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

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.

α-Proteobacterial RNA Degradosomes Assemble Liquid-Liquid Phase-Separated RNP Bodies.

Ribonucleoprotein (RNP) granules play an important role in organizing eukaryotic mRNA metabolism via liquid-liquid phase separation (LLPS) of mRNA decay factors into membrane-less organelles in the cytoplasm. Here we show that the bacterium Caulobacter crescentus Ribonuclease (RNase) E assembles RNP LLPS condensates that we term bacterial RNP-bodies (BR-bodies), similar to eukaryotic P-bodies and stress granules. RNase E requires RNA to assemble a BR-body, and disassembly requires RNA cleavage, suggesting BR-bodies provide localized sites of RNA degradation. The unstructured C-terminal domain of RNase E is both necessary and sufficient to assemble the core of the BR-body, is functionally conserved in related α-proteobacteria, and influences mRNA degradation. BR-bodies are rapidly induced under cellular stresses and provide enhanced cell growth under stress. To our knowledge, Caulobacter RNase E is the first bacterial protein identified that forms LLPS condensates, providing an effective strategy for subcellular organization in cells lacking membrane-bound compartments.

Regulated Formation of lncRNA-DNA Hybrids Enables Faster Transcriptional Induction and Environmental Adaptation.

Long non-coding (lnc)RNAs, once thought to merely represent noise from imprecise transcription initiation, have now emerged as major regulatory entities in all eukaryotes. In contrast to the rapidly expanding identification of individual lncRNAs, mechanistic characterization has lagged behind. Here we provide evidence that the GAL lncRNAs in the budding yeast S. cerevisiae promote transcriptional induction in trans by formation of lncRNA-DNA hybrids or R-loops. The evolutionarily conserved RNA helicase Dbp2 regulates formation of these R-loops as genomic deletion or nuclear depletion results in accumulation of these structures across the GAL cluster gene promoters and coding regions. Enhanced transcriptional induction is manifested by lncRNA-dependent displacement of the Cyc8 co-repressor and subsequent gene looping, suggesting that these lncRNAs promote induction by altering chromatin architecture. Moreover, the GAL lncRNAs confer a competitive fitness advantage to yeast cells because expression of these non-coding molecules correlates with faster adaptation in response to an environmental switch.

A role for CF1A 3' end processing complex in promoter-associated transcription.

The Cleavage Factor 1A (CF1A) complex, which is required for the termination of transcription in budding yeast, occupies the 3' end of transcriptionally active genes. We recently demonstrated that CF1A subunits also crosslink to the 5' end of genes during transcription. The presence of CF1A complex at the promoter suggested its possible involvement in the initiation/reinitiation of transcription. To check this possibility, we performed transcription run-on assay, RNAP II-density ChIP and strand-specific RT-PCR analysis in a mutant of CF1A subunit Clp1. As expected, RNAP II read through the termination signal in the temperature-sensitive mutant of clp1 at elevated temperature. The transcription readthrough phenotype was accompanied by a decrease in the density of RNAP II in the vicinity of the promoter region. With the exception of TFIIB and TFIIF, the recruitment of the general transcription factors onto the promoter, however, remained unaffected in the clp1 mutant. These results suggest that the CF1A complex affects the recruitment of RNAP II onto the promoter for reinitiation of transcription. Simultaneously, an increase in synthesis of promoter-initiated divergent antisense transcript was observed in the clp1 mutant, thereby implicating CF1A complex in providing directionality to the promoter-bound polymerase. Chromosome Conformation Capture (3C) analysis revealed a physical interaction of the promoter and terminator regions of a gene in the presence of a functional CF1A complex. Gene looping was completely abolished in the clp1 mutant. On the basis of these results, we propose that the CF1A-dependent recruitment of RNAP II onto the promoter for reinitiation and the regulation of directionality of promoter-associated transcription are accomplished through gene looping.

Caulobacter crescentus RNase E condensation contributes to autoregulation and fitness.

