Deletion of the mRNA endonuclease Regnase-1 promotes NK cell anti-tumor activity via OCT2-dependent transcription of Ifng.

Limited infiltration and activity of natural killer (NK) and T cells within the tumor microenvironment (TME) correlate with poor immunotherapy responses. Here, we examined the role of the endonuclease Regnase-1 on NK cell anti-tumor activity. NK cell-specific deletion of Regnase-1 (Reg1ΔNK) augmented cytolytic activity and interferon-gamma (IFN-γ) production in vitro and increased intra-tumoral accumulation of Reg1ΔNK-NK cells in vivo, reducing tumor growth dependent on IFN-γ. Transcriptional changes in Reg1ΔNK-NK cells included elevated IFN-γ expression, cytolytic effectors, and the chemokine receptor CXCR6. IFN-γ induced expression of the CXCR6 ligand CXCL16 on myeloid cells, promoting further recruitment of Reg1ΔNK-NK cells. Mechanistically, Regnase-1 deletion increased its targets, the transcriptional regulators OCT2 and IκBζ, following interleukin (IL)-12 and IL-18 stimulation, and the resulting OCT2-IκBζ-NF-κB complex induced Ifng transcription. Silencing Regnase-1 in human NK cells increased the expression of IFNG and POU2F2. Our findings highlight NK cell dysfunction in the TME and propose that targeting Regnase-1 could augment active NK cell persistence for cancer immunotherapy.

Ancestral ribonucleases back in motion for evolutionary-dynamics guided protein design.

The dynamics behavior of a protein is essential for its functionality. Here, Doucet et al. demonstrate how the evolutionary analysis of conformational pathways within a protein family serves to identify common core scaffolds that accommodate branch-specific functional regions controlled by flexibility switches, offering a model for evolutionary-dynamics based protein design.

Molecular mechanism of allosteric activation of the CRISPR ribonuclease Csm6 by cyclic tetra-adenylate.

Type III CRISPR systems are innate immune systems found in bacteria and archaea, which produce cyclic oligoadenylate (cOA) second messengers in response to viral infections. In these systems, Csm6 proteins serve as ancillary nucleases that degrade single-stranded RNA (ssRNA) upon activation by cOA. In addition, Csm6 proteins also possess cOA-degrading activity as an intrinsic off-switch to avoid degradation of host RNA and DNA that would eventually lead to cell dormancy or cell death. Here, we present the crystal structures of Thermus thermophilus (Tt) Csm6 alone, and in complex with cyclic tetra-adenylate (cA4) in both pre- and post-cleavage states. These structures establish the molecular basis of the long-range allosteric activation of TtCsm6 ribonuclease by cA4. cA4 binding induces significant conformational changes, including closure of the CARF domain, dimerization of the HTH domain, and reorganization of the R-X4-6-H motif within the HEPN domain. The cleavage of cA4 by the CARF domain restores each domain to a conformation similar to its apo state. Furthermore, we have identified hyperactive TtCsm6 variants that exhibit sustained cA4-activated RNase activity, showing great promise for their applications in genome editing and diagnostics.

The making and breaking of tRNAs by ribonucleases.

Ribonucleases (RNases) play important roles in supporting canonical and non-canonical roles of tRNAs by catalyzing the cleavage of the tRNA phosphodiester backbone. Here, we highlight how recent advances in cryo-electron microscopy (cryo-EM), protein structure prediction, reconstitution experiments, tRNA sequencing, and other studies have revealed new insight into the nucleases that process tRNA. This represents a very diverse group of nucleases that utilize distinct mechanisms to recognize and cleave tRNA during different stages of a tRNA's life cycle including biogenesis, fragmentation, surveillance, and decay. In this review, we provide a synthesis of the structure, mechanism, regulation, and modes of tRNA recognition by tRNA nucleases, along with open questions for future investigation.

Decreasing the intrinsically disordered protein α-synuclein levels by targeting its structured mRNA with a ribonuclease-targeting chimera.

