Biology subjects

Condon, C.

Publications and source records attributed to Condon, C..

15 recordsLinked to original sources

bioRxiv2026

EF-P-resolved ribosome stalling and collision trigger Rae1-mediated mRNA decay

Ribosome stalling compromises proteome integrity and cellular fitness, and requires efficient mechanisms to eliminate problematic mRNAs and rescue blocked ribosomes. We previously identified Rae1 as an endoribonuclease that cleaves mRNAs at the tail end of stalled ribosomes to promote mRNA degradation in Bacillus subtilis. Here, we show that Rae1 has a broader role in the quality control of mRNAs that encounter translation problems. Global 5-end sequencing identified 31 previously unknown Rae1 cleavage sites, many of them associated with ribosome pausing at polyproline motifs in the absence of elongation factor P (EF-P). We also demonstrate that Rae1 cleaves mRNAs with ribosomes stalled on truncated transcripts lacking a stop-codon (non-stop mRNAs), provided that the transcript is sufficiently long to accommodate multiple collided ribosomes. Rae1 cleavage depends on a cytosine-poor context at the cut site, showing that both ribosome collisions and RNA sequence at the cleavage site govern the ability of Rae1 to promote the removal of stalled ribosomes from mRNA.

microbiology↗

bioRxiv2026

RNase J2 is a specificity factor that governs global mRNA stability in Bacillus subtilis

Ribonucleases (RNases) play a key role in modulating gene expression at the post-transcriptional level, enabling bacteria to rapidly adapt to their environment. The B. subtilis genome encodes more than 20 RNases and understanding the role of each of these enzymes is crucial for a comprehensive knowledge of bacterial post-transcriptional adaptation mechanisms. Among these, RNase J1 and J2, belonging to the {beta}-lactamase enzyme family, form a heterodimer. While the primary function of RNase J1 as a 5'-exoribonuclease in B. subtilis has been known for some time, the role of RNase J2, encoded by the rnjB gene, has remained unclear. Indeed, it has been considered a minor degradation factor due its very weak 5'-exoribonuclease in vitro, the lack of a growth phenotype and the limited number of mRNAs with altered equilibrium levels in rnjB mutants. In this study, we demonstrate that the absence of RNase J2 influences pellicle and macro-colony biofilm formation, as well as swarming in B. subtilis. We show that RNase J2 plays a far more global role in governing mRNA half-lives than originally thought, by stimulating RNase J1-mediated degradation of a subset of RNase J1's mRNA substrates. An inter-subunit contact between RNase J1 and J2 is crucial for RNase J2 action. Altogether, our results suggest that RNase J2 acts primarily as a specificity co-factor for RNase J1 rather than as a ribonuclease per se.

microbiology↗

bioRxiv2025

Extensive splicing deficiency in a degenerating mating-type chromosome

Splicing deficiency may represent a critical yet underexplored form of genomic erosion in non-recombining regions. Across four phytoplankton species diverged [~]333-639 million years ago, genes within U (female) and V (male) "UV" mating-type regions--non-recombining chromosomal regions that determine mating compatibility--show strikingly elevated intron retention relative to genes in other genomic regions. Long-read data reveal abundant aberrant, likely non-functional mRNA isoforms despite preserved coding potential. This preservation suggests that splicing defects arose early in UV evolution and have persisted over deep time. We propose that these defects arise from evolutionary changes in sequence composition and chromatin organization that accompany recombination suppression, such as reduced GC content, altered nucleosome occupancy, and disrupted methylation, that collectively compromise splicing fidelity. Unlike sex chromosomes, which often degenerate through gene loss, splicing-deficient UV regions in green algae retain hundreds of genes, indicating that transcript-level dysfunction provides an alternative route to functional decay. Our results identify chromatin-mediated splicing deficiency as a novel axis of genomic erosion and position algal UV systems as models for studying how recombination suppression reshapes RNA processing fidelity in essential, non-recombining genomes.

