Biology subjects

Granneman, S.

Publications and source records attributed to Granneman, S..

15 recordsLinked to original sources

bioRxiv2026

Tripartite toxin-antitoxin system regulates the production and release of pneumolysin by Streptococcus pneumoniae.

The production of toxins is critical to the success of bacteria, with their targets and outcomes varying widely. Pneumolysin (Ply), produced by the major human pathogen Streptococcus pneumoniae, is a cytolytic pore forming toxin that plays many diverse roles including inducing tissue damage and inflammation, evasion of many aspects of the human immune system, invasion of the bacteria into deeper tissue, and transmission to new hosts. Despite its importance, many aspects of the biology of this toxin have to date remained unknown, in particular, what role of the three other genes that form part of the ply operon play in Ply production. Here we demonstrate that these genes encode a complex regulatory system, that includes a putative membrane destabilising protein (PlyA) and a MazEF-like toxin-antitoxin system (PlyB and PlyC), the toxin of which (PlyC) destabilises the Ply transcript. We propose a tripartite model whereby when the operon reaches relatively high levels of expression PlyA destabilises the S. pneumoniae membrane creating a stress that triggers a necessary negative feedback loop which results in the degradation of the PlyB antitoxin, release of the PlyC toxin and degradation of the ply transcript, thereby providing a means by which Ply production can be tightly regulated. Given the importance of Ply to the ability of S. pneumoniae to cause infection, identifying the factors that result in the shutdown of Ply production and release by the bacteria would seem promising target for future therapeutic strategies.

microbiology↗

bioRxiv2026

Interdependent mode of RNA recognition by the interacting KH-domain proteins KhpA and KhpB

RNA-binding proteins (RBPs) are central regulators of gene expression. In bacteria, two KH domain-containing proteins, KhpA and KhpB, are increasingly recognized as globally-acting RBPs, including in pathogens that lack well-studied RBPs, such as Hfq and ProQ. While KhpA and KhpB interact with various RNAs in diverse bacteria, their crosstalk and molecular basis of RNA recognition remain poorly understood. Here, we explored the mutual dependence between KhpA and KhpB in the food-borne pathogen Campylobacter jejuni. We demonstrate KhpA and KhpB interact in vivo, and their individual global RNA recognition is largely dependent on the presence of the partner protein. Using RNA co-immunoprecipitation, we reveal that KhpA requires a conserved KH domain motif for RNA binding, whereas KhpB relies on an atypical R3H domain. Moreover, mutating the RNA-binding domain of one protein compromises the RNA interactome of the other. Overall, we demonstrate an interdependent RNA-binding mode of KhpA and KhpB and anticipate that such KH-proteins have co-evolved to elicit joint RNA regulation in bacteria.

microbiology↗

bioRxiv2026

Characterisation of the RNA-Binding Properties of the MRSA β-lactam resistance enzyme PBP2a

Analysis of RNA-binding proteome data from different bacterial species revealed many cell wall metabolic enzymes cross-linking to RNA in vivo, hinting that these proteins directly bind RNA. Surprisingly, penicillin-binding proteins (PBPs) were also abundantly identified as putative RNA-binding proteins. The cell surface localisation properties of many of these proteins therefore beg the question at what stage of their cellular life cycle these proteins interact with RNA and what the functional significance is. Here, we characterised the RNA-binding activity of PBP2a, the alternative transpeptidase that confers {beta}-lactam resistance in MRSA. Using in vivo RNA-binding assays, we show that PBP2a interacts with hundreds of transcripts without apparent sequence specificity. Computational analyses identified a possible RNA-binding cleft in PBP2a proximal to its active site. Mutation of only two predicted positively charged residues located in this cleft substantially reduced cross-linking in vivo, implying that RNA recognition is largely dictated by RNA backbone interactions. While PBP2a does not regulate RNA steady-state levels, RNA-binding appears important for proper protein function: an RNA-binding deficient mutant exhibits reduced oxacillin resistance. These findings establish PBP2a as an RNA-binding protein in vivo and provide a framework to investigate how this non-canonical interaction may relate to cell wall biogenesis and {beta}-lactam resistance.