α-Synuclein is an important drug target for the treatment of Parkinson's disease (PD), but it is an intrinsically disordered protein lacking typical small-molecule binding pockets. In contrast, the encoding SNCA mRNA has regions of ordered structure in its 5' untranslated region (UTR). Here, we present an integrated approach to identify small molecules that bind this structured region and inhibit α-synuclein translation. A drug-like, RNA-focused compound collection was studied for binding to the 5' UTR of SNCA mRNA, affording Synucleozid-2.0, a drug-like small molecule that decreases α-synuclein levels by inhibiting ribosomes from assembling onto SNCA mRNA. This RNA-binding small molecule was converted into a ribonuclease-targeting chimera (RiboTAC) to degrade cellular SNCA mRNA. RNA-seq and proteomics studies demonstrated that the RiboTAC (Syn-RiboTAC) selectively degraded SNCA mRNA to reduce its protein levels, affording a fivefold enhancement of cytoprotective effects as compared to Synucleozid-2.0. As observed in many diseases, transcriptome-wide changes in RNA expression are observed in PD. Syn-RiboTAC also rescued the expression of ~50% of genes that were abnormally expressed in dopaminergic neurons differentiated from PD patient-derived iPSCs. These studies demonstrate that the druggability of the proteome can be expanded greatly by targeting the encoding mRNAs with both small molecule binders and RiboTAC degraders.

Regulation of the Drosophila transcriptome by Pumilio and the CCR4-NOT deadenylase complex.

The sequence-specific RNA-binding protein Pumilio (Pum) controls Drosophila development; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we use knockdown and knockout approaches coupled with RNA-seq to measure the impact of Pum on the transcriptome of Drosophila cells in culture. We also use an improved RNA coimmunoprecipitation method to identify Pum-bound mRNAs in Drosophila embryos. Integration of these data sets with the locations of Pum-binding motifs across the transcriptome reveals novel direct Pum target genes involved in neural, muscle, wing, and germ cell development and in cellular proliferation. These genes include components of Wnt, TGF-ß, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. We identify the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pum-mediated repression, and observe concordant regulation of Pum:CCR4-NOT target mRNAs. Computational modeling reveals that Pum binding, binding site number, clustering, and sequence context are important determinants of regulation. In contrast, we show that the responses of direct mRNA targets to Pum-mediated repression are not influenced by the content of optimal synonymous codons. Moreover, contrary to a prevailing model, we do not detect a role for CCR4-NOT in the degradation of mRNAs with low codon optimality. Together, the results of this work provide new insights into the Pum regulatory network and mechanisms and the parameters that influence the efficacy of Pum-mediated regulation.

IRE1 RNase controls CD95-mediated cell death.

Signalling by the Unfolded Protein Response (UPR) or by the Death Receptors (DR) are frequently activated towards pro-tumoral outputs in cancer. Herein, we demonstrate that the UPR sensor IRE1 controls the expression of the DR CD95/Fas, and its cell death-inducing ability. Both genetic and pharmacologic blunting of IRE1 activity increased CD95 expression and exacerbated CD95L-induced cell death in glioblastoma (GB) and Triple-Negative Breast Cancer (TNBC) cell lines. In accordance, CD95 mRNA was identified as a target of Regulated IRE1-Dependent Decay of RNA (RIDD). Whilst CD95 expression is elevated in TNBC and GB human tumours exhibiting low RIDD activity, it is surprisingly lower in XBP1s-low human tumour samples. We show that IRE1 RNase inhibition limited CD95 expression and reduced CD95-mediated hepatic toxicity in mice. In addition, overexpression of XBP1s increased CD95 expression and sensitized GB and TNBC cells to CD95L-induced cell death. Overall, these results demonstrate the tight IRE1-mediated control of CD95-dependent cell death in a dual manner through both RIDD and XBP1s, and they identify a novel link between IRE1 and CD95 signalling.

Structural insight into the Csx1-Crn2 fusion self-limiting ribonuclease of type III CRISPR system.

