BR-Bodies Provide Selectively Permeable Condensates that Stimulate mRNA Decay and Prevent Release of Decay Intermediates

Abstract

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.

Keywords: BR-bodies; RNP granules; Ribonuclease E; biomolecular condensates; mRNA decay; phase separation; ribonucleoprotein.

Figure 1.. RNase E cleavage is needed for rapid mRNA decay in C. crescentus.

A.) Cartoon of RNA degradosome mediated mRNA decay. The C. crescentus RNase E (black) degradosome components are shown in colored circles. The mRNA containing a 5’ terminal phosphate (red star) is shown in red. The first “entry” step of mRNA decay pathway is the rate limiting step performed by RNase E endonuclease cleavage. After initial cleavage, further decay occurs by 3’ to 5’ exoribonucleases. After exo decay into short RNA oligos, oligoRNases convert the small oligos into nucleotide mono-phosphates to complete decay. B.) RNA-seq measurement of wild type (WT, JS38) or active site mutant RNase E variant (ASM, JS299) after treatment with 200 μg/mL rifampicin for the indicated amount of time. Each row represents a different transcript whose RNA level is normalized to the level in untreated cells. Grey color represents a value too low to be determined. C.) Box-plots of RNA half-lives based on the data in panel A. Two-tailed T-test between FL and ASM mRNA decay rates (n=465 mRNAs) with uneven variance yield a p-value of 1.3 × 10⁻²³.

Figure 2.. BR-bodies are enriched for mRNAs.

A.) Differential centrifugation-based enrichment of BR-bodies. An aliquot of the cell lysate and enriched BR-bodies were RNA-extracted and libraries for RNA-seq were generated and sequenced. Red lines represent enriched mRNAs, black lines represent excluded RNAs. In the magnified view of the BR-body, RNase E tetramers are shown in green, with degradosome components as red circles, and Arg-rich regions in blue. B.) Normalized read density for the parB operon (top) and for a tRNA gene (bottom). C.) Fraction of RNA-seq reads mapping to each RNA category from whole-cell lysate (WCL, blue) or BR-body enriched samples (orange). Values are averages ± σ. D.) Log₂ ratio of the BR-body enriched sample RPKM compared to the cell lysate RPKM values of two biological replicates. RNAs with p-values >0.05 are colored in red Fraction of total reads mapping to non-coding RNA (blue) and mRNA (orange) in the lysate and in the enriched BR-body samples. p-values were determined using a negative binomial model in edgeR. E.) Box plots of BR-body enrichment for RNAs of each RNA category.

Figure 3.. Long poorly structured RNAs have enhanced association with BR-bodies.

A.) Log₂ ratio of the BR-body enriched RPKM vs cell lysate RPKM. RNAs were divided into bins based on their length and the medians of enrichment are highlighted (black bars). Conserved ncRNAs are indicated. Right, plot of RNA size distributions for BR-body enriched, depleted, or neither enriched nor depleted. Two-tailed T-tests with unequal variances resulting in p-values less than 0.05 are highlighted with asterisks. n=number of RNAs for depleted (n=51), neither (n=152), and enriched (n=2046). P-values are 2.6 × 10⁻⁶ (D to N), 1.5 × 10⁻⁵³ (E to N), and 1.0 × 10⁻³⁹ (D to E). B.) In vitro RNase E CTD-YFP biomolecular condensate recruitment assay with the indicated labeled RNAs. CTD-YFP was incubated with the indicated Cy-5 RNAs grouped by length before droplet formation was induced . Condensate assays were either performed in the presence of folded RNAs with Mg²⁺ (left) or with heat denatured RNAs lacking Mg²⁺ in the presence of PEG(8000) crowding agent (right). Quantification of droplet area is shown for each RNA on the right in the folded and denatured conditions. On the bottom, each sample is quantified compared to the protein only condition. Asterisks signify samples with p-values < 0.001 from one-way ANOVA. n displayed is the number of droplets analyzed.

