Biology subjects

Schrader, J. M.

Publications and source records attributed to Schrader, J. M..

At least 19 recordsLinked to original sources

bioRxiv2026

BR-bodies link RNA homeostasis to stress tolerance and intracellular fitness in Brucella

Bacterial gene expression depends on the coordinated regulation of RNA synthesis, processing, and decay. The conserved RNA degradosome scaffold, Ribonuclease E (RNase E), assembles into phase-separated bacterial ribonucleoprotein bodies (BR-bodies) through its C-terminal intrinsically disordered region (IDR). This IDR also scaffolds recruitment of degradosome client proteins that carry out post-transcriptional gene regulation. Whether BR-bodies regulate virulence programs to support infection, however, is largely undefined. We show that RNase E of the intracellular pathogen Brucella ovis forms RNA-dependent condensates in vivo and phase-separates with RNA in vitro, with the IDR necessary and sufficient for BR-body assembly. Deleting the IDR (rne({Delta}IDR)) did not impair growth but sensitized Brucella to host-relevant oxidative and cell-envelope stressors. Transcriptome-wide profiling that simultaneously resolved mRNA decay and processing revealed that BR-bodies primarily accelerate mRNA turnover while stabilizing a distinct subset of transcripts. Notably, BR-bodies control the processing and levels of virB type IV secretion system (T4SS) mRNA and the turnover of its key activators, linking condensate-based RNA regulation to a core virulence pathway. Consistent with virB dysregulation, the rne({Delta}IDR) mutant was severely attenuated in mammalian macrophages. To test whether phase separation is sufficient for BR-body function, we replaced the B. ovis IDR with the highly divergent Caulobacter crescentus IDR. This chimera assembled BR-bodies but rescued fitness incompletely, fully restoring oxidative-stress resistance but not cell-envelope stress resistance or intracellular fitness. BR-bodies therefore link RNA metabolism to Brucella stress resistance and infection, and their full function requires both phase separation and additional native IDR-specific activities such as degradosome interactions. ImportanceBiomolecular condensates are non-membrane bound organelles that organize biological processes across all domains of life, but their role in bacterial infection is not well characterized. We show that Brucella, an intracellular bacterial pathogen and causative agent of the disease brucellosis, relies on biomolecular condensates called BR-bodies to control RNA stability and regulate gene expression. Without BR-bodies, Brucella is sensitized to host-relevant chemical stresses, cannot properly regulate essential infection machinery, and has severely diminished fitness within mammalian host cells. These results indicate that control of RNA levels by biomolecular condensates is required for Brucella survival within animal hosts, providing evidence that phase separation is a fundamental mechanism underlying Brucella adaptation to the hostile environment encountered during infection.

microbiology↗

bioRxiv2026

The E. coli DEAD box ATPase CsdA, is a NAD+ capped RNA binding protein

5' nicotinamide adenine dinucleotide (NAD+) caps are one of the most common metabolites derived non-canonical caps reported on bacterial RNAs. Multiple decapping proteins are known to regulate the stability of NAD+ capped transcripts. However, no other proteins have been identified that preferentially interact with these NAD+ caps, and mechanistic details of the cap-dependent recognition remain poorly understood. Using an affinity capture approach, we identified multiple E. coli proteins that selectively recognize NAD+ caps, including the ATP-dependent RNA helicase, CsdA. CsdA preferentially interacts directly with NAD+ capped RNAs and can discriminate between 5' NAD+ capped and 5' triphosphate end transcripts. Binding to NAD+ capped RNA versus 5' triphosphate RNA more greatly enhances the ATPase activity of CsdA and the presence of NAD+ caps on transcripts modulates the ability of CsdA to form RNA condensates. Furthermore, we find that CsdA enhances the decapping activity of the NADH hydrolase NudC, suggesting CsdA plays a role in regulating the degradation of NAD+ capped transcripts. CsdA is the first identified NAD+ cap reader protein and its preference for binding NAD+ capped RNA provides a mechanism by which E. coli cells link RNA stability to the identity of the 5' cap. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/730015v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@115a057org.highwire.dtl.DTLVardef@d1c2c6org.highwire.dtl.DTLVardef@14a6d2dorg.highwire.dtl.DTLVardef@145ccff_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Defining the role of RNase E in the mycobacterial degradosome-like network