RNase E is the most common RNA decay nuclease in bacteria, setting the global mRNA decay rate and scaffolding formation of the RNA degradosome complex and BR-bodies. To properly set the global mRNA decay rate, RNase E from Escherichia coli and neighboring γ-proteobacteria were found to autoregulate RNase E levels via the decay of its mRNA's 5' untranslated region (UTR). While the 5' UTR is absent from other groups of bacteria in the Rfam database, we identified that the α-proteobacterium Caulobacter crescentus RNase E contains a similar 5' UTR structure that promotes RNase E autoregulation. In both bacteria, the C-terminal intrinsically disordered region (IDR) of RNase E is required for proper autoregulation to occur, and this IDR is also necessary and sufficient for RNase E to phase-separate, generating BR-bodies. Using in vitro purified RNase E, we find that the IDR's ability to promote phase separation correlates with enhanced 5' UTR cleavage, suggesting that phase separation of RNase E with the 5' UTR enhances autoregulation. Finally, using growth competition experiments, we find that a strain capable of autoregulation rapidly outcompetes a strain with a 5' UTR mutation that cannot autoregulate, suggesting autoregulation promotes optimal cellular fitness.

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.

Caulobacter crescentus RNase E condensation contributes to autoregulation and fitness.

RNase E is the most common RNA decay nuclease in bacteria, setting the global mRNA decay rate and scaffolding formation of the RNA degradosome complex and BR-bodies. To properly set the global mRNA decay rate, RNase E from Escherichia coli and neighboring γ-proteobacteria were found to autoregulate RNase E levels via the decay of its mRNA's 5' UTR. While the 5' UTR is absent from other groups of bacteria in the Rfam database, we identified that the α-proteobacterium Caulobacter crescentus RNase E contains a similar 5' UTR structure that promotes RNase E autoregulation. In both bacteria, the C-terminal IDR of RNase E is required for proper autoregulation to occur, and this IDR is also necessary and sufficient for RNase E to phase-separate, generating BR-bodies. Using in vitro purified RNase E, we find that the IDR's ability to promote phase-separation correlates with enhanced 5' UTR cleavage, suggesting that phase-separation of RNase E with the 5' UTR enhances autoregulation. Finally, using growth competition experiments we find that a strain capable of autoregulation rapidly outcompetes a strain with a 5' UTR mutation that cannot autoregulate, suggesting autoregulation promotes optimal cellular fitness.

Evidence for a complex of transcription factor IIB with poly(A) polymerase and cleavage factor 1 subunits required for gene looping.

Gene looping, defined as the interaction of the promoter and the terminator regions of a gene during transcription, requires transcription factor IIB (TFIIB). We have earlier demonstrated association of TFIIB with the distal ends of a gene in an activator-dependent manner (El Kaderi, B., Medler, S., Raghunayakula, S., and Ansari, A. (2009) J. Biol. Chem. 284, 25015-25025). The presence of TFIIB at the 3' end of a gene required its interaction with cleavage factor 1 (CF1) 3' end processing complex subunit Rna15. Here, employing affinity chromatography and glycerol gradient centrifugation, we show that TFIIB associates with poly(A) polymerase and the entire CF1 complex in yeast cells. The factors required for general transcription such as TATA-binding protein, RNA polymerase II, and TFIIH are not a component of the TFIIB complex. This holo-TFIIB complex was resistant to MNase digestion. The complex was observed only in the looping-competent strains, but not in the looping-defective sua7-1 strain. The requirement of Rna15 in gene looping has been demonstrated earlier. Here we provide evidence that poly(A) polymerase (Pap1) as well as CF1 subunits Rna14 and Pcf11 are also required for loop formation of MET16 and INO1 genes. Accordingly, cross-linking of TFIIB to the 3' end of genes was abolished in the mutants of Pap1, Rna14, and Pcf11. We further show that in sua7-1 cells, where holo-TFIIB complex is not formed, the kinetics of activated transcription is altered. These results suggest that a complex of TFIIB, CF1 subunits, and Pap1 exists in yeast cells. Furthermore, TFIIB interaction with the CF1 complex and Pap1 is crucial for gene looping and transcriptional regulation.

