Ribonuclease Activity Research Articles

Discovery of a Compound That Inhibits IRE1α S-Nitrosylation and Preserves the Endoplasmic Reticulum Stress Response under Nitrosative Stress.

Inositol-requiring enzyme 1α (IRE1α) is a sensor of endoplasmic reticulum (ER) stress and drives ER stress response pathways. Activated IRE1α exhibits RNase activity and cleaves mRNA encoding X-box binding protein 1, a transcription factor that induces the expression of genes that maintain ER proteostasis for cell survival. Previously, we showed that IRE1α undergoes S-nitrosylation, a post-translational modification induced by nitric oxide (NO), resulting in reduced RNase activity. Therefore, S-nitrosylation of IRE1α compromises the response to ER stress, making cells more vulnerable. We conducted virtual screening and cell-based validation experiments to identify compounds that inhibit the S-nitrosylation of IRE1α by targeting nitrosylated cysteine residues. We ultimately identified a compound (1ACTA) that selectively inhibits the S-nitrosylation of IRE1α and prevents the NO-induced reduction of RNase activity. Furthermore, 1ACTA reduces the rate of NO-induced cell death. Our research identified S-nitrosylation as a novel target for drug development for IRE1α and provides a suitable screening strategy.

Structural and Functional Insight Into YefM-YoeB Complex of Toxin-Antitoxin System From Streptococcus pneumoniae.

Streptococcus pneumonia is a Gram-positive and facultative anaerobic bacterium that causes a number of diseases, including otitis media, community-acquired pneumonia, sepsis, and meningitis. With the emergence of antibiotic-resistant strains, there is an urgent need to develop antibiotics with a novel mechanism. The toxin-antitoxin (TA) system, which is primarily found in prokaryotes, consists of a toxin and its equivalent antitoxin genes. The YefM-YoeB module is a Type II TA system, where the YoeB toxin functions as a putative mRNA interferase upon activation, while the YefM antitoxin acts as a transcription repressor by binding to its promoter region along with YoeB. In this study, we determined the crystal structure of the YefM-YoeB complex from S. pneumoniae TIGR4 to comprehend the binding mechanism of the TA system. Furthermore, an in vitro ribonuclease activity assay was conducted to identify the ribonuclease activity of the YoeB toxin. Additionally, furthermore, the oligomeric state of the YefM-YoeB complex in solution was investigated, and a DNA-binding mode was proposed. These structural and functional insights into the YefM-YoeB complex could provide valuable information for the development of novel antibiotics targeting S. pneumonia-associated diseases.

Emerging role of IRE1α in vascular diseases

AbstractA mounting body of evidence suggests that the endoplasmic reticulum stress and the unfolded protein response are involved in the underlying mechanisms responsible for vascular diseases. Inositol‐requiring protein 1α (IRE1α), the most ancient branch among the UPR‐related signaling pathways, can possess both serine/threonine kinase and endoribonuclease (RNase) activity and can perform physiological and pathological functions. The IRE1α‐signaling pathway plays a critical role in the pathology of various vascular diseases. In this review, we provide a general overview of the physiological function of IRE1α and its pathophysiological role in vascular diseases.

Preferential cleavage of the coronavirus defective viral genome by cellular endoribonuclease with characteristics of RNase L

In testing whether coronavirus defective viral genome 12.7 (DVG12.7) with transcription regulating sequence (TRS) can synthesize subgenomic mRNA (sgmRNA) in coronavirus-infected cells, it was unexpectedly found by Northern blot assay that not only sgmRNA (designated sgmDVG 12.7) but also an RNA fragment with a size less than sgmDVG 12.7 was identified. A subsequent study demonstrated that the identified RNA fragment (designated clvDVG) was a cleaved RNA product originating from DVG12.7, and the cleaved sites were located in the loop region of stem‒loop structure and after UU and UA dinucleotides. clvDVG was also identified in mock-infected HRT-18 cells transfected with DVG12.7 transcript, indicating that cellular endoribonuclease is responsible for the cleavage. In addition, the sequence and structure surrounding the cleavage sites can affect the cleavage efficiency of DVG12.7. The cleavage features are therefore consistent with the general criteria for RNA cleavage by cellular RNase L. Furthermore, both the cleavage of rRNA and the synthesis of clvDVG were also identified in A549 cells. Because (i) the cleavage sites occurred predominantly after single-stranded UA and UU dinucleotides, (ii) the sequence and structure surrounding the cleavage sites affected the cleavage efficiency, (iii) the cleavage of rRNA is an index of the activation of RNase L, and (iv) the cleavage of both rRNA and DVG12.7 was identified in A549 cells, the results together indicated that the preferential cleavage of DVG12.7 is correlated with cellular endoribonuclease with the characteristics of RNase L and such cleavage features have not been previously characterized in coronaviruses.