mRNA degradation is a fundamentally important process that is regulated in response to stress in the globally important pathogen Mycobacterium tuberculosis. Several mycobacterial ribonucleases (RNases) are hypothesized to function together to coordinate mRNA degradation, but the interactions among them are mostly undefined. One of the rate-limiting enzymes, RNase E, contains intrinsically disordered regions (IDRs). Here, we have defined the interactions among major mycobacterial mRNA degradation enzymes and identified the functions of the two IDRs of RNase E in the nonpathogenic model Mycolicibacterium smegmatis. We found that the two IDRs differentially impact mRNA degradation rates in vivo but are largely functionally redundant in their impact on steady-state transcript abundance. In vitro, the IDRs are uninvolved in catalysis but play major roles in RNA binding and interactions with other mRNA degradation enzymes, namely PNPase, RNase J, and RhlE1, including stimulation of RhlE1 activity. In vivo, these enzymes localize with RNase E, but its IDRs play only a minor role, suggesting substantial redundancy in subcellular localization mechanisms. Collectively, we propose a degradosome-like network model in mycobacteria, held together by dynamic, transient interactions among RNA degradation enzymes and RNA that can be disrupted during physiologically relevant stress to allow for adaptability.

molecular biology↗

bioRxiv2025

Rif-seq reveals Caulobacter crescentus mRNA decay is globally coordinated with transcription and translation

While transcription and translation have been shown to be coordinated with mRNA decay across various single-gene studies, their global coordination remains poorly defined. Therefore, we performed Rif-seq experiments in C. crescentus to measure genome-wide mRNA lifetimes and analyzed the impact of transcription and translation. Based upon the RNAP elongation speed, we identified that approximately 20% of mRNAs were cotranscriptionally degraded. We generated absolute quantitative estimates of mRNA copy numbers in C. crescentus, a useful systems biology resource, revealing that some gene categories have coordinated mRNA turnover. To investigate translations impact on mRNA decay, we found that translation efficiency measured by ribosome profiling correlates with mRNA lifetime. We compared the 5 P cleavage sites to ribosome occupancy and found that cleavage sites occur preferentially in regions of low ribosome occupancy. Using the translation initiation inhibitor retapamulin, which traps ribosomes at the start codon, and chloramphenicol, which arrests elongating ribosomes, shows that chloramphenicol leads to global mRNA stabilization. Surprisingly, we find that the codon adaptation index is inversely correlated with mRNA lifetime, suggesting slow translation elongation may be stabilizing mRNAs from decay. We confirmed the roles of translation initiation and elongation on mRNA lifetimes by generating synthetic YFP and mCherry reporter mRNAs. Taken together, mRNA decay is globally interconnected with transcription and translation. Highlights[bullet] Quantitative analysis yields absolute mRNA abundance and half-lives for C. crescentus [bullet]Identification of 47 new stable ncRNAs [bullet]Both transcription and translation are globally coordinated with mRNA decay [bullet]Translation initiation and elongation impact mRNA decay through ribosome occupancy

systems biology↗

bioRxiv2025

BR-Bodies Facilitate Adaptive Responses and Survival During Copper Stress in Caulobacter crescentus

Microbes must rapidly adapt to environmental stresses, including toxic heavy metals like copper, by sensing and mitigating their harmful effects. Here, we demonstrate that the phase separation properties of bacterial ribonucleoprotein bodies (BR-bodies) enhance Caulobacter crescentus fitness under copper stress. To uncover the underlying mechanism, we identified two key interactions between copper and the central scaffold of BR-bodies, RNase E. First, biochemical assays and fluorescence microscopy experiments show that reductive chelation of Cu{superscript 2} leads to cysteine oxidation, driving the transition of BR-bodies into more solid-like condensates. Second, tryptophan fluorescence and EPR assays reveal that RNase E binds Cu{superscript 2} at histidine sites, creating a protective microenvironment that prevents mismetallation and preserves PNPase activity. More broadly, this example highlights how metal-condensate interactions can regulate condensate material properties and establish specialized chemical environments that safeguard enzyme function.

microbiology↗

bioRxiv2025

Bacterial IF2's N-terminal IDR drives cold-induced phase separation and promotes fitness during cold stress