biochemistry↗

bioRxiv2026

Non-coding RNA RsaE regulates biofilm thickness, viability and dissemination in methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a formidable human pathogen responsible for life-threatening infections worldwide. Central to its pathogenic success is the tightly coordinated regulation of virulence factors, including the phenol soluble modulins (PSM), short amphipathic toxins that drive cytolysis, immune evasion and biofilm maturation. We previously identified the conserved non-coding RNA RsaE as a putative regulator of the psm operon, but the biological significance of this interaction remained unclear. Here we show that RsaE and endoribonuclease Y jointly regulate psm transcript abundance at the post-transcriptional and transcriptional levels, the latter primarily through activation of the agr quorum sensing system. We show that the psm transcript is unusually stable and highly structured, with Shine-Dalgarno sequences of individual toxin-coding sequences differentially accessible, providing a mechanism for translational fine-tuning of individual PSM peptides. In vitro biofilm analyses revealed that RsaE deletion produces thinner biofilms with reduced extracellular DNA accumulation on the surface and transiently elevated cell viability. Strikingly, in a murine catheter infection model,{Delta} rsaE biofilms exhibited structural abnormalities and significantly reduced dissemination to kidneys. These findings identify the RsaE non-coding RNA as a key regulator linking central metabolism and quorum sensing to toxin expression, biofilm maturation and infection in MRSA. Author summaryMethicillin-resistant Staphylococcus aureus (MRSA) causes severe, often device-associated infections that are hard to treat. A central reason for its success is tight control over toxins such as the -phenol-soluble modulins (PSM), which help bacteria damage host cells and build/disperse biofilms. Small RNAs (sRNAs) are fast-acting genetic regulators in bacteria, that also regulate toxin production. We show that the conserved sRNA RsaE and the endoribonuclease RNase Y jointly tune the level and lifetime of the psm RNA transcript, which encodes four PSM peptides. The psm RNA is unusually stable and highly structured with its design favouring translation of PSM4 over the other peptides. Removing RsaE or RNase Y increases psm transcription via the agr quorum-sensing system and further stabilises the psm RNA. In biofilms, loss of RsaE reduces the thickness and extracellular DNA layer, while increasing early cell viability. In a mouse catheter model, RsaE deletion leads to structurally altered biofilms and reduced early dissemination to kidneys. These results identify RsaE as a key regulator connecting metabolism and quorum sensing to toxin expression, biofilm maturation and disease progression, and point to RNA-centred strategies to regulate MRSA spread from biofilms.

biochemistry↗

bioRxiv2025

A simple, reversible and non-toxic anchor-away system for effective nuclear depletion of proteins

The anchor-away (AA) technique enables rapid depletion of nuclear proteins by tethering them to cytoplasmic anchors through rapamycin-induced heterodimerisation. Albeit powerful, in Saccharomyces cerevisiae, this system is restricted to rapamycin-resistant strains, as the drug inhibits TOR signalling and hinders the heat-shock response, limiting its application to stress-related studies. Moreover, this AA method is not fully reversible, limiting studies of dynamic cellular processes that require transient perturbation and functional restoration. To overcome these constraints, we developed an alternative AA system that uses the plant hormone abscisic acid (ABA) to induce conditional association of the target to its cytoplasmic anchor. The ABA-AA system enables efficient and fully reversible depletion of highly abundant nuclear proteins. Unlike rapamycin, ABA does not cause major gene expression changes and is suitable for diverse genetic backgrounds. The ABA-AA system provides a fully reversible, non-toxic, and broadly applicable alternative for nuclear protein depletion across eukaryotic systems.

molecular biology↗

bioRxiv2025

RNMT is recruited to RNA by interaction with RNA G-quadraplexes

The 7-methylguanosine (m7G) cap protects RNA pol II transcripts from exonucleases and allows interaction with cap binding proteins which direct processing and translation. Duringm7G cap formation in mammals, nascent transcripts receive a guanosine cap which is methylated by RNA guanine-7 methyltransferase (RNMT). Unlike other capping enzymes which interact directly with RNA pol II, RNMT is recruited to the guanosine cap by interactions with RNA and the cap itself. RNMT is regulated during cell differentiation with significant impact on which RNAs are expressed and translated. The gene-specificity of RNMT was unexplained since RNMT in complex with activating subunit, RAM, binds to RNA without sequence preference. Through the development of improved RNA-protein detection, CLIP-ART, we report that RNMT interacts directly with RNA G4 quadraplexes (rG4). RNMT predominantly interacts with rG4s in the 5 UTR (untranslated regions) of mRNAs, indicating a mechanism for anchoring RNMT adjacent to the guanosine cap substrate. RNMT interacts with rG4s in transcripts which encode proteins involved in growth and proliferation. The RNMT-rG4 interaction is specific to the RNMT monomer, rather than RNMT-RAM, indicating a mechanism by which differential regulation of RNMT and RAM, can lead to cap methylation of specific RNAs involved in growth control. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/688685v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@f546a1org.highwire.dtl.DTLVardef@f000d4org.highwire.dtl.DTLVardef@c665f5org.highwire.dtl.DTLVardef@14fcfd0_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

