Crosstalk between Regnase-1 and -3 shapes mast cell survival and cytokine expression.

Post-transcriptional regulation of immune-related transcripts by RNA-binding proteins (RBPs) impacts immune cell responses, including mast cell functionality. Despite their importance in immune regulation, the functional role of most RBPs remains to be understood. By manipulating the expression of specific RBPs in murine mast cells, coupled with mass spectrometry and transcriptomic analyses, we found that the Regnase family of proteins acts as a potent regulator of mast cell physiology. Specifically, Regnase-1 is required to maintain basic cell proliferation and survival, whereas both Regnase-1 and -3 cooperatively regulate the expression of inflammatory transcripts upon activation, with Tnf being a primary target in both human and mouse cells. Furthermore, Regnase-3 directly interacts with Regnase-1 in mast cells and is necessary to restrain Regnase-1 expression through the destabilization of its transcript. Overall, our study identifies protein interactors of endogenously expressed Regnase factors, characterizes the regulatory interplay between Regnase family members in mast cells, and establishes their role in the control of mast cell homeostasis and inflammatory responses.

The role of promiscuous molecular recognition in the evolution of RNase-based self-incompatibility in plants.

How do biological networks evolve and expand? We study these questions in the context of the plant collaborative-non-self recognition self-incompatibility system. Self-incompatibility evolved to avoid self-fertilization among hermaphroditic plants. It relies on specific molecular recognition between highly diverse proteins of two families: female and male determinants, such that the combination of genes an individual possesses determines its mating partners. Though highly polymorphic, previous models struggled to pinpoint the evolutionary trajectories by which new specificities evolved. Here, we construct a novel theoretical framework, that crucially affords interaction promiscuity and multiple distinct partners per protein, as is seen in empirical findings disregarded by previous models. We demonstrate spontaneous self-organization of the population into distinct "classes" with full between-class compatibility and a dynamic long-term balance between class emergence and decay. Our work highlights the importance of molecular recognition promiscuity to network evolvability. Promiscuity was found in additional systems suggesting that our framework could be more broadly applicable.

Murine Ribonuclease 6 Limits Bacterial Dissemination during Experimental Urinary Tract Infection.

INTRODUCTION: The ribonuclease (RNase) A superfamily encodes cationic antimicrobial proteins with potent microbicidal activity toward uropathogenic bacteria. Ribonuclease 6 (RNase6) is an evolutionarily conserved, leukocyte-derived antimicrobial peptide with potent microbicidal activity toward uropathogenic Escherichia coli (UPEC), the most common cause of bacterial urinary tract infections (UTIs). In this study, we generated Rnase6-deficient mice to investigate the hypothesis that endogenous RNase 6 limits host susceptibility to UTI. METHODS: We generated a Rnase6EGFP knock-in allele to identify cellular sources of Rnase6 and determine the consequences of homozygous Rnase6 deletion on antimicrobial activity and UTI susceptibility. RESULTS: We identified monocytes and macrophages as the primary cellular sources of Rnase6 in bladders and kidneys of Rnase6EGFP/+ mice. Rnase6 deficiency (i.e., Rnase6EGFP/EGFP) resulted in increased upper urinary tract UPEC burden during experimental UTI, compared to Rnase6+/+ controls. UPEC displayed increased intracellular survival in Rnase6-deficient macrophages. CONCLUSION: Our findings establish that RNase6 prevents pyelonephritis by promoting intracellular UPEC killing in monocytes and macrophages and reinforce the overarching contributions of endogenous antimicrobial RNase A proteins to host UTI defense.

Prezygotic mate selection is only partially correlated with the expression of NaS-like RNases and affects offspring phenotypes.