Regulation and Dynamics of IFN-β Expression Revealed with a Knockin Reporter Mouse.

IFN-β is a potent antiviral cytokine and the first member of the type I IFN family of cytokines to be induced during the antiviral response. IFN-β plays an essential protective role in host defense against virus infections, as well as a pathogenic role in numerous autoimmune and autoinflammatory disorders. However, contemporary tools to study the induction, kinetics, and behavior of IFN-β are lacking. In this study, we describe a knockin Ifnb-IRES-TdTomato-Cre reporter mouse to track IFN-β-expressing cells. We demonstrate pathway-specific induction of the TdTomato reporter and show that the linked Cre recombinase enables permanent marking of cells that express IFN-β. We identify a robust MAVS-dependent IFN-β response in lung epithelial cells following Sendai virus infection invivo. Finally, we find that activation of RNase L in macrophages by RNA ligands of the RIG-I-like receptors prevents protein translation of IFN-β and the TdTomato reporter. Our mouse model provides a powerful tool to study the biology of type I IFN induction and the antiviral response.

A CRISPR-Cas9 knockout screening identifies IRF2 as a key driver of OAS3/RNase L-mediated RNA decay during viral infection

OAS-RNase L is a double-stranded RNA-induced antiviral pathway triggered in response to diverse viral infections. Upon activation, OAS-RNase L suppresses virus replication by promoting the decay of host and viral RNAs and inducing translational shutdown. However, whether OASs and RNase L are the only factors involved in this pathway remains unclear. Here, we develop CRISPR-Translate, a FACS-based genome-wide CRISPR-Cas9 knockout screening method that uses translation levels as a readout and identifies IRF2 as a key regulator of OAS3. Mechanistically, we demonstrate that IRF2 promotes basal expression of OAS3 in unstressed cells, allowing a rapid activation of RNase L following viral infection. Furthermore, IRF2 works in concert with the interferon response through STAT2 to further enhance OAS3 expression. We propose that IRF2-induced RNase L is critical in enabling cells to mount a rapid antiviral response immediately after viral infection, serving as the initial line of defense. This rapid response provides host cells the necessary time to activate additional antiviral signaling pathways, forming secondary defense waves.

RNase W, a conserved ribonuclease family with a novel active site.

Ribosome biogenesis is a complex process requiring multiple precursor ribosomal RNA (rRNA) cleavage steps. In archaea, the full set of ribonucleases (RNases) involved in rRNA processing remains to be discovered. A previous study suggested that FAU-1, a conserved protein containing anRNase G/E-like protein domain fused to a domain of unknown function (DUF402), acts as an RNase in archaea. However, the molecular basis of this activity remained so far elusive. Here, we report two X-ray crystallographic structures of RNase G/E-like-DUF402 hybrid proteins from Pyrococcus furiosus and Sulfolobus acidocaldarius, at 2.1 and 2.0 Å, respectively. The structures highlight a structural homology with the 5' RNA recognition domain of Escherichia coli RNase E but no homology with other known catalytic nuclease domains. Surprisingly, we demonstrate that the C-terminal domain of this hybrid protein, annotated as a putative diphosphatase domain, harbors the RNase activity. Our functional analysis also supports a model by which the RNase G/E-like domain acts as a regulatory subunit of the RNase activity. Finally, in vivo experiments in Haloferax volcanii suggest that this RNase participates in the maturation of pre-16S rRNA. Together, our study defines a new RNase family, which we termed the RNase W family, as the first archaea-specific contributor to archaeal ribosome biogenesis.

New Catalytic Residues and Catalytic Mechanism of the RNase T1 Family.