biochemistry↗

bioRxiv2024

A co-transcriptional mechanism for tightly controlling RNA homeostasis in yeast

Transcription termination by the Nrd1-Nab3-Sen1 (NNS) complex is key in repressing pervasive transcription in Saccharomyces cerevisiae. Counterintuitively, during starvation, multiple mRNAs that are upregulated are also increasingly bound and prematurely terminated and degraded via NNS. Here we demonstrate that this NNS-mediated attenuation is important for controlling the expression and protein concentration of an evolutionarily conserved mitochondrial transporter, Pic2. Strikingly, we find that even a modest increase in Pic2 protein levels caused by defective NNS regulation has major phenotypical consequences, increasing cell volume and intracellular stress, prolonging cell cycle and decreasing growth rate. Disrupting Nab3 binding to PIC2 globally redistributed Nrd1 binding, changing the levels of other NNS-regulated transcripts. We propose that imbalances in the availability of the subunits constituting the NNS complex underlie the cell volume and cycle anomalies. Collectively our results illustrate that even subtle changes in how RNA-binding proteins interact with a single RNA substrate can cause global defects and they emphasise the crucial role of the NNS complex in preserving microbial fitness during stress. HighlightsO_LINNS regulates the expression and protein concentration of a stress-response protein-coding gene (PIC2), improving cell fitness and adaptability to environmental challenges. C_LIO_LICreating an imbalance in RNA binding of Nab3 and Nrd1 for PIC2 mRNA disturbs the homeostasis of co-regulated transcripts. C_LIO_LIEven a modest defect in NNS regulation of PIC2 elicits severe defects in cell growth, increases cell size and intracellular stress, and prolongs the cell cycle. C_LI

molecular biology↗

bioRxiv2024

Integrative structural analysis of NF45-NF90 heterodimers reveals architectural rearrangements and oligomerisation on binding dsRNA.

Complexes of nuclear factors 45 and 90 (NF45-NF90) play a multitude of roles in co- and post-transcriptional RNA processing, including regulating adenosine-to-inosine editing, cassette exon and back splicing, and splicing fidelity. NF45-NF90 complexes recognise dsRNA and, in human cells, primarily interact with inverted Alu repeats (AluIRs) that are commonly inserted into introns and other non-coding RNA regions. Intronic AluIRs of [~]300 bp can regulate splicing outcomes, such as generation of circRNAs. We examined domain reorganisation of NF45-NF90 domains on dsRNAs exceeding 50 bp to gain insight into its RNA recognition properties on longer dsRNAs. Using a combination of phylogenetic analysis, solution methods (including small angle X-ray scattering and quantitative cross-linking mass spectrometry), machine learning and negative stain electron microscopy, we generated a model of NF45-NF90 complex formation on dsRNA. Our data reveal that different interactions of NF45-NF90 complexes allow these proteins to coat long stretches of dsRNA. This property of the NF45-NF90 complex has important implications for how long, nuclear dsRNAs are recognised in the nucleus and how this might promote (co)-regulation of specific RNA splicing and editing events that shape the mammalian transcriptome.

biochemistry↗

bioRxiv2023

pyRBDome: A comprehensive computational platform for enhancing and interpreting RNA-binding proteome data

High-throughput proteomics approaches have revolutionised the identification of RNA-binding proteins (RBPome) and RNA-binding sequences (RBDome) across organisms. Yet the extent of noise, including false-positives, associated with these methodologies, is difficult to quantify as experimental approaches for validating the results are generally low throughput. To address this, we introduce pyRBDome, a pipeline for enhancing RNA-binding proteome data in silico. It aligns the experimental results with RNA-binding site (RBS) predictions from distinct machine learning tools and integrates high-resolution structural data when available. Its statistical evaluation of RBDome data enables quick identification of likely genuine RNA-binders in experimental datasets. Furthermore, by leveraging the pyRBDome results, we have enhanced the sensitivity and specificity of RBS detection through training new ensemble machine learning models. pyRBDome analysis of a human RBDome dataset, compared with known structural data, revealed that while UV cross-linked amino acids were more likely to contain predicted RBSs, they infrequently bind RNA in high-resolution structures. This discrepancy underscores the limitations of structural data as benchmarks, positioning pyRBDome as a valuable alternative for increasing confidence in RBDome datasets.

systems biology↗

bioRxiv2021

Fine-tuning of RecBCD expression by post-transcriptional regulation is required for optimal DNA repair in Escherichia coli

All living organisms have developed strategies to respond to chromosomal damage and preserve genome integrity. One such response is the repair of DNA double-strand breaks (DSBs), one of the most toxic forms of DNA lesions. In Escherichia coli, DSBs are repaired via RecBCD-dependent homologous recombination. RecBCD is essential for accurate chromosome maintenance, but its over-expression can lead to reduced DNA repair ability. This apparent paradox suggests that RecBCD copy numbers may need to be tightly controlled within an optimal range. Using single-molecule fluorescence microscopy, we have established that RecB is present in very low abundance at mRNA and protein levels. RecB transcription shows high fluctuations, yet cell-to-cell protein variability remains remarkably low. Here, we show that the post-transcriptional regulator Hfq binds to recB mRNA and down-regulates RecB protein translation in vivo. Furthermore, specific disruption of the Hfq-binding site leads to more efficient translation of recB mRNAs. In addition, we observe a less effective reduction of RecB protein fluctuations in the absence of Hfq. This fine-tuning Hfq-mediated mechanism might have the underlying physiological function of maintaining RecB protein levels within an optimal range.

