Role Of RNase Research Articles

The Impact of the Major Endoribonucleases RNase E and RNase III and of the sRNA StsR on Photosynthesis Gene Expression in Rhodobacter sphaeroides Is Growth-Phase-Dependent.

Rhodobacter sphaeroides is a facultative phototrophic bacterium that performs aerobic respiration when oxygen is available. Only when oxygen is present at low concentrations or absent are pigment-protein complexes formed, and anoxygenic photosynthesis generates ATP. The regulation of photosynthesis genes in response to oxygen and light has been investigated for decades, with a focus on the regulation of transcription. However, many studies have also revealed the importance of regulated mRNA processing. This study analyzes the phenotypes of wild type and mutant strains and compares global RNA-seq datasets to elucidate the impact of ribonucleases and the small non-coding RNA StsR on photosynthesis gene expression in Rhodobacter. Most importantly, the results demonstrate that, in particular, the role of ribonuclease E in photosynthesis gene expression is strongly dependent on growth phase.

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

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. 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. 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. PWH with tuberculosis disease have elevated plasma ribonuclease activity, which is also associated with HIV disease severity and systemic inflammation.

A novel role of RNase L in the development of nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and affects about 25% of the population globally. NAFLD has the potential to cause significant liver damage in many patients because it can progress to nonalcoholic steatohepatitis (NASH) and cirrhosis, which substantially increases disease morbidity and mortality. Despite the key role of innate immunity in the disease progression, the underlying molecular and pathogenic mechanisms remain to be elucidated. RNase L is a key enzyme in interferon action against viral infection and displays pleiotropic biological functions such as control of cell proliferation, apoptosis, and autophagy. Recent studies have demonstrated that RNase L is involved in innate immunity. In this study, we revealed that RNase L contributed to the development of NAFLD, which further progressed to NASH in a time-dependent fashion after RNase L wild-type (WT) and knockout mice were fed with a high-fat and high-cholesterol diet. RNase L WT mice showed significantly more severe NASH, evidenced by widespread macro-vesicular steatosis, hepatocyte ballooning degeneration, inflammation, and fibrosis, although physiological and biochemical data indicated that both types of mice developed obesity, hyperglycemia, hypercholesterolemia, dysfunction of the liver, and systemic inflammation at different extents. Further investigation demonstrated that RNase L was responsible for the expression of some key genes in lipid metabolism, inflammation, and fibrosis signaling. Taken together, our results suggest that a novel therapeutic intervention for NAFLD may be developed based on regulating the expression and activity of RNase L.

Ribonuclease E strongly impacts bacterial adaptation to different growth conditions

ABSTRACT Adaptation of bacteria to changes in their environment is often accomplished by changes of the transcriptome. While we learned a lot on the impact of transcriptional regulation in bacterial adaptation over the last decades, much less is known on the role of ribonucleases. This study demonstrates an important function of the endoribonuclease RNase E in the adaptation to different growth conditions. It was shown previously that RNase E activity does not influence the doubling time of the facultative phototroph Rhodobacter sphaeroides during chemotrophic growth, however, it has a strong impact on phototrophic growth. To better understand the impact of RNase E on phototrophic growth, we now quantified gene expression by RNA-seq and mapped 5’ ends during chemotrophic growth under high oxygen or low oxygen levels and during phototrophic growth in the wild type and a mutant expressing a thermosensitive RNase E. Based on the RNase E-dependent expression pattern, the RNAs could be grouped into different classes. A strong effect of RNase E on levels of RNAs for photosynthesis genes was observed, in agreement with poor growth under photosynthetic conditions. RNase E cleavage sites and 5’ ends enriched in the rne ts mutant were differently distributed among the gene classes. Furthermore, RNase E affects the level of RNAs for important transcription factors thus indirectly affecting the expression of their regulons. As a consequence, RNase E has an important role in the adaptation of R. sphaeroides to different growth conditions.

New insights into the role of ribonuclease P protein subunit p30 from tumor to internal reference

Ribonuclease P protein subunit p30 (RPP30) is a highly conserved housekeeping gene that exists in many species and tissues throughout the three life kingdoms (archaea, bacteria, and eukaryotes). RPP30 is closely related to a few types of tumors in human diseases but has a very stable transcription level in most cases. Based on this feature, increasing number of studies have used RPP30 as an internal reference gene. Here, the structure and basic functions of RPP30 are summarized and the likely relationship between RPP30 and various diseases in plants and human is outlined. Finally, the current application of RPP30 as an internal reference gene and its advantages over traditional internal reference genes are reviewed. RPP30 characteristics suggest that it has a good prospect of being selected as an internal reference; more work is needed to develop this research avenue.