The ribonuclease T1 family, including RNase Po1 secreted by Pleurotus ostreatus, exhibits antitumor activity. Here, we resolved the Po1/guanosine-3'-monophosphate complex (3'GMP) structure at 1.75 Å. Structure comparison and fragment molecular orbital (FMO) calculation between the apo form and the Po1/3'GMP complex identified Phe38, Phe40, and Glu42 as the key binding residues. Two types of the RNase/3'GMP complex in RNasePo1 and RNase T1 were homologous to Po1, and FMO calculations elucidated that the biprotonated histidine on the β3 sheet (His36) on the β3 sheet and deprotonated Glu54 on the β4 sheet were advantageous to RNase activity. Moreover, tyrosine (Tyr34) on the β3 sheet was elucidated as a crucial catalytic residues. Mutation of Tyr34 with phenylalanine decreased RNase activity and diminished antitumor efficacy compared to that in the wild type. This suggests the importance of RNase activity in antitumor mechanisms.

Changes in the Repertoire of tRNA-Derived Fragments in Different Blood Cell Populations.

tRNA-derived fragments function as markers in addition to playing the key role of signalling molecules in a number of disorders. It is known that the repertoire of these molecules differs greatly in different cell types and varies depending on the physiological condition. The aim of our research was to compare the pattern of tRF expression in the main blood cell types and to determine how the composition of these molecules changes during COVID-19-induced cytokine storms. Erythrocytes, monocytes, lymphocytes, neutrophils, basophils and eosinophils from control donors and patients with severe COVID-19 were obtained by fluorescence sorting. We extracted RNA from FACS-sorted cells and performed NGS of short RNAs. The composition of tRNA-derived fragments was analysed by applying a semi-custom bioinformatic pipeline. In this study, we assessed the length and type distribution of tRFs and reported the 150 most prevalent tRF sequences across all cell types. Additionally, we demonstrated a significant (p < 0.05, fold change >16) change in the pattern of tRFs in erythrocytes (21 downregulated, 12 upregulated), monocytes (53 downregulated, 38 upregulated) and lymphocytes (49 upregulated) in patients with severe COVID-19. Thus, different blood cell types exhibit a significant variety of tRFs and react to the cytokine storm by dramatically changing their differential expression patterns. We suppose that the observed phenomenon occurs due to the regulation of nucleotide modifications and alterations in activity of various Rnases.

AtC3H3, an Arabidopsis Non-TZF Gene, Enhances Salt Tolerance by Increasing the Expression of Both ABA-Dependent and -Independent Stress-Responsive Genes.

Salinity causes widespread crop loss and prompts plants to adapt through changes in gene expression. In this study, we aimed to investigate the function of the non-tandem CCCH zinc-finger (non-TZF) protein gene AtC3H3 in response to salt stress in Arabidopsis. AtC3H3, a gene from the non-TZF gene family known for its RNA-binding and RNase activities, was up-regulated under osmotic stress, such as high salt and drought. When overexpressed in Arabidopsis, AtC3H3 improved tolerance to salt stress, but not drought stress. The expression of well-known abscisic acid (ABA)-dependent salt stress-responsive genes, namely Responsive to Desiccation 29B (RD29B), RD22, and Responsive to ABA 18 (RAB18), and representative ABA-independent salt stress-responsive genes, namely Dehydration-Responsive Element Binding protein 2A (DREB2A) and DREB2B, was significantly higher in AtC3H3-overexpressing transgenic plants (AtC3H3 OXs) than in wild-type plants (WT) under NaCl treatment, indicating its significance in both ABA-dependent and -independent signal transduction pathways. mRNA-sequencing (mRNA-Seq) analysis using NaCl-treated WT and AtC3H3 OXs revealed no potential target mRNAs for the RNase function of AtC3H3, suggesting that the potential targets of AtC3H3 might be noncoding RNAs and not mRNAs. Through this study, we conclusively demonstrated that AtC3H3 plays a crucial role in salt stress tolerance by influencing the expression of salt stress-responsive genes. These findings offer new insights into plant stress response mechanisms and suggest potential strategies for improving crop resilience to salinity stress.