genomics↗

bioRxiv2025

The endoribonuclease Rae1 from Bacillus subtilis cleaves mRNA upstream of stalled ribosomes

The ribosome-associated endoribonuclease 1 (Rae1) cleaves mRNAs in a translation-dependent manner. Here, we identify a new Rae1 target, the fliY mRNA, which is cleaved by Rae1 in the absence of the elongation factor P (EF-P). The Rae1 site was mapped 12 nucleotides upstream of the second proline codon of an SPP stalling motif in fliY. Remarkably, Rae1 cleavages also occur 12 nucleotides upstream of the stop codon within two validated Rae1 mRNA targets, bmrX and spyA (S1025). Shifting the stop codon relative to the Rae1 cutting site abolished Rae1 sensitivity of bmrX and spyA mRNAs. We show that ribosome pausing occurs at the spyA stop codon, confirming its crucial role, and positioning the Rae1 cleavage at the tail end of the stalled ribosome, rather than in the A-site as previously proposed. These findings reveal a compelling novel mechanism by which Rae1 mediates mRNA cleavage in coordination with immobile ribosomes.

molecular biology↗

bioRxiv2024

Real-Time Nanoscale Investigation of Spore Coat Assembly in Bacillus subtilis

Spores of Bacillaceae, ubiquitous in soil, can withstand extreme conditions virtually indefinitely. The spore coat, one of the most sophisticated multiprotein complexes built by bacteria, protects the spore from environmental stresses and predators. In Bacillus subtilis, assembly of the multilayered coat initiates at the forespore pole and is perceived as a stable process that progresses continuously until covering the entire forespore surface. In contrast, in Bacillus cereus, coat formation initiates in the midspore region and extends outward to the spore poles. Using Structured Illumination Microscopy, we monitored B. subtilis coat development in real-time at the single-sporangium level with lateral resolution of 70 nm. We found that late-synthesized proteins from the innermost coat layers first assemble in the midspore region and are subsequently displaced towards the poles. This process is coupled with a unique redistribution of pre-assembled coat material across the forespore surface and influenced by outer coat development, highlighting a dynamic interplay in coat layers co-construction.

microbiology↗

bioRxiv2024

Diverging Arabidopsis populations quickly accumulate pollen-acting genetic incompatibilities

The process by which species diverge from one another, gradually accumulate genetic incompatibilities and eventually reach full-fledged reproductive isolation is a key question in evolutionary biology. However, the nature of reproductive barriers, the pace at which they accumulate and their genomic distribution remain poorly documented. The disruption of co-adapted epistatic interactions in hybrids and the accumulation of selfish genetic elements are proposed contributors to this process, and can lead to the distortion of the mendelian segregation of the affected loci across the genome. In this study we detect and quantify segregation distortion across the genomes of crosses produced from a diverse sampling of Arabidopsis lyrata and A. halleri populations, two species at the early stages of speciation and that can still interbreed. We show that both the frequency of occurrence and the magnitude of distortion loci increase as the parents genetic distance from one another increases. We also observe that distorter loci evolve rapidly, as they occur not only within interspecific hybrids, but also in intraspecific hybrids produced from isolated population crosses. Finally, we identify both genome-wide non-independence and two specific genomic regions on different chromosomes where opposite distortion effects are repeatedly observed across multiple F1 individuals, suggesting negative epistasis is a major contributor to the evolution of hybrid segregation distortion. Our study demonstrates that pollen-acting segregation distortion is ubiquitous, and contributes not only to the ongoing reproductive isolation between A. halleri and A. lyrata, but also between very recently diverged populations of the same species.

genomics↗

bioRxiv2023

Complex sporulation-specific expression of transcription termination factor Rho highlights its involvement in Bacillus subtilis cell differentiation