RNase III, Ribosome Biogenesis and Beyond.

The ribosome is the universal catalyst for protein synthesis. Despite extensive studies, the diversity of structures and functions of this ribonucleoprotein is yet to be fully understood. Deciphering the biogenesis of the ribosome in a step-by-step manner revealed that this complexity is achieved through a plethora of effectors involved in the maturation and assembly of ribosomal RNAs and proteins. Conserved from bacteria to eukaryotes, double-stranded specific RNase III enzymes play a large role in the regulation of gene expression and the processing of ribosomal RNAs. In this review, we describe the canonical role of RNase III in the biogenesis of the ribosome comparing conserved and unique features from bacteria to eukaryotes. Furthermore, we report additional roles in ribosome biogenesis re-enforcing the importance of RNase III.

620 RNase L is a regeneration repressor gene

Mammalian injury responses are characterized by fibrosis and scarring rather than functional regeneration. Limited regenerative capacity in mammals could reflect a loss of pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we performed comprehensive transcript screening in human subjects after laser rejuvenation treatment and cross-referenced these transcripts to those found in mice with enhanced Wound Induced Hair Neogenesis (WIHN), a rare example of mammalian organogenesis. We find the anti-viral endoribonuclease RNase L to be a powerful suppressor of regeneration. Rnasel-/- mice exhibit remarkable regenerative capacity, with elevated WIHN (n=10, p<0.0001) through enhanced IL-36α (n=3, p<0.01). Consistent with the known role of RNase L to stimulate caspase-1, we find that pharmacologic inhibition of caspases promotes regeneration (n=3 versus 4, p<0.001) in a novel IL-36-dependent manner (n=4, n.s.= not significant). Additionally, these responses are not limited to skin but extend to other organs, such as the colon (n=4, p<0.05), suggesting that suppression of regeneration is a fundamental characteristic of epithelial wound healing. Taken together, this work suggests that RNaseL functions as a regeneration repressor gene in a functional tradeoff that prioritizes host antiviral abilities and is a target to enhance healing in multiple epithelial organs, perhaps even during viral infection.

The Role of Ribonuclease 1 and Ribonuclease Inhibitor 1 in Acute Kidney Injury after Open and Endovascular Thoracoabdominal Aortic Aneurysm Repair.

Acute kidney injury (AKI) is one of the most common post-operative complications and is closely associated with increased mortality after open and endovascular thoracoabdominal aortic aneurysm (TAAA) repair. Ribonuclease (RNase) 1 belongs to the group of antimicrobial peptides elevated in septic patients and indicates the prediction of two or more organ failures. The role of RNase 1 and its antagonist RNase inhibitor 1 (RNH1) after TAAA repair is unknown. In this study, we analyzed RNase 1 and RNH1 serum levels in patients undergoing open (n = 14) or endovascular (n = 19) TAAA repair to determine their association with post-operative AKI and in-hospital mortality. Increased RNH1 serum levels after open TAAA repair as compared with endovascular TAAA repair immediately after surgery and 12, 48, and 72 h after surgery (all p < 0.05) were observed. Additionally, elevated RNase 1 and RNH1 serum levels 12, 24, and 48 h after surgery were shown to be significantly associated with AKI (all p < 0.05). RNH1 serum levels before and RNase 1 serum levels 12 h after TAAA repair were significantly correlated with in-hospital mortality (both p < 0.05). On the basis of these findings, RNase 1 and RNH1 may be therapeutically relevant and may represent biomarkers for post-operative AKI and in-hospital mortality.

The Viral Defense Gene RNaseL Acts as a Regeneration Repressor

Mammalian injury responses are characterized by fibrosis and scarring rather than functional regeneration. Limited regenerative capacity in mammals could reflect a loss of pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we performed comprehensive transcript screening in human subjects after laser rejuvenation treatment. We searched for overlapping transcripts also associated with Wound Induced Hair Neogenesis (WIHN), a rare example of regeneration in mice. We found the anti-viral endoribonuclease RNase L to be a powerful suppressor of regeneration. Rnasel-/- mice exhibit remarkable regenerative capacity and accelerated wound healing following injury, through the production of IL-36α. Consistent with the known role of RNase L to stimulate caspase-1 signaling, we find that pharmacologic inhibition of caspases promotes regeneration in a novel IL-36-dependent manner. We also find a gene set of inner root sheath keratins, such as KRT71, that is associated with poor wound healing in mouse and man, and decreases with caspase inhibition. Finally, these responses are not limited to skin, but occur following intestinal injury as well, suggesting that suppression of regeneration is a general characteristic of epithelial wound healing. In all, this work suggests that RNaseL functions as a regeneration repressor gene in a functional tradeoff that prioritizes host antiviral abilities and is a target to enhance healing in multiple epithelial organs, perhaps even during viral infection.