Changes in the genome of tick-borne encephalitis virus during cultivation

The tick-borne encephalitis virus (TBEV) of the Siberian genotype was previously isolated from the brain of a deceased person. TBEV variants obtained at passages 3 and 8 on SPEV cells were inoculated into the brains of white mice for subsequent passages. Full-genome sequences of all virus variants were analyzed by high-throughput sequencing. The analysis showed the presence of 41 point nucleotide substitutions, which were mainly localized in the genes of non-structural proteins NS3 and NS5 of TBEV. In the deduced virus protein sequences, 12 amino acid substitutions were identified. After three passages through mouse brains, reverse nucleotide and amino acid substitutions were detected. Most of them were mapped in the NS5 protein gene, where 5 new nucleotide substitutions also appeared. At the same time, there was an increase in the length of the 3′-untranslated region (3′-UTR) of the viral genome by 306 nucleotides. The Y3 and Y2 3’-UTR elements were found to contain imperfect L and R repeats, which probably associated with inhibition of the activity of cellular XRN1 RNase and thus involved in the formation of sfRNA1 and sfRNA2. For all TBEV variants, high levels of infectious virus were detected both in cell culture and in the brain of white mice. The revealed changes in the genome that occur during successive passages of TBEV are most likely due to the significant genetic variability of the virus, which ensures its efficient reproduction in different hosts and active circulation in nature.

Characterization of Escherichia coli chaperonin GroEL as a ribonuclease

Chaperonins are evolutionarily conserved proteins that facilitate polypeptide assemblies. The most extensively studied chaperonin is GroEL, which plays a crucial role in Escherichia coli. In addition to its chaperone activity, the RNA cleavage activity of GroEL has also been proposed. However, direct evidence of GroEL as a ribonuclease (RNase) and its physiological significance has not been fully elucidated. Here, we characterized the role of GroEL in E. coli as an RNase distinct from RNase E/G activity using in vivo reporter assays, in vitro cleavage assays with varying reaction times, divalent ions, and 5′ phosphorylation status. GroEL bound to single-stranded RNA at nanomolar concentrations. Functional analysis of GroEL chaperonin-defective mutants and segments identified specific regions, and the chaperone active status of GroEL is not a necessary factor for RNase activity. Additionally, RNase activity of GroEL was attenuated by co-overexpression with GroES. Finally, we characterized potential transcripts regulated by GroEL and the conserved RNase activity of GroEL in Shigella flexneri. Our findings indicate that GroEL is a novel post-transcriptional regulator in bacteria.

Selective degradation of phage RNAs by the Csm6 ribonuclease provides robust type III CRISPR immunity in Streptococcus thermophilus.

Type III CRISPR immune systems bind viral or plasmid RNA transcripts and activate Csm3/Cmr4 and Cas10 nucleases to uniquely cleave both invader RNA and DNA, respectively. Additionally, type III effector complexes generate cyclic oligoadenylate (cOA) signaling molecules to activate trans-acting, auxiliary Csm6/Csx1 ribonucleases, previously proposed to be non-specific in their in vivo RNA cleavage preference. Despite extensive in vitro studies, the nuclease requirements of type III systems in their native contexts remain poorly understood. Here we systematically investigated the in vivo roles for immunity of each of the three Streptococcus thermophilus (Sth) type III-A Cas nucleases and cOA signaling by challenging nuclease defective mutant strains with plasmid and phage infections. Our results reveal that RNA cleavage by Csm6 is both sufficient and essential for maintaining wild-type levels of immunity. Importantly, Csm6 RNase activity leads to immunity against even high levels of phage challenge without causing host cell dormancy or death. Transcriptomic analyses during phage infection indicated Csm6-mediated and crRNA-directed preferential cleavage of phage transcripts. Our findings highlight the critical role of Csm6 RNase activity in type III immunity and demonstrate specificity for invader RNA transcripts by Csm6 to ensure host cell survival upon phage infection.

MCPIP1 promotes atrial remodeling by exacerbating miR‐26a‐5p/FRAT/Wnt axis–mediated atrial fibrosis in a rat model susceptible to atrial fibrillation

AbstractAtrial fibrosis plays a critical role in the pathogenesis of atrial fibrillation (AF). Monocyte chemotactic protein–induced protein‐1 (MCPIP1), recognized as a functional ribonuclease (RNase), exacerbates cardiac remodeling and contributes to a range of cardiovascular diseases. However, the involvement of MCPIP1 in atrial fibrosis and development of AF, along with its underlying biological mechanisms, remains poorly understood. This study demonstrated that knockdown of MCPIP1 significantly reduced AF inducibility, decreased left atrial diameter, and ameliorated atrial fibrosis, coinciding with reduced FRAT1/2/Wnt/β‐catenin signaling. Furthermore, the MCPIP1‐D141N mutation attenuated AF vulnerability and atrial remodeling compared to MCPIP1 overexpression in an acetylcholine and calcium chloride (ACh‐CaCl2)–induced rat model of AF. Conversely, overexpression of FRAT1/2 partially reversed the cardioprotective effects of MCPIP1‐D141N mutation. Using H9C2 cell lines, we observed that MCPIP1 may induce a transcriptional effect that downregulates miR‐26a‐5p expression, and luciferase and RNA immunoprecipitation (RIP) assays substantiated the direct interaction between miR‐26a‐5p and FRAT1/2. Moreover, overexpression of miR‐26a‐5p countered MCPIP1‐induced atrial remodeling and attenuated the progression of AF. In conclusion, these findings indicate that MCPIP1 facilitates atrial remodeling and the progression of AF by exacerbating miR‐26a‐5p/FRAT/Wnt axis–mediated atrial fibrosis through its RNase activity in an ACh‐CaCl2–induced rat model of AF.