Nicotiana attenuata styles preferentially select pollen from among accessions with corresponding expression patterns of NaS-like-RNases (SLRs), and the postpollination ethylene burst (PPEB) is an accurate predictor of seed siring success. However, the ecological consequences of mate selection, its effect on the progeny, and the role of SLRs in the control of ethylene signaling remain unknown. We explored the link between the magnitude of the ethylene burst and expression of the SLRs in a set of recombinant inbred lines (RILs), dissected the genetic underpinnings of mate selection through genome-wide association study (GWAS), and examined its outcome for phenotypes in the next generation. We found that high levels of PPEB are associated with the absence of SLR2 in most of the tested RILs. We identified candidate genes potentially involved in the control of mate selection and showed that pollination of maternal genotypes with their favored pollen donors produces offspring with longer roots. When the maternal genotypes are only able to select against nonfavored pollen donors, the selection for such positive traits is abolished. We conclude that plants' ability of mate choice contributes to measurable changes in progeny phenotypes and is thus likely a target of selection.

Self-(in)compatibility in Tunisian apple accessions [Malus domestica. Borkh]: S-genotypes identification and pollen tube growth analysis.

MAIN CONCLUSION: Self-incompatibility studies have revealed a potential use of Tunisian apple resources for crop improvement and modern breeding programs and a likely correlation between the pollen tube growth and flowering period. Apples [Malus domestica. Borkh] exhibit an S-RNase-based gametophytic self-incompatibility (GSI) system. Four primer combinations were used to S-genotype eighteen Tunisian local apple accessions and twelve introduced accessions that served as references. Within the Tunisian local accessions, S2, S3, S7, and S28 S-alleles were the most frequent and were assigned to 14 S-genotypes; among them, S7S28, S3S7, S2S5, and S2S3 were the most abundant. PCA plot showed that population structuring was affected by the S-alleles frequencies and revealed a modern origin of the Tunisian varieties rather than being ancient ones. Nonetheless, the results obtained with 17 SSR markers showed a separate grouping of local Tunisian accessions that calls into question the hypothesis discussed. Pollination experiments showed that the pollen started to germinate within 24 h of pollination but 48 h after pollination in the "El Fessi" accession. The first pollen tubes arrived in the styles within 36 h of pollination in two early flowering accessions known as "Arbi" and "Bokri", and after 72 h of pollination in late flowering "El Fessi" and 48 h after pollination in remaining accessions. The first pollen tube arrests were observed in accessions "Arbi" and "Bokri" within 84 h of pollination, within 108 h of pollination in "El Fessi" and within 108 h of pollination in remaining accessions. In the apple accession called "Boutabgaya," the pollen tubes reached the base of the style within 120 h of pollination without being aborted. Nevertheless, the self-compatible nature of "Boutabgaya" needs more studies to be confirmed. However, our results revealed the malfunction of the female component of the GSI in this accession. To conclude, this work paved the path for further studies to enhance the insight (i) into the relation between the flowering period and the pollen tube growth, (ii) self-compatible nature of "Boutabgaya", and (iii) the origin of the Tunisian apple.

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.

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.

Suppression of TCF4 promotes a ZC3H12A-mediated self-sustaining inflammatory feedback cycle involving IL-17RA/IL-17RE epidermal signaling.

IL-17C is an epithelial cell-derived proinflammatory cytokine whose transcriptional regulation remains unclear. Analysis of the IL17C promoter region identified TCF4 as putative regulator, and siRNA knockdown of TCF4 in human keratinocytes (KCs) increased IL17C. IL-17C stimulation of KCs (along with IL-17A and TNF-α stimulation) decreased TCF4 and increased NFKBIZ and ZC3H12A expression in an IL-17RA/RE-dependent manner, thus creating a feedback loop. ZC3H12A (MCPIP1/Regnase-1), a transcriptional immune-response regulator, also increased following TCF4 siRNA knockdown, and siRNA knockdown of ZC3H12A decreased NFKBIZ, IL1B, IL36G, CCL20, and CXCL1, revealing a proinflammatory role for ZC3H12A. Examination of lesional skin from the KC-Tie2 inflammatory dermatitis mouse model identified decreases in TCF4 protein concomitant with increases in IL-17C and Zc3h12a that reversed following the genetic elimination of Il17c, Il17ra, and Il17re and improvement in the skin phenotype. Conversely, interference with Tcf4 in KC-Tie2 mouse skin increased Il17c and exacerbated the inflammatory skin phenotype. Together, these findings identify a role for TCF4 in the negative regulation of IL-17C, which, alone and with TNF-α and IL-17A, feed back to decrease TCF4 in an IL-17RA/RE-dependent manner. This loop is further amplified by IL-17C-TCF4 autocrine regulation of ZC3H12A and IL-17C regulation of NFKBIZ to promote self-sustaining skin inflammation.

