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.

FGD5 regulates endothelial cell PI3 kinase-ß to promote neo-angiogenesis.

Angiogenesis is required in embryonic development and tissue repair in the adult. Vascular endothelial growth factor (VEGF) initiates angiogenesis, and VEGF or its receptor is targeted therapeutically to block pathological angiogenesis. Additional pro-angiogenic cues, such as CXCL12 acting via the CXCR4 receptor, co-operate with VEGF/VEGFR2 to cue vascular patterning. We studied the role of FGD5, an endothelial Rho GTP/GDP exchange factor (RhoGEF), to regulate CXCR4-dependent signals in the endothelial cell (EC). Patient-derived renal cell carcinomas produce a complex milieu of growth factors that stimulated sprouting angiogenesis and endothelial tip cell differentiation ex vivo that was blocked by EC FGD5 loss. In a simplified model, CXCL12 augmented sprouting and tip gene expression under conditions where VEGF was limiting. CXCL12-stimulated tip cell differentiation was dependent on PI3 kinase (PI3K)-ß activity. Knockdown of EC FGD5 abolished CXCR4 signaling to PI3K-ß and Akt. Further, inhibition of Rac1, a Rho GTPase required for PI3K-ß activity, recapitulated the signaling defects of FGD5 deficiency, suggesting that FGD5 may regulate PI3K-ß activity through Rac1. Overexpression of a RhoGEF deficient, Dbl domain-deleted FGD5 mutant reduced CXCL12-stimulated Akt phosphorylation and failed to rescue PI3K signaling in native FGD5-deficient EC, indicating that FGD5 RhoGEF activity is required for FDG5 function. Endothelial expression of mutant PI3K-ß with an inactivated Rho binding domain confirmed that CXCL12-stimulated PI3K activity in EC requires Rac1-GTP co-regulation. Together, this data identify the role of FGD5 to generate Rac1-GTP to regulate pro-angiogenic CXCR4-dependent PI3K-ß signaling in EC. Inhibition of FGD5 activity may complement current angiogenesis inhibitor drugs.

SMORE: Synteny Modulator of Repetitive Elements.

Several families of multicopy genes, such as transfer ribonucleic acids (tRNAs) and ribosomal RNAs (rRNAs), are subject to concerted evolution, an effect that keeps sequences of paralogous genes effectively identical. Under these circumstances, it is impossible to distinguish orthologs from paralogs on the basis of sequence similarity alone. Synteny, the preservation of relative genomic locations, however, also remains informative for the disambiguation of evolutionary relationships in this situation. In this contribution, we describe an automatic pipeline for the evolutionary analysis of such cases that use genome-wide alignments as a starting point to assign orthology relationships determined by synteny. The evolution of tRNAs in primates as well as the history of the Y RNA family in vertebrates and nematodes are used to showcase the method. The pipeline is freely available.