Long Dynamic β1-β2 Loops in M. tb MazF Toxins Affect the Interaction Modes and Strengths of the Toxin-Antitoxin Pairs.

Tuberculosis is a worldwide plague caused by the pathogen Mycobacterium tuberculosis (M. tb). Toxin-antitoxin (TA) systems are genetic elements abundantly present in prokaryotic organisms and regulate important cellular processes. MazEF is a TA system implicated in the formation of "persisters cells" of M. tb, which contain more than 10 such members. However, the exact function and inhibition mode of each MazF are not fully understood. Here we report crystal structures of MazF-mt3 in its apo form and in complex with the C-terminal half of MazE-mt3. Structural analysis suggested that two long but disordered β1-β2 loops would interfere with the binding of the cognate MazE-mt3 antitoxin. Similar loops are also present in the MazF-mt1 and -mt9 but are sustainably shortened in other M. tb MazF members, and these TA pairs behave distinctly in terms of their binding modes and their RNase activities. Systematic crystallographic and biochemical studies further revealed that the biochemical activities of M. tb toxins were combined results between the interferences from the characteristic loops and the electrostatic interactions between the cognate TA pairs. This study provides structural insight into the binding mode and the inhibition mechanism of the MazE/F TA pairs, which facilitate the structure-based peptide designs.

Paper‐Based RNase Digestion toward Viral Nucleic Acid Self‐Tests

AbstractHome‐based Nucleic Acid Amplification Tests (NAATs) for viral infections would be an important step to improve public health, but sample preparation remains an important obstacle, particularly for the protection of target RNA from RNases in samples. Here, a new method for RNase deactivation in saliva samples is presented. This method uses Proteinase K (PK) immobilized on nitrocellulose membrane to store and deliver the enzyme, capable of digesting nucleases present in the sample. The immobilized PK is also separated from the amplification reagents, so that it does not disrupt DNA amplification, thus omitting the need for heat‐deactivation or dilution steps. Treatment by PK nitrocellulose at 50 °C dramatically decreases the RNase activity of RNase A, in diluted saliva samples, and even shows promising results at 42 °C. The potential of this method to protect RNA from digestion is further demonstrated, by pretreating diluted saliva samples spiked with Influenza Virus A (IVA) genomic RNA, which allows its amplification and colorimetric detection by lateral flow strips. In the absence of PK pretreatment, the RNA is digested by RNase, which leads to false negative results. These findings show that immobilized PK enables the integration of sample preparation of viral samples toward home‐based NAATs.

RPA transforms RNase H1 to a bidirectional exoribonuclease for processive RNA–DNA hybrid cleavage

RNase H1 has been acknowledged as an endoribonuclease specializing in the internal degradation of the RNA moiety within RNA–DNA hybrids, and its ribonuclease activity is indispensable in multifaceted aspects of nucleic acid metabolism. However, the molecular mechanism underlying RNase H1-mediated hybrid cleavage remains inadequately elucidated. Herein, using single-molecule approaches, we probe the dynamics of the hybrid cleavage by Saccharomyces cerevisiae RNase H1. Remarkably, a single RNase H1 enzyme displays 3′-to-5′ exoribonuclease activity. The directional RNA degradation proceeds processively and yet discretely, wherein unwinding approximately 6-bp hybrids as a prerequisite for two consecutive 3-nt RNA excisions limits the overall rate within each catalytic cycle. Moreover, Replication Protein A (RPA) reinforces RNase H1’s 3′-to-5′ nucleolytic rate and processivity and stimulates its 5′-to-3′ exoribonuclease activity. This stimulation is primarily realized through the pre-separation of the hybrids and consequently transfers RNase H1 to a bidirectional exoribonuclease, further potentiating its cleavage efficiency. These findings unveil unprecedented characteristics of an RNase and provide a dynamic view of RPA-enhanced processive hybrid cleavage by RNase H1.