Are all VapC toxins of Mycobacterium tuberculosis endowed with enigmatic RNase activity?

Mycobacterium tuberculosis (M. tb) employs an extensive network of more than 90 toxin-antitoxin systems, and among them, VapC toxins are the most abundant. While most VapCs function as classical RNases with toxic effects, a significant number of them do not exhibit toxicity. However, these non-toxic VapCs may retain specific RNA binding abilities as seen in case of VapC16, leading to ribosome stalling at specific codons and reprofiling M. tb's proteome to aid in the bacterium's survival under different stressful conditions within the host. Here, we challenge the conventional classification of all VapC toxins as RNases and highlight the complexity of M. tb's strategies for survival and adaptation during infection.

MCPIP1 Inhibits Hepatic Stellate Cell Activation in Autocrine and Paracrine Manners, Preventing Liver Fibrosis.

BACKGROUND & AIMS: Hepatic fibrosis is characterized by enhanced deposition of extracellular matrix (ECM), which results from the wound healing response to chronic, repeated injury of any etiology. Upon injury, hepatic stellate cells (HSCs) activate and secrete ECM proteins, forming scar tissue, which leads to liver dysfunction. Monocyte-chemoattractant protein-induced protein 1 (MCPIP1) possesses anti-inflammatory activity, and its overexpression reduces liver injury in septic mice. In addition, mice with liver-specific deletion of Zc3h12a develop features of primary biliary cholangitis. In this study, we investigated the role of MCPIP1 in liver fibrosis and HSC activation. METHODS: We analyzed MCPIP1 levels in patients' fibrotic livers and hepatic cells isolated from fibrotic murine livers. In vitro experiments were conducted on primary HSCs, cholangiocytes, hepatocytes, and LX-2 cells with MCPIP1 overexpression or silencing. RESULTS: MCPIP1 levels are induced in patients' fibrotic livers compared with their nonfibrotic counterparts. Murine models of fibrosis revealed that its level is increased in HSCs and hepatocytes. Moreover, hepatocytes with Mcpip1 deletion trigger HSC activation via the release of connective tissue growth factor. Overexpression of MCPIP1 in LX-2 cells inhibits their activation through the regulation of TGFB1 expression, and this phenotype is reversed upon MCPIP1 silencing. CONCLUSIONS: We demonstrated that MCPIP1 is induced in human fibrotic livers and regulates the activation of HSCs in both autocrine and paracrine manners. Our results indicate that MCPIP1 could have a potential role in the development of liver fibrosis.

Lung Epithelial Regnase-1 Dampens Local Immune Response but Does Not Worsen Susceptibility to Klebsiella pneumoniae.

Klebsiella pneumoniae (KP) presents a global health threat, leading to significant morbidity and mortality due to its multidrug-resistant profile and the limited availability of therapeutic options. To eliminate KP lung infection, the host initiates a robust inflammatory response. One of the host's mechanisms for mitigating excessive inflammation involves the RNA-binding protein regnase-1 (Reg1, MCPIP1, or ZC3H12A). Reg1 has an RNA binding domain that recognizes stem-loop structures in the 3' untranslated region of various proinflammatory transcripts, leading to mRNA decay. However, excessive suppression of inflammation by Reg1 results in suboptimal KP control. Reg1 deficiency within the nonhematopoietic compartment confers resistance to KP in the lung. Given that lung epithelium is crucial for KP resistance, we hypothesized that selective deletion of Reg1 in lung epithelial cells might enhance proinflammatory signals, leading to a better control of KP. Our transcriptomic analysis of epithelial cells in KP-infected wild-type mice revealed the presence of three distinct alveolar type 2 cell (AT2) subpopulations (conventional, inflammatory, and cycling) and enrichment of Reg1 in inflammatory AT2 cells. We conditionally deleted Reg1 in lung AT2 cells (ΔReg1), which amplified the local inflammatory response in the lung and increased macrophage cell numbers compared with controls. However, when ΔReg1 mice were subjected to KP infection, there were no significant differences in bacterial burden or survival compared with controls. These findings suggest that the local inflammatory response enhanced by Reg1 deletion in AT2 cells is insufficient to control KP infection.

