects of RNA metabolism, contributing to cellular fitness. However, the mechanism by which abiotic-stress modulation of protein stability regulates bacterial helicase abundance has not been extensively characterized. Here, we provide in vivo evidence that proteolytic degradation of the cyanobacterial DEAD-box RNA helicase CrhR is conditional, being initiated by a temperature upshift from 20 to 30 °C in the model cyanobacterium, Synechocystis sp. PCC 6803. We show degradation requires a unique, highly conserved, inherently bipartite degron located in the C-terminal extension found only in CrhR-related RNA helicases in the phylum Cyanobacteria. However, although necessary, the degron is not sufficient for proteolysis, as disruption of RNA helicase activity and/or translation inhibits degradation. These results suggest a positive feedback mechanism involving a role for CrhR in expression of a crucial factor required for degradation. Furthermore, AlphaFold structural prediction indicated the C-terminal extension is a homodimerization domain with homology to other bacterial RNA helicases, and mass photometry data confirmed that CrhR exists as a dimer in solution at 22 °C. These structural data suggest a model wherein the CrhR degron is occluded at the dimerization interface but could be exposed if dimerization was disrupted by nonpermissive conditions.

Genera specific distribution of DEAD-box RNA helicases in cyanobacteria.

Although RNA helicases are essentially ubiquitous and perform roles in all stages of RNA metabolism, phylogenetic analysis of the DEAD (Asp-Glu-Ala-Asp)-box RNA helicase family in a single phylum has not been performed. Here, we performed a phylogenetic analysis on DEAD-box helicases from all currently available cyanobacterial genomes, comprising a total of 362 helicase protein sequences from 280 strains. DEAD-box helicases belonging to three distinct clades were observed. Two clades, the CsdA (cold shock DEAD-box A)-like and RhlE (RNA helicase E)-like helicases, cluster with the homologous proteins from Escherichia coli. The third clade, the CrhR (cyanobacterial RNA helicase Redox)-like helicases, is unique to cyanobacteria and characterized by a conserved sequence motif in the C-terminal extension. Restricted distribution is observed across cyanobacterial diversity with respect to both helicase type and strain. CrhR-like and CsdA-like helicases essentially never occur together, while RhlE always occurs with either a CrhR-like or CsdA-like helicase. CrhR-like and RhlE-like proteins occurred in filamentous cyanobacteria of the orders Nostocales, Oscillatoriales and Synechococcales. Similarly, CsdA- and RhlE-like proteins are restricted to unicellular cyanobacteria of the genera Cyanobium and Synechococcus. In addition, the unexpected occurrence of RhlE in two Synechococcus strains suggests recent acquisition and evolutionary divergence. This study, therefore, raises physiological and evolutionary questions as to why DEAD-box RNA helicases encoded in cyanobacterial lineages display restricted distributions, suggesting niches that require either CrhR or CsdA RNA helicase activity but not both. Extensive conservation of gene synteny surrounding the previously described rimO-crhR operon is also observed, indicating a role in the maintenance of photosynthesis. The analysis provides insights into the evolution, origin and dissemination of sequences within a single gene family to yield divergent functional roles.