systems biology↗

bioRxiv2020

Massive genome decay and expansion of insertion sequences drive the evolution of a novel host-restricted bacterial pathogen

BackgroundThe emergence of new pathogens is a major threat to public and veterinary health. Changes in bacterial habitat such as those associated with a switch in host or disease tropism are often accompanied by genetic adaptation. Staphylococcus aureus is a multi-host bacterial species comprising strains with distinct tropisms for human and livestock species. A microaerophilic subspecies, Staphylococcus aureus subsp. anaerobius, is responsible for outbreaks of Morels disease, a lymphadenitis in small ruminants. However, the evolutionary history of S. aureus subsp. anaerobius and its relatedness to S. aureus are unknown. ResultsEvolutionary genomic analyses of clinical S. aureus subsp. anaerobius isolates revealed a highly conserved clone that descended from a S. aureus progenitor about 1000 years ago before differentiating into distinct lineages representing African and European isolates. S. aureus subsp. anaerobius has undergone limited clonal expansion, with a restricted population size, and an evolutionary rate 10-fold slower than S. aureus. The transition to its current restricted ecological niche involved acquisition of a pathogenicity island encoding a ruminant host-specific effector of abscess formation, several large chromosomal re-arrangements, and the accumulation of at least 205 pseudogenes resulting in a highly fastidious metabolism. Importantly, expansion of [~]87 insertion sequences (IS) located largely in intergenic regions provided distinct mechanisms for the control of expression of flanking genes, representing a novel concept of the IS regulon. ConclusionsOur findings reveal the remarkable evolutionary trajectory of a host-restricted bacterial pathogen that resulted from extensive remodelling of the S. aureus genome through an array of parallel mechanisms.

microbiology↗

bioRxiv2020

Differential BUM-HMM: a robust statistical modelling approach for detecting RNA flexibility changes in high-throughput structure probing data

Combining RNA structure probing with high-throughput sequencing technologies has greatly enhanced our ability to analyze RNA structure at transcriptome scale. However, the high level of noise and variability encountered in these data call for the development of computational tools that robustly extract RNA structural information. Here we present diffBUM-HMM, a noise-aware model that enables accurate detection of RNA flexibility and conformational changes from high-throughput RNA structure-probing data. DiffBUM-HMM is compatible with a wide variety of high-throughput RNA structure probing data, taking into consideration biological variation, sequence coverage and sequence representation biases. We demonstrate that, compared to the existing approaches, diffBUM-HMM displays higher sensitivity while calling virtually no false positives. DiffBUM-HMM analysis of ex vivo and in vivo Xist SHAPE-MaP data detected many more RNA structural differences, involving mostly single-stranded nucleotides located at or near protein-binding sites. Collectively, our analyses demonstrate the value of diffBUM-HMM for quantitatively detecting RNA structural changes and reinforce the notion that RNA structure probing is a very powerful tool for identifying protein-binding sites.

systems biology↗

bioRxiv2018

Hfq CLASH uncovers sRNA-target interaction networks enhancing adaptation to nutrient availability

By shaping gene expression profiles, small RNAs (sRNAs) enable bacteria to very efficiently adapt to constant changes in their environment. To better understand how Escherichia coli acclimatizes to changes in nutrient availability, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) to uncover sRNA-target interactions. Strikingly, we uncovered hundreds of novel Hfq-mediated sRNA-target interactions at specific growth stages, involving many novel 3UTR-derived sRNAs and a plethora of sRNA-sRNA interactions. We discovered sRNA-target interaction networks that play a role in adaptation to changes in nutrient availability. We characterized a novel 3UTR-derived sRNA (MdoR), which is part of a regulatory cascade that enhances maltose uptake by (a) inactivating repressive pathways that block the accumulation of maltose transporters and (b) by reducing the flux of general porins to the outer membrane. Our work provides striking examples of how bacteria utilize sRNAs to integrate multiple regulatory pathways to enhance nutrient stress adaptation.

systems biology↗

bioRxiv2018

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). To overcome these limitations, we have developed a novel protocol, Phenol Toluol extraction (PTex), that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a first global RNA-bound proteome of human HEK293 cells and Salmonella Typhimurium as a bacterial species.

molecular biology↗

bioRxiv2017

RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination

TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. Almost nothing is known about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. Here, we reveal that RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25s endogenous RNA targets and protein binding partners. Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase function. These results reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor for their biological functions.

molecular biology↗