In the type III CRISPR system, cyclic oligoadenylate (cOA) molecules act as second messengers, activating various promiscuous ancillary nucleases that indiscriminately degrade host and viral DNA/RNA. Conversely, ring nucleases, by specifically cleaving cOA molecules, function as off-switches to protect host cells from dormancy or death, and allow viruses to counteract immune responses. The fusion protein Csx1-Crn2, combining host ribonuclease with viral ring nuclease, represents a unique self-limiting ribonuclease family. Here, we describe the structures of Csx1-Crn2 from the organism of Marinitoga sp., in both its full-length and truncated forms, as well as in complex with cA4. We show that Csx1-Crn2 operates as a homo-tetramer, a configuration crucial for preserving the structural integrity of the HEPN domain and ensuring effective ssRNA cleavage. The binding of cA4 to the CARF domain triggers significant conformational changes across the CARF, HTH, and into the HEPN domains, leading the two R-X4-6-H motifs to form a composite catalytic site. Intriguingly, an acetate ion was found to bind at this composite site by mimicking the scissile phosphate. Further molecular docking analysis reveals that the HEPN domain can accommodate a single ssRNA molecule involving both R-X4-6-H motifs, underscoring the importance of HEPN domain dimerization for its activation.

Prokaryotic Argonaute nuclease cooperates with co-encoded RNase to acquire guide RNAs and target invader DNA.

Argonautes are an evolutionary conserved family of programmable nucleases that identify target nucleic acids using small guide oligonucleotides. In contrast to eukaryotic Argonautes (eAgos) that act on RNA, most studied prokaryotic Argonautes (pAgos) recognize DNA targets. Similarly to eAgos, pAgos can protect prokaryotic cells from invaders, but the biogenesis of guide oligonucleotides that confer them specificity to their targets remains poorly understood. Here, we have identified a new group of RNA-guided pAgo nucleases and demonstrated that a representative pAgo from this group, AmAgo from the mesophilic bacterium Alteromonas macleodii, binds guide RNAs of varying lengths for specific DNA targeting. Unlike most pAgos and eAgos, AmAgo is strictly specific to hydroxylated RNA guides containing a 5'-adenosine. AmAgo and related pAgos are co-encoded with a conserved RNA endonuclease from the HEPN superfamily (Ago-associated protein, Agap-HEPN). In vitro, Agap cleaves RNA between guanine and adenine nucleotides producing hydroxylated 5'-A guide oligonucleotides bound by AmAgo. In vivo, Agap cooperates with AmAgo in acquiring guide RNAs and counteracting bacteriophage infection. The AmAgo-Agap pair represents the first example of a pAgo system that autonomously produces RNA guides for DNA targeting and antiviral defense, which holds promise for programmable DNA targeting in biotechnology.

Ancestral sequence reconstruction dissects structural and functional differences among eosinophil ribonucleases.

Evolutionarily conserved structural folds can give rise to diverse biological functions, yet predicting atomic-scale interactions that contribute to the emergence of novel activities within such folds remains challenging. Pancreatic-type ribonucleases illustrate this complexity, sharing a core structure that has evolved to accommodate varied functions. In this study, we used ancestral sequence reconstruction to probe evolutionary and molecular determinants that distinguish biological activities within eosinophil members of the RNase 2/3 subfamily. Our investigation unveils functional, structural, and dynamical behaviors that differentiate the evolved ancestral ribonuclease (AncRNase) from its contemporary eosinophil RNase orthologs. Leveraging the potential of ancestral reconstruction for protein engineering, we used AncRNase predictions to design a minimal 4-residue variant that transforms human RNase 2 into a chimeric enzyme endowed with the antimicrobial and cytotoxic activities of RNase 3 members. This work provides unique insights into mutational and evolutionary pathways governing structure, function, and conformational states within the eosinophil RNase subfamily, offering potential for targeted modulation of RNase-associated functions.

Postbiotic lactobacilli induce cutaneous antimicrobial response and restore the barrier to inhibit the intracellular invasion of Staphylococcus aureus in vitro and ex vivo.