Translation initiation factor 2 (IF2) plays an essential role in bacterial cells by delivering the fMet-tRNAfMet to the ribosome pre-initiation complex. IF2 is known to have an N-terminal disordered region which is present across bacterial species, yet its function is not fully understood. Deletion of the IDR in E. coli showed no phenotypes at normal growth temperature (37{degrees}C); however, this IDR was found to be required for growth at cold temperatures (15{degrees}C). Since large IDRs can drive phase separation of various RNA binding proteins into biomolecular condensates, we investigated whether E. coli IF2 could phase separate. We discovered that IF2s N-terminal IDR drives phase separation in E. coli and C. crescentus, suggesting that IF2 condensation is a conserved property. Finally, using E. coli, we found that the IDR strongly drives phase separation in the cold, suggesting IF2 condensates promote fitness during cold stress. HighlightsO_LIIF2s IDR promotes phase separation with RNA. C_LIO_LICold temperature promotes IF2 condensation with RNA. C_LIO_LIIF2s IDR promotes fitness during cold shock. C_LI

microbiology↗

bioRxiv2024

Stress Changes the Bacterial Biomolecular Condensate Material State and Shifts Function from mRNA Decay to Storage

Bacterial ribonucleoprotein bodies (BR-bodies) are dynamic biomolecular condensates that play a pivotal role in RNA metabolism. We investigated how BR-bodies significantly influence mRNA fate by transitioning between liquid- and solid-like states in response to stress. With a combination of single-molecule and bulk fluorescence microscopy, biochemical assays, and quantitative analyses, we determine that BR-bodies promote efficient mRNA decay in a liquid-like condensate during exponential growth. On the other hand, BR-bodies are repurposed from sites of mRNA decay to reservoirs for mRNA storage under stress; a functional change that is enabled by their transition to a more rigid state, marked by reduced internal dynamics, increased molecular density, and prolonged residence time of ribonuclease E. Furthermore, we manipulated ATP levels and translation rates, and we conclude that the accumulation of ribosome-depleted mRNA is a key factor driving BR-body rigification, and that condensate maturation further contributes to this process. Upon nutrient replenishment, stationary-phase BR-bodies disassemble, releasing stored mRNAs for rapid translation, demonstrating that BR-body function is governed by a reversible mechanism for resource management. These findings reveal adaptive strategies by which bacteria regulate RNA metabolism through condensate-mediated control of mRNA decay and storage.

biophysics↗

bioRxiv2024

APEX2 proximity labeling of RNA in bacteria

Rapid spatially controlled methods are needed to investigate RNA localization in bacterial cells. APEX2 proximity labeling was shown to be adaptable to rapid RNA labeling in eukaryotic cells, and through the fusion of APEX2 to different proteins targeted to different subcellular locations, has been useful to identify RNA localization in these cells. Therefore, we adapted APEX2 proximity labeling of RNA to bacterial cells by generating an APEX2 fusion to the RNase E gene, which is necessary and sufficient for BR-body formation. APEX2 fusion is minimally perturbative and RNA can be rapidly labeled on the sub-minute timescale with Alkyne-Phenol, outpacing the rapid speed of mRNA decay in bacteria. Alkyne-Phenol provides flexibility in the overall downstream application with copper catalyzed click-chemistry for downstream applications, such as fluorescent dye-azides or biotin-azides for purification. Altogether, APEX2 proximity labeling of RNA provides a useful method for studying RNA localization in bacteria. MotivationStudies over the past several years have shown that distinct RNAs can be targeted to subcellular locations in bacterial cells. The ability to investigate localized RNAs in bacteria is currently limited to imaging-based approaches or to laborious procedures to isolate ribonucleoprotein complexes by grad-seq, HITS-CLIP, or Rloc-seq. However, a major challenge in studying mRNA localization in bacterial cells is that bacterial mRNAs typically last for only a few minutes in the cell, while experiments to investigate their localization or interaction partners can take much longer. Therefore, rapid methods of studying RNA localization are needed to bridge this technical challenge. HighlightsO_LIAPEX2 proximity labeling can be applied to RNA in bacteria C_LIO_LIAPEX2 RNA labeling reactions occur on the sub-minute timescale. C_LIO_LIAPEX2 workflow requires less material and time than current methods. C_LIO_LIAlkyne-Phenol APEX2 substrate provides flexibility with click-chemistry. C_LI

microbiology↗

bioRxiv2024

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. SignificanceWhile eukaryotes often organize their biochemical pathways in membrane-bound organelles, bacteria generally lack such subcellular structures. Instead, membraneless compartments called biomolecular condensates have recently been found in bacteria to enhance biochemical activities. Bacterial ribonucleoprotein bodies (BR-bodies), as one of the most widespread biomolecular condensates identified to date, assemble the mRNA decay machinery via the intrinsically disordered regions (IDRs) of proteins. However, the implications of such assemblies are unclear. Using a plant-associated symbiont, we show that the IDR of its mRNA degradation protein is necessary for condensate formation. Absence of BR-bodies results in slower mRNA decay and ineffective symbiosis, suggesting that BR-bodies play critical roles in regulating biochemical pathways and promoting fitness during host colonization.