Transcription termination factor Rho controls pervasive, mainly antisense, transcription initiated at cryptic signals or resulting from read-through at weak terminators in various bacterial species. In Bacillus subtilis, Rho is intricately involved in the regulation of phenomena associated with the adaptation to stationary phase and cell differentiation including the ultimate survival program of sporulation. While knockout or overexpression of the rho gene alters global transcription and modifies cell physiology, in wild-type B. subtilis cells, the reduction of Rho levels during the transition to stationary phase is necessary for both initiation and implementation of the sporulation program. However, the mechanisms that govern Rho expression throughout the cell cycle remain largely unknown. Here, we demonstrate that, besides the previously identified vegetative SigA-dependent promoter active during exponential growth, two distinct mechanisms ensure a spatiotemporal expression of the rho gene during sporulation. In the mother cell of the sporangium, rho expression occurs through the read-through transcription initiated at the distal SigH-dependent and Spo0A[~]P-regulated promoter of the spo0F gene. In the forespore, rho is transcribed from a genuine promoter recognized by the alternative sigma factor SigF. These regulatory elements compensate for the inactivation of SigA-dependent rho expression at the end of exponential growth and allow the critical "refueling" of Rho protein in both compartments of the sporangium. We show that altering rho expression in the mother cell or in the forespore affects differently the properties and the morphology of mature spores. Moreover, spores formed in the absence of Rho are impaired in their ability to revive under favorable growth conditions, exhibiting accelerated germination and slow outgrowth. Finally, we show that optimal outgrowth of the wild-type spores requires the expression of rho during spore maturation and additionally after spore germination.

microbiology↗

bioRxiv2023

Critical factors for precise and efficient RNA cleavage by RNase Y in Staphylococcus aureus

Cellular processes require precise and specific gene regulation, in which continuous mRNA degradation is a major element. The mRNA degradation mechanisms should be able to degrade a wide range of different RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y. Author summaryIn order to correctly regulate the level of RNAs, bacteria require their RNA to be continuously synthesised and degraded. However, even related bacterial species can have different sets of ribonucleases, each with their own target criteria. Here we explore which sequence elements of an RNA are important for being targeted by the endoribonuclese RNase Y in the two bacteria, Staphylococcus aureus and Bacillus subtilis. We specifically examine the RNase Y dependent cleavage of two transcripts that have homologs in both bacteria. We identify a short single-stranded regions immediately downstream of the cleavage position can be modified to change the cleavage efficiency up to 20-fold. We furthermore discover that a secondary structure a few nucleotides downstream of the cleavage is required for cleavage and that the positioning of the cleavage can be modulated by moving this structure.

molecular biology↗

bioRxiv2023

A high-resolution view of RNA endonuclease cleavage in Bacillus subtilis

RNA endonucleases are the rate-limiting initiator of decay for many bacterial mRNAs. However, the positions of cleavage and their sequence determinants remain elusive even for the well-studied Bacillus subtilis. Here we present two complementary approaches - transcriptome-wide mapping of endoribonucleolytic activity and deep mutational scanning of RNA cleavage sites - that reveal distinct rules governing the specificity among B. subtilis endoribonucleases. Detection of RNA terminal nucleotides in both 5'- and 3'-exonuclease-deficient cells revealed >103 putative endonucleolytic cleavage sites with single-nucleotide resolution. We found a surprisingly weak consensus for RNase Y targets, a contrastingly strong primary sequence motif for EndoA targets, and long-range intramolecular secondary structures for RNase III targets. Deep mutational analysis of RNase Y cleavage sites showed that the specificity is governed by many disjointed sequence features, each with mild contributions. Our results highlight the delocalized nature of mRNA stability determinants and provide a strategy for elucidating endoribonuclease specificity in vivo.

molecular biology↗

bioRxiv2023

Phased nanopore assembly with Shasta and modular graph phasing with GFAse

As a step towards simplifying and reducing the cost of haplotype resolved de novo assembly, we describe new methods for accurately phasing nanopore data with the Shasta genome assembler and a modular tool for extending phasing to the chromosome scale called GFAse. We test using new variants of Oxford Nanopore Technologies (ONT) PromethION sequencing, including those using proximity ligation and show that newer, higher accuracy ONT reads substantially improve assembly quality.