Crosstalk of promoter and terminator during RNA polymerase II transcription cycle.

The transcription cycle of RNAPII is comprised of three consecutive steps; initiation, elongation and termination. It has been assumed that the initiation and termination steps occur in spatial isolation, essentially as independent events. A growing body of evidence, however, has challenged this dogma. First, factors involved in initiation and termination exhibit both a genetic and a physical interaction during transcription. Second, the initiation and termination factors have been found to occupy both ends of a transcribing gene. Third, physical interaction of initiation and termination factors occupying distal ends of a gene sometime results in the entire terminator region of a genes looping back and contact its cognate promoter, thereby forming a looped gene architecture during transcription. A logical interpretation of these findings is that the initiation and termination steps of transcription do not occur in isolation. There is extensive communication of factors occupying promoter and terminator ends of a gene during transcription cycle. This review entails a discussion of the promoter-terminator crosstalk and its implication in the context of transcription.

Differential Centrifugation to Enrich Bacterial Ribonucleoprotein Bodies (BR bodies) from Caulobacter crescentus.

Bacterial RNP bodies (BR bodies) contain the mRNA decay machinery, but the collection of associated RNAs and proteins are poorly defined. Here, we present a protocol for the rapid differential centrifugation-based enrichment of BR bodies from Caulobacter crescentus cells. As native BR bodies are highly labile and dissociate by degrading internal mRNAs, an active site mutant of RNase E, which blocks dissolution of BR bodies, allows BR-body stabilization during enrichment. For complete details on the use and execution of this protocol, please refer to Al-Husini et al. (2020).

Methodology for Ribosome Profiling of Key Stages of the Caulobacter crescentus Cell Cycle.

Bacterial cell division is the result of a productive round of the cell cycle to yield two daughter cells. The cell cycle is highly coordinated in Caulobacter crescentus where it is driven by a cell cycle gene-regulatory network that coordinates gene expression with the major cell cycle events such as chromosome replication and cell division. Recent ribosomes profiling data showed that 484 genes undergo changes in translation efficiency during the cell cycle, suggesting a broad role for translational control in cell cycle regulation. In this chapter, we focus on how to perform ribosome profiling to measure the translation efficiency across cellular mRNAs at key stages in the Caulobacter cell cycle. This methodology relies on the high-yield ludox gradient synchronization of Caulobacter cells followed by ribosome profiling to measure ribosome density and total RNA-seq to measure mRNA levels.

Genomewide Analysis of Clp1 Function in Transcription in Budding Yeast.

In budding yeast, the 3' end processing of mRNA and the coupled termination of transcription by RNAPII requires the CF IA complex. We have earlier demonstrated a role for the Clp1 subunit of this complex in termination and promoter-associated transcription of CHA1. To assess the generality of the observed function of Clp1 in transcription, we tested the effect of Clp1 on transcription on a genomewide scale using the Global Run-On-Seq (GRO-Seq) approach. GRO-Seq analysis showed the polymerase reading through the termination signal in the downstream region of highly transcribed genes in a temperature-sensitive mutant of Clp1 at elevated temperature. No such terminator readthrough was observed in the mutant at the permissive temperature. The poly(A)-independent termination of transcription of snoRNAs, however, remained unaffected in the absence of Clp1 activity. These results strongly suggest a role for Clp1 in poly(A)-coupled termination of transcription. Furthermore, the density of antisense transcribing polymerase upstream of the promoter region exhibited an increase in the absence of Clp1 activity, thus implicating Clp1 in promoter directionality. The overall conclusion of these results is that Clp1 plays a general role in poly(A)-coupled termination of RNAPII transcription and in enhancing promoter directionality in budding yeast.