Intracellular pathogens including Staphylococcus aureus contribute to the non-healing phenotype of chronic wounds. Lactobacilli, well known as beneficial bacteria, are also reported to modulate the immune system, yet their role in cutaneous immunity remains largely unknown. We explored the therapeutic potential of bacteria-free postbiotics, bioactive lysates of lactobacilli, to reduce intracellular S. aureus colonization and promote healing. Fourteen postbiotics derived from various lactobacilli species were screened, and Latilactobacillus curvatus BGMK2-41 was selected for further analysis based on the most efficient ability to reduce intracellular infection by S. aureus diabetic foot ulcer clinical isolate and S. aureus USA300. Treatment of both infected keratinocytes in vitro and infected human skin ex vivo with BGMK2-41 postbiotic cleared S. aureus. Keratinocytes treated in vitro with BGMK2-41 upregulated expression of antimicrobial response genes, of which DEFB4, ANG, and RNASE7 were also found upregulated in treated ex vivo human skin together with CAMP exclusively upregulated ex vivo. Furthermore, BGMK2-41 postbiotic treatment has a multifaceted impact on the wound healing process. Treatment of keratinocytes stimulated cell migration and the expression of tight junction proteins, while in ex vivo human skin BGMK2-41 increased expression of anti-inflammatory cytokine IL-10, promoted re-epithelialization, and restored the epidermal barrier via upregulation of tight junction proteins. Together, this provides a potential therapeutic approach for persistent intracellular S. aureus infections.

RNA processing by the CRISPR-associated NYN ribonuclease.

CRISPR-Cas systems confer adaptive immunity in prokaryotes, facilitating the recognition and destruction of invasive nucleic acids. Type III CRISPR systems comprise large, multisubunit ribonucleoprotein complexes with a catalytic Cas10 subunit. When activated by the detection of foreign RNA, Cas10 generates nucleotide signalling molecules that elicit an immune response by activating ancillary effector proteins. Among these systems, the Bacteroides fragilis type III CRISPR system was recently shown to produce a novel signal molecule, SAM-AMP, by conjugating ATP and SAM. SAM-AMP regulates a membrane effector of the CorA family to provide immunity. Here, we focus on NYN, a ribonuclease encoded within this system, probing its potential involvement in crRNA maturation. Structural modelling and in vitro ribonuclease assays reveal that NYN displays robust sequence-nonspecific, Mn2+-dependent ssRNA-cleavage activity. Our findings suggest a role for NYN in trimming crRNA intermediates into mature crRNAs, which is necessary for type III CRISPR antiviral defence. This study sheds light on the functional relevance of CRISPR-associated NYN proteins and highlights the complexity of CRISPR-mediated defence strategies in bacteria.

Plasma Ribonuclease Activity in Antiretroviral Treatment-Naive People With Human Immunodeficiency Virus and Tuberculosis Disease.

BACKGROUND: The role of ribonucleases in tuberculosis among people with human immunodeficiency virus (HIV; PWH) is unknown. We explored ribonuclease activity in plasma from PWH with and without tuberculosis. METHODS: Participants were identified from a cohort of treatment-naive PWH in Ethiopia who had been classified for tuberculosis disease (HIV positive [HIV+]/tuberculosis positive [tuberculosis+] or HIV+/tuberculosis negative [tuberculosis-]). Ribonuclease activity in plasma was investigated by quantification of synthetic spike-in RNAs using sequencing and quantitative polymerase chain reaction and by a specific ribonuclease activity assay. Quantification of ribonuclease 1, 2, 3, 6, 7, and T2 proteins was performed by enzyme-linked immunosorbent assay. Ribonuclease activity and protein concentrations were correlated with markers of tuberculosis and HIV disease severity and with concentrations of inflammatory mediators. RESULTS: Ribonuclease activity was significantly higher in plasma of HIV+/tuberculosis+ (n = 51) compared with HIV+/tuberculosis- (n = 78), causing reduced stability of synthetic spike-in RNAs. Concentrations of ribonucleases 2, 3, and T2 were also significantly increased in HIV+/tuberculosis+ compared with HIV+/tuberculosis-. Ribonuclease activity was correlated with HIV viral load, and inversely correlated with CD4 cell count, mid-upper arm circumference, and body mass index. Moreover, ribonuclease activity was correlated with concentrations of interleukin 27, procalcitonin and the kynurenine-tryptophan ratio. CONCLUSIONS: PWH with tuberculosis disease have elevated plasma ribonuclease activity, which is also associated with HIV disease severity and systemic inflammation.