bioinformatics↗

bioRxiv2023

New RoxS sRNA targets identified in B. subtilis by pulsed SILAC

Non-coding RNAs (sRNA) play a key role in controlling gene expression in bacteria, typically by base-pairing with ribosome binding sites to block translation. The modification of ribosome traffic along the mRNA generally affects its stability. However, a few cases have been described in bacteria where sRNAs can affect translation without a major impact on mRNA stability. To identify new sRNA targets in B. subtilis potentially belonging to this class of mRNAs, we used pulsed-SILAC (stable isotope labeling by amino acids in cell culture) to label newly synthesized proteins after short expression of the RoxS sRNA, the best characterized sRNA in this bacterium. RoxS sRNA was previously shown to interfere with the expression of genes involved in central metabolism, permitting control of the NAD+/NADH ratio in B. subtilis. In this study, we confirmed most of the known targets of RoxS, showing the efficiency of the method. We further expanded the number of mRNA targets encoding enzymes of the TCA cycle and identified new targets primarily regulated at the translational level. One of these is YcsA, a tartrate dehydrogenase that uses NAD+ as co-factor, in excellent agreement with the proposed role of RoxS in Firmicutes. ImportanceNon-coding RNA (sRNA) play an important role in bacterial adaptation and virulence. The identification of the most complete set of targets for these regulatory RNAs is key to fully identify the perimeter of its function(s). Most sRNAs modify both the translation (directly) and mRNA stability (indirectly) of their targets. However, sRNAs can also influence the translation efficiency of the target primarily, with little or no impact on mRNA stability. The characterization of these targets is challenging. We describe here the application of the pulsed SILAC method to identify these targets and obtain the most complete list of targets for a defined sRNA.

microbiology↗

bioRxiv2023

Identification of a new substrate for the ribosome associated endoribonuclease Rae1 reveals a link to the B. subtilis response and sensitivity to chloramphenicol

Rae1 is a well-conserved endoribonuclease among Gram-positive bacteria, cyanobacteria and the chloroplasts of higher plants. We have previously shown that Rae1 cleaves the Bacillus subtilis yrzI operon mRNA in a translation-dependent manner, within a short open reading frame (ORF) called S1025, encoding a 17-amino acid (aa) peptide of unknown function. Here, we map a new Rae1 cleavage site in the bmrBCD operon mRNA encoding a multidrug transporter, within a previously unannotated 26-aa short ORF that we have named bmrX. Similar to S1025, Rae1 cleavage within bmrX is both translation- and reading frame-dependent. Both mRNAs were previously shown to be induced by the presence of the protein synthesis inhibitor chloramphenicol (Cm). Strikingly, a rae1 deletion strain shows greater resistance to Cm than the wild-type strain, while its over-expression leads to increased Cm sensitivity, suggesting a link to translation quality control. Consistent with this, we show that cleavage by Rae1 promotes ribosome rescue by the tmRNA. Globally, our data point to a role of Rae1 in mRNA surveillance by eliminating mRNAs that encounter problems with translation.

microbiology↗

bioRxiv2022

Effect of tRNA maturase depletion on the levels and stabilities of ribosome assembly cofactor mRNAs in Bacillus subtilis