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.

Escherichia coli strains with precise domain deletions in the ribonuclease RNase E can achieve greatly enhanced levels of membrane protein production.

Escherichia coli is one of the most widely utilized hosts for production of recombinant membrane proteins (MPs). Bacterial MP production, however, is usually accompanied by severe toxicity and low-level volumetric accumulation. In previous work, we had discovered that co-expression of RraA, an inhibitor of the RNA-degrading activity of RNase E, can efficiently suppress the cytotoxicity associated with the MP overexpression process and, simultaneously, enhance significantly the cellular accumulation of membrane-incorporated recombinant MPs in bacteria. Based on this, we constructed the specialized MP-producing E. coli strain SuptoxR, which can achieve dramatically enhanced volumetric yields of well-folded recombinant MPs. Ιn the present work, we have investigated whether domain deletions in the E. coli RNase E, which exhibit reduced ribonucleolytic activity, can result in suppressed MP-induced toxicity and enhanced recombinant MP production, in a manner resembling the conditions of rraA overexpression in E. coli SuptoxR. We have found that some strains encoding specific RNase E truncation variants can achieve significantly enhanced levels of recombinant MP production. Among these, we have found a single RNase E variant strain, which can efficiently suppress MP-induced toxicity and achieve greatly enhanced levels of recombinant MP production for proteins of both prokaryotic and eukaryotic origin. Based on its properties, and in analogy to the original SuptoxR strain, we have termed this strain SuptoxRNE22. E. coli SuptoxRNE22 can perform better than commercially available bacterial strains, which are frequently utilized for recombinant MP production. We anticipate that SuptoxRNE22 will become a widely utilized host for recombinant MP production in bacteria.

The p30 protein of the African swine fever virus behaves as an RNase.

The African Swine Fever Virus (ASFV) is responsible for causing African Swine Fever (ASF), a severe contagious disease characterized by hemorrhagic symptoms. The p30 protein of ASFV is the most abundantly expressed viral protein. It is reported to be antigenic and has recognized phosphorylation, glycosylation, and membrane attachment sites, which also shows that the C-terminal region of p30 is more active than the N-terminal region. The present study reports the unique RNase activity of recombinant p30. The RNase activity of p30 was stable at an optimum temperature of 37 °C, and the maximum activity was recorded at pH 7-9 in the presence of monovalent salts. The mutant of p30 (p30m), where cysteine was mutated to alanine at position 109, showed a loss of RNase activity. Our understanding of ASFV biology is significantly less; until now, we have little knowledge about the functions of its proteins. The results of the present study will assist in exploring the biology of ASFV and the role of its protein in counteracting the host immune response.

Hepatic Mcpip1 regulates adaptation to food restriction in mice.

Monocyte-chemoattractant protein-induced protein 1 (MCPIP1, or Regnase-1) is an endoribonuclease that degrades translationally active mRNA molecules. MCPIP1 is mostly known for its anti-inflammatory actions, but it is also an important regulator of adipogenesis and lipid metabolism. Its overexpression impairs adipogenesis by reducing mRNA levels of C/EBPß and PPARγ, key transcription factors regulating this process. Although adipocytes overexpressing MCPIP1 are characterised by impaired glucose uptake, the function of MCPIP1 in hepatocyte metabolism remains unknown. In this study, conditional deletion of Zc3h12a in murine liver epithelial cells was used to characterise the role of Mcpip1 in adaptation to 24-hour food restriction. We found that Mcpip1 deficiency in liver epithelial cells (Mcpip1fl/flAlbCre mice) resulted in higher blood glucose levels in response to fasting in comparison to Mcpip1fl/fl counterparts. Hepatic proteome analysis showed 26 down-regulated and 117 up-regulated proteins in Mcpip1fl/flAlbCre animals that were involved in cellular adhesion, extracellular matrix and metabolic processes. In conclusion, our studies provide new insight into the hepatic function of Mcpip1 and its involvement in metabolic control.