Figure 4.. Ribosomes and the nucleoid are excluded from BR-bodies.

A.) Dual labeled strain expressing ribosomal protein L1-eYFP (yellow) and BR-body scaffold RNase E (RNE)-eCFP (blue) as the sole copies (JS350). Red line is the source of the signal intensity plot for both fluorophores on right. B.) Super-resolution images of RNE-eYFP (green) and L1-PAmCherry (magenta) (JS545). White cell outlines from phase image. Red arrow indicates position of BR-body. In merged image, overlapping eYFP/PAmCherry signal is displayed in white. Scale bar is 500nm. C.) ASM-eYFP (JS299) (top), CTD-eYFP (JS230) middle, and NTD-eYFP (JS231) bottom colocalized with the DAPI stained nucleoid. Signal intensity plot for both fluorophores on right was generated from the red line. For E. coli cells, the black bar denotes the ribosomes rich cell pole which is known to exclude the nucleoid (Bakshi et al., 2012). Scale bars are 2 μm.

Figure 5.. RNase E endonuclease activity limits rsaA mRNA colocalization in BR-bodies.

A.) rsaA mRNA weakly colocalizes with BR-bodies. Left, rsaA mRNA visualization in fixed cells by mRNA FISH or by the Ms2 tagged system in living cells. Fluorescein mRNA FISH probes were probed with either RNE-mCherry (JS403) or mCherry-PopZ (JP369) as a negative control. In live cells the Ms2-coat protein double mutant (Ms2DM)-mCherry fusion with an array of 96 tandem Ms2 RNA hairpins fused to the 3’ end of the rsaA gene was imaged with RNE-msfGFP (JS287). Right, quantitation of the fraction of rsaA mRNA foci colocalized with RNase E or PopZ foci. B.) Left, mRNA FISH (Quasar 670 fluorophore) in either wild type RNE-eYFP (JS38) or with ASM-eYFP (JS299). Right, quantitation of the fraction of rsaA mRNA foci colocalized with RNase E or ASM foci.

Figure 6.. BR-bodies accelerate initial cleavage and exonucleolytic steps of mRNA decay.

A.) RNA-seq measurement of wild type (JS38), NTD truncation (JS221), or DBS mutant (JS233) strains after treatment with 200 μg/mL rifampicin for the indicated amount of time. Each row represents a different transcript whose RNA level is normalized to the level in untreated cells. B.) mRNA half-lives for each RNase E mutant across four bins of BR-body enrichment. Only simple mRNAs with a single ORF and TSS are shown. Asterisks indicate samples with p-values ≤0.05 based on a T-test with unequal variance. p-values can be found in Table S1. C.) qRT-PCR (>100nt fragments) and RNA-seq (<50nt fragments) RNA half-life measurements of the rne mRNA for the indicated strains. Each half-life measurement was performed on the same RNA samples split between the two assays.

Figure 7.. Model of BR-body mediated mRNA decay.

As translation levels drop, RNA degradosomes (RNase E catalytic region (green), Arg-rich CTD patches (blue), and degradosome proteins (red) are highlighted) can engage on the mRNA to initiate decay. On the left, is the pathway of decay for soluble mRNA decay where initiation of decay by RNase E (green spheres) and exoribonuclease (red spheres) steps are indicated and can occur slowly. On the right, is the pathway that can occur inside BR-bodies. First, the RNA degradosome and mRNAs phase-separate into a biomolecular condensate via the multivalent interactions with the mRNA and the RNase E CTD. Inside the condensate RNase E endonuclease and degradosome exoribonuclease activity are both accelerated from the high-local concentration. Once the mRNA fragments are cut to a small size to lower valence of the interaction with RNase E’s CTD, the BR-body can dissolve releasing both RNA degradosomes and oligonucleotides that can be converted into nucleotides by oligoribonuclease.