Sinorhizobium meliloti BR-bodies promote fitness during host colonization.

Biomolecular condensates, such as the nucleoli or P-bodies, are non-membrane-bound assemblies of proteins and nucleic acids that facilitate specific cellular processes. Like eukaryotic P-bodies, the recently discovered bacterial ribonucleoprotein bodies (BR-bodies) organize the mRNA decay machinery, yet the similarities in molecular and cellular functions across species have been poorly explored. Here, we examine the functions of BR-bodies in the nitrogen-fixing endosymbiont Sinorhizobium meliloti, which colonizes the roots of compatible legume plants. Assembly of BR-bodies into visible foci in S. meliloti cells requires the C-terminal intrinsically disordered region (IDR) of RNase E, and foci fusion is readily observed in vivo, suggesting they are liquid-like condensates that form via mRNA sequestration. Using Rif-seq to measure mRNA lifetimes, we found a global slowdown in mRNA decay in a mutant deficient in BR-bodies, indicating that compartmentalization of the degradation machinery promotes efficient mRNA turnover. While BR-bodies are constitutively present during exponential growth, the abundance of BR-bodies increases upon cell stress, whereby they promote stress resistance. Finally, using Medicago truncatula as host, we show that BR-bodies enhance competitiveness during colonization and appear to be required for effective symbiosis, as mutants without BR-bodies failed to stimulate plant growth. These results suggest that BR-bodies provide a fitness advantage for bacteria during infection, perhaps by enabling better resistance against the host immune response.

Measuring mRNA Decay in Budding Yeast Using Single Molecule FISH.

Cellular mRNA levels are determined by the rates of mRNA synthesis and mRNA decay. Typically, mRNA degradation kinetics are measured on a population of cells that are either chemically treated or genetically engineered to inhibit transcription. However, these manipulations can affect the mRNA decay process itself by inhibiting regulatory mechanisms that govern mRNA degradation, especially if they occur on short time-scales. Recently, single molecule fluorescent in situ hybridization (smFISH) approaches have been implemented to quantify mRNA decay rates in single, unperturbed cells. Here, we provide a step-by-step protocol that allows quantification of mRNA decay in single Saccharomyces cerevisiae using smFISH. Our approach relies on fluorescent labeling of single cytoplasmic mRNAs and nascent mRNAs found at active sites of transcription, coupled with mathematical modeling to derive mRNA half-lives. Commercially available, single-stranded smFISH DNA oligonucleotides (smFISH probes) are used to fluorescently label mRNAs followed by the quantification of cellular and nascent mRNAs using freely available spot detection algorithms. Our method enables quantification of mRNA decay of any mRNA in single, unperturbed yeast cells and can be implemented to quantify mRNA turnover in a variety of cell types as well as tissues.

Method for measuring mRNA decay rate in Saccharomyces cerevisiae.

Eukaryotic mRNA degradation is an essential aspect of gene regulation. Properly turning off transcript generation ensures that protein synthesis does not occur indefinitely. By ensuring that all mRNAs are destroyed, cells can adapt quickly to changing physiological and environmental conditions. Eukaryotic cytoplasmic mRNA degradation is predominately initiated by removal of the poly(A) tail at the 3' end (deadenylation). Following deadenylation, either the mRNA is degraded in a 3'-5' manner or the cap is removed and the mRNA is degraded 5'-3' (reviewed in Coller and Parker, 2004). Determining mRNA decay rates, as indicated by mRNA half-life, is vital to understand how mRNA stability is modulated under various physiological conditions.