The SC10-RNase promoter displays changes in DNA methylation patterns through pistil development in self-incompatible Nicotiana alata.

In Solanaceae, self-incompatibility is a genetic mechanism that prevents endogamy in plant populations. Expression of the S-determinants, S-RNase, and SLF, is tightly regulated during pistil and pollen development. However, the molecular mechanism of gene expression regulation in S-RNase-based self-incompatibility systems must be better understood. Here, we identified a 1.3 Kbp sequence upstream to the coding region of the functional SC10-RNase allele from the self-incompatible Nicotiana alata, which directs SC10-RNase expression in mature pistils. This SC10-RNase promoter includes a 300 bp region with minimal elements that sustain the SC10-RNase expression. Likewise, a fragment of a transposable element from the Gypsy family of retrotransposons is also present at the -320 bp position. Nevertheless, its presence does not affect the expression of the SC10-RNase in mature pistils. Additionally, we determined that the SC10-RNase promoter undergoes different DNA methylation states during pistil development, being the mCHH methylation context the most frequent close to the transcription start site at pistil maturity. We hypothesized that the Gypsy element at the SC10-RNase promoter might contribute to the DNA methylation remodeling on the three sequence contexts analyzed here. We propose that mCHH methylation enrichment and other regulatory elements in the S-RNase coding region regulate the specific and abundant SC10-RNase expression in mature pistils in N. alata.

Knockdown of double-stranded RNases (dsRNases) enhances oral RNA interference (RNAi) in the corn leafhopper, Dalbulus maidis.

The leafhopper Dalbulus maidis is a harmful pest that causes severe damage to corn crops. Conventional chemical pesticides have negative environmental impacts, emphasizing the need for alternative solutions. RNA interference (RNAi) is a more specific and environmentally friendly method for controlling pests and reducing the negative impacts of current pest management practices. Previous studies have shown that orally administered double-stranded RNA (dsRNA) is less effective than injection protocols in silencing genes. This study focuses on identifying and understanding the role of double-stranded ribonucleases (dsRNases) in limiting the efficiency of oral RNAi in D. maidis. Three dsRNases were identified and characterized, with Dmai-dsRNase-2 being highly expressed in the midgut and salivary glands. An ex vivo degradation assay revealed significant nuclease activity, resulting in high instability of dsRNA when exposed to tissue homogenates. Silencing Dmai-dsRNase-2 improved the insects' response to the dsRNA targeting the gene of interest, providing evidence of dsRNases involvement in oral RNAi efficiency. Therefore, administering both dsRNase-specific and target gene-specific-dsRNAs simultaneously is a promising approach to increase the efficiency of oral RNAi and should be considered in future control strategies.

Human Ribonuclease 6 Has a Protective Role during Experimental Urinary Tract Infection.

Mounting evidence suggests that antimicrobial peptides and proteins (AMPs) belonging to the RNase A superfamily have a critical role in defending the bladder and kidney from bacterial infection. RNase 6 has been identified as a potent, leukocyte-derived AMP, but its impact on urinary tract infection (UTI) in vivo has not been demonstrated. To test the functional role of human RNase 6, we generated RNASE6 transgenic mice and studied their susceptibility to experimental UTI. In addition, we generated bone marrow-derived macrophages to study the impact of RNase 6 on antimicrobial activity within a cellular context. When subjected to experimental UTI, RNASE6 transgenic mice developed reduced uropathogenic Escherichia coli (UPEC) burden, mucosal injury, and inflammation compared to non-transgenic controls. Monocytes and macrophages were the predominant cellular sources of RNase 6 during UTI, and RNASE6 transgenic macrophages were more proficient at intracellular UPEC killing than non-transgenic controls. Altogether, our findings indicate a protective role for human RNase 6 during experimental UTI.