The impact of translation on mRNA stability can be varied, ranging from a protective effect of ribosomes that shield mRNA from ribonucleases (RNases), to preferentially exposing sites of RNase cleavage. These effects can change depending on whether ribosomes are actively moving along the mRNA or whether they are stalled at particular sequences, structures or awaiting charged tRNAs. We recently observed that depleting B. subtilis cells of its tRNA maturation enzymes RNase P or RNase Z, led to altered mRNA levels of a number of assembly factors involved in the biogenesis of the 30S ribosomal subunit. Here, we extend this study to other assembly factor mRNAs and identify multiple transcriptional and translational layers of regulation of the rimM operon mRNA that occur in response to the depletion of functional tRNAs. ImportanceThe passage of ribosomes across individual mRNAs during translation can have different effects on their degradation, ranging from a protective effect by shielding from ribonucleases, to in some cases, making the mRNA more vulnerable to RNase action. We recently showed that some mRNAs coding for proteins involved in ribosome assembly were highly sensitive to the availability of functional tRNA. Using strains depleted for the major tRNA processing enzymes RNase P and RNase Z, we expanded this observation to a wider set of mRNAs, including some unrelated to ribosome biogenesis. We characterize the impact of tRNA maturase depletion on the rimM operon mRNA and show it is highly complex, with multiple levels of transcriptional and post-transcriptional effects coming into play.

microbiology↗

bioRxiv2022

Ablative radiotherapy improves survival in autochthonous cancer mouse models

Genetically engineered mouse models (GEMMs) of cancer are powerful tools to study mechanisms of disease progression and therapy response, yet little is known about how these models respond to multimodality therapy used for curative intent in patients. Radiation therapy (RT) is frequently used to treat localized cancers with curative intent, delay progression of oligometastases, and palliate symptoms of metastatic disease. RT can be combined with surgery, systemic therapy, and/or immunotherapy for definitive treatment of solid tumors. Here we report the development, testing, and validation of a murine platform that faithfully emulates human stereotactic ablative radiotherapy (SABR). We demonstrate that SABR regimens used in clinical practice can be effectively delivered in mouse models and establish the intestinal tract as the dose limiting organ for abdominopelvic radiation with notable, but transient, lymphodepletion. SABR alters tumor stroma and immune environment, improves survival in GEMMs of primary prostate and colorectal cancer, and synergizes with androgen deprivation in prostate cancer. While SABR is capable of fully ablating xenografts it is unable to completely eradicate disease in GEMMs. These data show that some GEMMs are resistant to curative therapy suggesting the existence of treatment-resistant persister cells in these models.

cancer biology↗

bioRxiv2019

Novel regulation from novel interactions: Identification of an RNA sponge that controls the levels, processing and efficacy of the RoxS riboregulator of central metabolism in Bacillus subtilis

Small RNAs (sRNAs) are a taxonomically-restricted but transcriptomically-abundant class of post-transcriptional regulators. While potentially of importance, we know the function of few. This is in no small part because we lack global-scale methodology enabling target identification, this being especially acute in species without known RNA meeting point proteins (e.g. Hfq). We apply a combination of psoralen RNA cross-linking and Illumina-sequencing to identify RNA-RNA interacting pairs in vivo in Bacillus subtilis, resolving previously well-described interactants. Although sRNA-sRNA pairings are rare (compared with sRNA/mRNA), we identify a robust example involving the unusually conserved sRNA (RoxS/RsaE) and an unstudied sRNA that we term Regulator of small RNA A (RosA). This interaction is found in independent samples across multiple conditions. Given the possibility of a novel associated regulatory mechanism, and the rarity of well-characterised bacterial sRNA-sRNA interactions, we mechanistically dissect RosA and its interactants. RosA we show to be a sponge RNA, the first to be described in a Gram-positive bacterium. RosA interacts with at least two sRNAs, RoxS and FsrA. Unexpectedly, it acts differently on each. As expected of a sponge RNA, FsrA is sequestered by RosA. The RosA/RoxS interaction is more complex affecting not only the level of RoxS but also its processing and efficacy. Importantly, RosA provides the condition-dependent intermediary between CcpA, the key regulator of carbon metabolism, and RoxS. This not only provides evidence for a novel, and functionally important, regulatory mechanism, but in addition, provides the missing link between transcriptional and post-transcriptional regulation of central metabolism.

microbiology↗