microbiology↗

bioRxiv2024

TFIIB-termination factor interaction affects termination of transcription on a genomewide scale

Apart from its well-established role in initiation of transcription, the general transcription factor TFIIB has been implicated in the termination step as well. The ubiquity of TFIIB involvement in termination as well as mechanistic details of its termination function, however, remains largely unexplored. To determine the prevalence of TFIIBs role in termination, we performed GRO-seq analyses in sua7-1 mutant (TFIIBsua7-1) and the isogenic wild type (TFIIBWT) strains of yeast. Almost a three-fold increase in readthrough of the poly(A)-termination signal was observed in TFIIBsua7-1 mutant compared to the TFIIBWT cells. Of all genes analyzed in this study, nearly 74% genes exhibited a statistically significant increase in terminator readthrough in the mutant. To gain an understanding of the mechanistic basis of TFIIB involvement in termination, we performed mass spectrometry of TFIIB, affinity purified from chromatin and soluble cellular fractions, from TFIIBsua7-1 and TFIIBWT cells. TFIIB purified from the chromatin fraction of TFIIBWT cells exhibited significant enrichment of CF1A and Rat1 termination complexes. There was, however, a drastic decrease in TFIIB interaction with both CF1A and Rat1 termination complexes in TFIIBsua7-1 mutant. ChIP assay revealed that the recruitment of Pta1 subunit of CPF complex, Rna15 subunit of CF1 complex and Rat1 subunit of Rat1 complex registered nearly 90% decline in the mutant over wild type cells. The overall conclusion of these results is that TFIIB affects termination of transcription on a genome-wide scale, and TFIIB-termination factor interaction may play a crucial role in the process.

molecular biology↗

bioRxiv2023

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 {gamma}-proteobacteria were found to autoregulate RNase E levels via the decay of its mRNAs 5 UTR. While the 5 UTR is absent from other groups of bacteria in the Rfam database, we identified that the -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 IDRs 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.

biochemistry↗

bioRxiv2023

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

Bacterial RNP 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 found 111 BR-body enriched proteins, including several RNA binding proteins, many of which are also recruited directly to in vitro reconstituted RNase E droplets, showing BR-bodies are more complex than previously assumed. While most BR-body enriched proteins that were tested cannot phase separate, we identified five that undergo RNA-dependent phase separation in vitro, showing other RNP condensates interface with BR-bodies. RNA degradosome protein clients are recruited more strongly to RNase E droplets than droplets of other RNP condensates, implying that client specificity is largely achieved through direct protein-protein interactions. We observe that some RNP condensates assemble with preferred directionally, 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. Finally, we find that RNA dramatically stimulates the rate of RNase E phase separation in vitro, explaining the dissolution of BR-bodies after cellular mRNA depletion observed previously. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing. HighlightsO_LIBR-body proteomics identified 111 proteins enriched in BR-bodies. C_LIO_LIBR-bodies associate with an interconnected network of RNP condensates. C_LIO_LIBR-body condensation is modulated by its interaction network. C_LIO_LIRNA is required for rapid BR-body condensation. C_LI Graphical AbstractSummary of the BR-body protein interactome. Lines between two protein circles represent a direct interaction.

microbiology↗

bioRxiv2022

Initiator AUGs are discriminated from Elongator AUGs predominantly through mRNA accessibility in C. crescentus

Translation initiation in bacteria is thought to occur upon base-pairing between the Shine-Dalgarno site in the mRNA and anti-Shine-Dalgarno site in the rRNA. However, in many bacterial species, such as Caulobacter crescentus, a minority of mRNAs have Shine-Dalgarno sites. To examine the functional importance of Shine-Dalgarno sites in C. crescentus, we analyzed the transcriptome and found more Shine-Dalgarno sites exist in the coding sequence than preceding start codons. To examine the function of Shine-Dalgarno sites in initiation we designed a series of mutants with altered ribosome accessibility and Shine-Dalgarno content in translation initiation regions (TIRs) and elongator AUG regions (EARs). A lack of mRNA structure content is required for initiation in TIRs, and when introduced into EARs, can stimulate initiation, suggesting that low mRNA structure content is a major feature 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, this suggests 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.

