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.

The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions.

Bacterial ribonucleoprotein bodies (BR-bodies) are non-membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here, we define the protein components of BR-bodies through enrichment of the bodies followed by mass spectrometry-based proteomic analysis. We find 111 BR-body-enriched proteins showing that BR-bodies are more complex than previously assumed. We identify five BR-body-enriched proteins that undergo RNA-dependent phase separation in vitro with a complex network of condensate mixing. We observe that some RNP condensates co-assemble with preferred directionality, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body-associated proteins have the capacity to dissolve the condensate. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing.

RNase E biomolecular condensates stimulate PNPase activity.

Bacterial Ribonucleoprotein bodies (BR-bodies) play an essential role in organizing RNA degradation via phase separation in the cytoplasm of bacteria. BR-bodies mediate multi-step mRNA decay through the concerted activity of the endoribonuclease RNase E coupled with the 3'-5' exoribonuclease Polynucleotide Phosphorylase (PNPase). In vivo, studies indicated that the loss of PNPase recruitment into BR-bodies led to a significant build-up of RNA decay intermediates in Caulobacter crescentus. However, it remained unclear whether this is due to a lack of colocalized PNPase and RNase E within BR-bodies or whether PNPase's activity is stimulated within the BR-body. We reconstituted RNase E's C-terminal domain with PNPase towards a minimal BR-body in vitro to distinguish these possibilities. We found that PNPase's catalytic activity is accelerated when colocalized within the RNase E biomolecular condensates, partly due to scaffolding and mass action effects. In contrast, disruption of the RNase E-PNPase protein-protein interaction led to a loss of PNPase recruitment into the RNase E condensates and a loss of ribonuclease rate enhancement. We also found that RNase E's unique biomolecular condensate environment tuned PNPase's substrate specificity for poly(A) over poly(U). Intriguingly, a critical PNPase reactant, phosphate, reduces RNase E phase separation both in vitro and in vivo. This regulatory feedback ensures that under limited phosphate resources, PNPase activity is enhanced by recruitment into RNase E's biomolecular condensates.

Initiator AUGs Are Discriminated from Elongator AUGs Predominantly through mRNA Accessibility in C. crescentus.

The initiation of translation in bacteria is thought to occur upon base pairing between the Shine-Dalgarno (SD) site in the mRNA and the anti-SD site in the rRNA. However, in many bacterial species, such as Caulobacter crescentus, a minority of mRNAs have SD sites. To examine the functional importance of SD sites in C. crescentus, we analyzed the transcriptome and found that more SD sites exist in the coding sequence than in the preceding start codons. To examine the function of SD sites in initiation, we designed a series of mutants with altered ribosome accessibility and SD content in translation initiation regions (TIRs) and in elongator AUG regions (EARs). A lack of mRNA structure content is required for initiation in TIRs, and, when introduced into EARs, can stimulate initiation, thereby suggesting that low mRNA structure content is a major feature that is required for initiation. SD sites appear to stimulate initiation in TIRs, which generally lack structure content, but SD sites only stimulate initiation in EARs if RNA secondary structures are destabilized. Taken together, these results suggest that the difference in secondary structure between TIRs and EARs directs ribosomes to start codons where SD base pairing can tune the efficiency of initiation, but SDs in EARs do not stimulate initiation, as they are blocked by stable secondary structures. This highlights the importance of studying translation initiation mechanisms in diverse bacterial species. IMPORTANCE Start codon selection is an essential process that is thought to occur via the base pairing of the rRNA to the SD site in the mRNA. This model is based on studies in E. coli, yet whole-genome sequencing revealed that SD sites are absent at start codons in many species. By examining the transcriptome of C. crescentus, we found more SD-AUG pairs in the CDS of mRNAs than preceding start codons, yet these internal sites do not initiate. Instead, start codon regions have lower mRNA secondary structure content than do internal SD-AUG regions. Therefore, we find that start codon selection is not controlled by the presence of SD sites, which tune initiation efficiency, but by lower mRNA structure content surrounding the start codon.

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.

Roles of liquid-liquid phase separation in bacterial RNA metabolism.

While bacteria typically lack membrane bound organelles, the mechanisms of subcellular organization have been unclear. Bacteria have recently been found to harbor membraneless organelles containing enzymes of many biochemical pathways. These organelles, called biomolecular condensates, have been found to commonly form through the process of liquid-liquid phase separation and are typically enriched in nucleic acid binding proteins. Interestingly, eukaryote and bacterial transcription and RNA decay machinery have been found to form biomolecular condensates. Additionally, DEAD Box ATPases from eukaryotes and bacteria have also been found to modulate biomolecular condensates. The shared ability of RNA metabolic enzymes to assemble into biomolecular condensates across domains suggests that this mode of subcellular organization aids in the control of RNA metabolism.

Procerain B, a cysteine protease from Calotropis procera, requires N-terminus pro-region for activity: cDNA cloning and expression with pro-sequence.

We have previously reported isolation and characterization of a novel plant cysteine protease, Procerain B, from the latex of Calotropis procera. Our initial attempts for active recombinant Procerain B in Escherichiacoli expression system was not successful. The reason for inactive enzyme production was attributed to the absence of 5' pro-region in the Procerain B cDNA that may be involved in proper folding and production of mature active protein. The current manuscript reports the cloning of full length Procerain B for the production of the active protein. The complete cDNA sequence of Procerain B with pro-region sequence was obtained by using RNA ligase mediated rapid amplification of 5' cDNA ends (RLM-RACE). The N-terminus pro-sequence region consists of 127 amino acids and characterized as the member of inhibitory I29 family. Further the three dimensional structure of full length Procerain B was modelled by homology modelling using X-ray crystal structure of procaricain (PDB ID: 1PCI). N-terminus pro-sequence of full length Procerain B runs along the active site cleft. Full length Procerain B was expressed in prokaryotic system and activated in vitro at pH 4.0. This is the first study reporting the production of active recombinant cysteine protease from C.procera.