RNS2 is required for the biogenesis of a wounding responsive 16 nts tsRNA in Arabidopsis thaliana.

tRNA-derived small RNAs (tsRNAs), a new category of regulatory small non-coding RNA existing in almost all branches of life, have recently attracted broad attention. Increasing evidence has shown that tsRNAs are not random degradation debris of tRNAs, but products cleaved by specific endoribonucleases, with versatile functions in response to various developmental and environmental cues. However, it is still unclear about the diversity, biogenesis and function of tsRNAs in plants. In this study, we comprehensively profiled 10-60 nts small RNAs in Arabidopsis thaliana leaf with or without wounding stress and identified four 16 nts tiny tRFs (tRNA-derived fragments) sharply increased after wounding, namely tRF5'Ala. Notably, genetic, biochemical and bioinformatic data indicated that RNS2, a member of class II RNase T2 enzymes, was the main endoribonuclease responsible for the biogenesis of tRF5'Ala. Moreover, tRF5'Ala was highly abundant and conserved in Arabidopsis and rice pollen. However, tRF5'Ala did not associate with AGO 1 in vivo or display any inhibitory effect on the translation of a luciferase mRNA in vitro. Altogether, our study highlights the discovery of a novel class of tiny tsRNAs drastically increased under wounding stress as well as their generation by RNS2, which provides a new insight into tsRNAs research in plants.

Involvement of CgHSFB1 in the regulation of self-incompatibility in 'Shatian' pummelo.

As self-incompatibility is a major issue in pummelo breeding and production, its mechanism in citrus was analyzed to improve breeding efficiency and reduce production costs. Rutaceae belongs to S-RNase type of gametophytic self-incompatibility. While the function of S-RNase/SLF and the mechanism of self-incompatibility have been studied extensively, the transcriptional regulation of S-RNase has been less studied. We performed transcriptome sequencing with the styles of 'Shatian' pummelo on the day of anthesis and 1-5 days before anthesis, and found that the transcript level of S-RNase gradually decreased with flower development. By analyzing differentially expressed genes and correlation with the expression trend of S-RNase, we identified a candidate gene, CgHSFB1, and utilized biochemical experiments such as yeast one-hybrid assay, electrophoretic mobility shift assay and dual-luciferase assay, as well as transient transformation of citrus calli and Citrus microcarpa and demonstrated that CgHSFB1 could directly bind to the S1-RNase promoter and repress the expression of S1-RNase, which is involved in the pummelo self-incompatibility response. In contrast, CgHSFB1 did not bind to the promoter of S2-RNase, and there was specificity in the regulation of S-RNase.

G-Quadruplex mRNAs Silencing with Inducible Ribonuclease Targeting Chimera for Precision Tumor Therapy.

Ribonuclease targeting chimera (RIBOTAC) represents an emerging strategy for targeted therapy. However, RIBOTAC that is selectively activated by bio-orthogonal or cell-specific triggers has not been explored. We developed a strategy of inducible RIBOTAC (iRIBOTAC) that enables on-demand degradation of G-quadruplex (G4) RNAs for precision cancer therapy. iRIBOTAC is designed by coupling an RNA G4 binder with a caged ribonuclease recruiter, which can be decaged by a bio-orthogonal reaction, tumor-specific enzyme, or metabolite. A bivalent G4 binder is engineered by conjugating a near-infrared (NIR) fluorescence G4 ligand to a noncompetitive G4 ligand, conferring fluorescence activation on binding G4s with synergistically enhanced affinity. iRIBOTAC is demonstrated to greatly knockdown G4 RNAs upon activation under bio-orthogonal or cell-specific stimulus, with dysregulation of gene expressions involving cell killing, channel regulator activity, and metabolism as revealed by RNA sequencing. This strategy also shows a crucial effect on cell fate with remarkable biochemical hallmarks of apoptosis. Mice model studies demonstrate that iRIBOTAC allows selective imaging and growth suppression of tumors with bio-orthogonal and tumor-specific controls, highlighting G4 RNA targeting and inducible silencing as a valuable RIBOTAC paradigm for cancer therapy.

Transcriptome-Wide, Unbiased Profiling of Ribonuclease Targeting Chimeras.