microbiology↗

bioRxiv2022

RNase E biomolecular condensates stimulate PNPase activity

Bacterial Ribonucleoprotein bodies (BR-bodies) play an essential role in organizing RNA degradation via liquid-liquid 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 exonuclease Polynucleotide Phosphorylase (PNPase). Our past 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. We reconstituted RNase Es C-terminal domain together with PNPase to understand how RNase E biomolecular condensates can tailor the functions of PNPase. We found that PNPase catalytic activity is accelerated when colocalized with the RNase E biomolecular condensates. In contrast, disruption of the RNase E-PNPase protein-protein interaction led to a loss of PNPase recruitment into the BR-bodies and a loss of ribonuclease rate enhancement. We also found that BR-bodies could enhance the decay of select RNA substrates, as we observed a 3.4-fold enhancement of polyadenylic acid (poly(A)) degradation and no impact upon poly(U) degradation. Our investigation into the origins of the 3.4-fold rate enhancement for poly(A) decay indicates a combination of scaffolding and mass action effects impact due to the concentrated biomolecular condensate environment accelerating RNA decay. Consistent with our past in vivo work, these studies suggest BR-bodies are sites of accelerated RNA decay that can shape the available transcriptome.

biochemistry↗

bioRxiv2022

Rif-Correct, a software tool for bacterial mRNA half-life estimation

BackgroundGenome-wide measurement of bacterial mRNA lifetimes using the antibiotic rifampicin has provided new insights into the control of bacterial mRNA decay. However, for long polycistronic mRNAs, the estimation of mRNA half-life can be confounded with transcriptional runoff caused by rifampicins inhibition of initiating RNA polymerases, and not elongating RNA polymerases. ResultsWe present the Rif-correct software package, a free open-source tool that uses transcriptome models of transcript architecture to allow for more accurate mRNA half-life estimates that account for the transcriptional runoff. Rif-correct is implemented as a customizable python script that allows for users to control all the analysis parameters to achieve improved mRNA half-life estimates. ConclusionsRif-correct is the first free open-source computational analysis pipeline for Rif-seq dataset mRNA half-life estimation. It is simple to run, fast, and easy to run with a detailed instruction manual and example datasets.

microbiology↗

bioRxiv2021

ΔGunfold leaderless, a package for high-throughput analysis of translation initiation regions (TIRs) at the transcriptome scale and for leaderless mRNA optimization

BackgroundTranslation initiation is an essential step for fidelity of gene expression, in which the ribosome must bind to the translation initiation region (TIR) and position the initiator tRNA in the P-site (1). For this to occur correctly, the TIR encompassing the ribosome binding site (RBS) needs to be highly accessible (2-5). {Delta}Gunfold is a metric for computing accessibility of the TIR, but there is no automated way to compute it manually with existing software/tools limiting throughput. Results{Delta}Gunfold leaderless allows users to automate the {Delta}Gunfold calculation to perform high-throughput analysis. Importantly,{Delta} Gunfold leaderless allows calculation of TIRs of both leadered mRNAs and leaderless mRNAs which lack a 5 UTR and which are abundant in bacterial, archaeal, and mitochondrial transcriptomes (4, 6, 7). The ability to analyze leaderless mRNAs also allows one additional feature where users can computationally optimize leaderless mRNA TIRs to maximize their gene expression (8, 9). ConclusionsThe {Delta}Gunfold leaderless package facilitates high-throughput calculations of TIR accessibility, is designed to calculate TIR accessibility for leadered and leaderless mRNA TIRs which are abundant in bacterial/archaeal/organellar transcriptomes and allows optimization of leaderless mRNA TIRs for biotechnology.

bioinformatics↗

bioRxiv2020

A combinatorial model predicts leaderless mRNA start codon selection in C. crescentus

Bacterial translation is thought to initiate by base-pairing of the 16S rRNA and the Shine-Dalgarno sequence in the mRNAs 5UTR. However, transcriptomics has revealed that leaderless mRNAs, which completely lack any 5UTR, are broadly distributed across bacteria and can initiate translation in the absence of the Shine-Dalgarno sequence. To investigate the mechanism of leaderless mRNA translation initiation, synthetic in vivo translation reporters were designed that systematically tested the effects of start codon accessibility, leader length, and start codon identity on leaderless mRNA translation initiation. Using this data, a simple computational model was built based on the combinatorial relationship of these mRNA features which can accurately classify leaderless mRNAs and predict the translation initiation efficiency of leaderless mRNAs. Thus, start codon accessibility, leader length, and start codon identity combine to define leaderless mRNA translation initiation in bacteria.

systems biology↗

bioRxiv2019

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-seq. We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved 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 build-up 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.

biochemistry↗