Biofilms from the Kapova Cave Walls as a Source of Hydrolase Producers

The studies of bacterial communities from extreme econiches are presently aimed mainly at analyzing the biodiversity of microorganisms using molecular biology methods. Cultivated bacteria from karst caves represent a unique group of microorganisms, the biochemical potential of which has been poorly studied. In the present work, bacteria from biofilms on the walls of the Kapova Cave (Shulgan-Tash Nature Reserve, Bashkortostan) were isolated and characterized in order to assess the ability of identified isolates to produce extracellular hydrolytic enzymes. Most of the isolates (89%) were members of the phylum Proteobacteria, with the remaining ones belonging to the phyla Actinobacteria, Firmicutes, and Bacteroidetes, which accounted for 5, 4, and 2% of the isolates, respectively. Strains with high levels of secreted protease, RNase, and amylase activity were identified as Stenotrophomonas rhizophila, Lysinibacillus fusiformis, and Pseudomonas stutzeri, respectively.

S. mansoni -derived omega-1 prevents OVA-specific allergic airway inflammation via hampering of cDC2 migration.

Chronic infection with Schistosoma mansoni parasites is associated with reduced allergic sensitization in humans, while schistosome eggs protects against allergic airway inflammation (AAI) in mice. One of the main secretory/excretory molecules from schistosome eggs is the glycosylated T2-RNAse Omega-1 (ω1). We hypothesized that ω1 induces protection against AAI during infection. Peritoneal administration of ω1 prior to sensitization with Ovalbumin (OVA) reduced airway eosinophilia and pathology, and OVA-specific Th2 responses upon challenge, independent from changes in regulatory T cells. ω1 was taken up by monocyte-derived dendritic cells, mannose receptor (CD206)-positive conventional type 2 dendritic cells (CD206+ cDC2), and by recruited peritoneal macrophages. Additionally, ω1 impaired CCR7, F-actin, and costimulatory molecule expression on myeloid cells and cDC2 migration in and ex vivo, as evidenced by reduced OVA+ CD206+ cDC2 in the draining mediastinal lymph nodes (medLn) and retainment in the peritoneal cavity, while antigen processing and presentation in cDC2 were not affected by ω1 treatment. Importantly, RNAse mutant ω1 was unable to reduce AAI or affect DC migration, indicating that ω1 effects are dependent on its RNAse activity. Altogether, ω1 hampers migration of OVA+ cDC2 to the draining medLn in mice, elucidating how ω1 prevents allergic airway inflammation in the OVA/alum mouse model.

Visual Evidence for the Recruitment of Four Enzymes with RNase Activity to the Bacillus subtilis Replication Forks.

Removal of RNA/DNA hybrids for the maturation of Okazaki fragments on the lagging strand, or due to misincorporation of ribonucleotides by DNA polymerases, is essential for all types of cells. In prokaryotic cells such as Escherichia coli, DNA polymerase 1 and RNase HI are supposed to remove RNA from Okazaki fragments, but many bacteria lack HI-type RNases, such as Bacillus subtilis. Previous work has demonstrated in vitro that four proteins are able to remove RNA from RNA/DNA hybrids, but their actual contribution to DNA replication is unclear. We have studied the dynamics of DNA polymerase A (similar to Pol 1), 5'->3' exonuclease ExoR, and the two endoribonucleases RNase HII and HIII in B. subtilis using single-molecule tracking. We found that all four enzymes show a localization pattern similar to that of replicative DNA helicase. By scoring the distance of tracks to replication forks, we found that all four enzymes are enriched at DNA replication centers. After inducing UV damage, RNase HIII was even more strongly recruited to the replication forks, and PolA showed a more static behavior, indicative of longer binding events, whereas RNase HII and ExoR showed no response. Inhibition of replication by 6(p hydroxyphenylazo)-uracil (HPUra) demonstrated that both RNase HII and RNase HIII are directly involved in the replication. We found that the absence of ExoR increases the likelihood of RNase HIII at the forks, indicating that substrate availability rather than direct protein interactions may be a major driver for the recruitment of RNases to the lagging strands. Thus, B. subtilis replication forks appear to be an intermediate between E. coli type and eukaryotic replication forks and employ a multitude of RNases, rather than any dedicated enzyme for RNA/DNA hybrid removal.