Various approaches have been developed to target RNA and modulate its function with modes of action including binding and cleavage. Herein, we explored how small molecule binding is correlated with cleavage induced by heterobifunctional ribonuclease targeting chimeras (RiboTACs), where RNase L is recruited to cleave the bound RNA target, in a transcriptome-wide, unbiased fashion. Only a fraction of bound targets was cleaved by RNase L, induced by RiboTAC binding. Global analysis suggested that (i) cleaved targets generally form a region of stable structure that encompasses the small molecule binding site; (ii) cleaved targets have preferred RNase L cleavage sites nearby small molecule binding sites; (iii) RiboTACs facilitate a cellular interaction between cleaved targets and RNase L; and (iv) the expression level of the target influences the extent of cleavage observed. In one example, we converted a binder of LGALS1 (galectin-1) mRNA into a RiboTAC. In MDA-MB-231 cells, the binder had no effect on galectin-1 protein levels, while the RiboTAC cleaved LGALS1 mRNA, reduced galectin-1 protein abundance, and affected galectin-1-associated oncogenic cellular phenotypes. Using LGALS1, we further assessed additional factors including the length of the linker that tethers the two components of the RiboTAC, cellular uptake, and the RNase L-recruiting module on RiboTAC potency. Collectively, these studies may facilitate triangulation of factors to enable the design of RiboTACs.

Pathogenic Roles for RNASET2 in Clear Cell Renal Cell Carcinoma.

A specific splicing isoform of RNASET2 is associated with worse oncologic outcomes in clear cell renal cell carcinoma (ccRCC). However, the interplay between wild-type RNASET2 and its splice variant and how this might contribute to the pathogenesis of ccRCC remains poorly understood. We sought to better understand the relationship of RNASET2 in the pathogenesis of ccRCC and the interplay with a pathogenic splicing isoform (RNASET2-SV) and the tumor immune microenvironment. Using data from The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium, we correlated clinical variables to RNASET2 expression and the presence of a specific RNASET2-SV. Immunohistochemical staining with matched RNA sequencing of ccRCC patients was then utilized to understand the spatial relationships of RNASET2 with immune cells. Finally, in vitro studies were performed to demonstrate the oncogenic role of RNASET2 and highlight its potential mechanisms. RNASET2 gene expression is associated with higher grade tumors and worse overall survival in The Cancer Genome Atlas cohort. The presence of the RNASET2-SV was associated with increased expression of the wild-type RNASET2 protein and epigenetic modifications of the gene. Immunohistochemical staining revealed increased intracellular accumulation of RNASET2 in patients with increased RNA expression of RNASET2-SV. In vitro experiments reveal that this accumulation results in increased cell proliferation, potentially from altered metabolic pathways. RNASET2 exhibits a tumor-promoting role in the pathogenesis of ccRCC that is increased in the presence of a specific RNASET2-SV and associated with changes in the cellular localization of the protein.

Translational arrest and mRNA decay are independent activities of alphaherpesvirus virion host shutoff proteins.

The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek's disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with poly(A)+ in situ hybridization indicating a global depletion of cytoplasmic poly(A)+ RNA and a concomitant increase in nuclear poly(A)+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and poly(A)+ depletion, but rather, induced cytoplasmic G3BP1 and poly(A)+ mRNA- containing granules and phosphorylation of the stress response proteins eIF2α and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single-cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.

Treatment with RNase alleviates brain injury but not neuroinflammation in neonatal hypoxia/ischemia.

There is a need for new treatments to reduce brain injuries derived from neonatal hypoxia/ischemia. The only viable option used in the clinic today in infants born at term is therapeutic hypothermia, which has a limited efficacy. Treatments with exogenous RNase have shown great promise in a range of different adult animal models including stroke, ischemia/reperfusion injury, or experimental heart transplantation, often by conferring vascular protective and anti-inflammatory effects. However, any neuroprotective function of RNase treatment in the neonate remains unknown. Using a well-established model of neonatal hypoxic/ischemic brain injury, we evaluated the influence of RNase treatment on RNase activity, gray and white matter tissue loss, blood-brain barrier function, as well as levels and expression of inflammatory cytokines in the brain up to 6 h after the injury using multiplex immunoassay and RT-PCR. Intraperitoneal treatment with RNase increased RNase activity in both plasma and cerebropinal fluids. The RNase treatment resulted in a reduction of brain tissue loss but did not affect the blood-brain barrier function and had only a minor modulatory effect on the inflammatory response. It is concluded that RNase treatment may be promising as a neuroprotective regimen, whereas the mechanistic effects of this treatment appear to be different in the neonate compared to the adult and need further investigation.