Ribonuclease 1 attenuates septic cardiomyopathy and cardiac apoptosis in a murine model of polymicrobial sepsis.

Septic cardiomyopathy is a life-threatening organ dysfunction caused by sepsis. Ribonuclease 1 (RNase 1) belongs to a group of host-defense peptides that specifically cleave extracellular RNA (eRNA). The activity of RNase 1 is inhibited by ribonuclease-inhibitor 1 (RNH1). However, the role of RNase 1 in septic cardiomyopathy and associated cardiac apoptosis is completely unknown. Here, we show that sepsis resulted in a significant increase in RNH1 and eRNA serum levels compared with those of healthy subjects. Treatment with RNase 1 resulted in a significant decrease of apoptosis, induced by the intrinsic pathway, and TNF expression in murine cardiomyocytes exposed to either necrotic cardiomyocytes or serum of septic patients for 16 hours. Additionally, treatment of septic mice with RNase 1 resulted in a reduction in cardiac apoptosis, TNF expression, and septic cardiomyopathy. These data demonstrate that eRNA plays a crucial role in the pathophysiology of the organ (cardiac) dysfunction in sepsis and that RNase and RNH1 may be new therapeutic targets and/or strategies to reduce the cardiac injury and dysfunction caused by sepsis.

Editorial: Role of Ribonucleases in Immune Response Regulation During Infection and Cancer

Editorial: Role of Ribonucleases in Immune Response Regulation During Infection and Cancer

RNase L Is Involved in Liposaccharide-Induced Lung Inflammation.

RNase L mediates interferon (IFN) function during viral infection and cell proliferation. Furthermore, the role of RNase L in the regulation of gene expression, cell apoptosis, autophagy, and innate immunity has been well established in the last decade. Tissue distribution reveals that RNase L is highly expressed in the lung and other organs. However, the physiological roles of RNase L in the lung are largely unknown. In this study, we found that polysaccharide (LPS)-induced acute lung injury (ALI) was remarkably intensified in mice deficient in RNase L compared to wild type mice under the same condition. Furthermore, we found that RNase L mediated the TLR4 signaling pathway, and regulated the expression of various pro- and anti-inflammatory genes in the lung tissue and blood. Most importantly, RNase L function in macrophages during LPS stimulation may be independent of the 2-5A system. These findings demonstrate a novel role of RNase L in the immune response via an atypical molecular mechanism.

Actinomycin D-Activated RNase L Promotes H2A.X/H2B-Mediated DNA Damage and Apoptosis in Lung Cancer Cells.

Background: Chemotherapy is an essential component for comprehensive cancer treatment, while drug resistance usually fails therapy. DNA repair mechanism of cancer cells restrains the efficacy of therapeutics targeting DNA damage. Investigating target-inducing irreversible cell death of cancer cells may be promising.Methods: The present study used lung cancer cell lines, transplanted tumor model of lung cancers derived from patients with lung adenocarcinoma, and molecular experiments to investigate the effects and mechanism of Actinomycin D (Act D)-activated RNase L in lung canceers.Results: We report that RNase L, when activated by Act D, induces Caspase-3/PARP activation. The latter further enables ROCK-1 to initiate subsequent membrane blebbing and, meanwhile, result in DNA cleavage and cell cycle arrest mediated by H2A.X/H2B-p21 axis, leading to irreversible DNA damage, and apoptosis of lung cancer cells. The present study highlighted the crucial role of RNase L in triggering apoptosis mechanism through the Caspase-3/ROCK-1/PARP/H2A.X+H2B/p21 axis during Act D treatment. Moreover, activation of RNase L suppressed the tumor formation and the induction of lung cancer stem cells.Conclusion: This study unveiled the regulatory function and related mechanism of RNase L and implied the promising application of therapeutics targeting RNase L in lung cancer.

Role of RNase H enzymes in maintaining genome stability in Escherichia coli expressing a steric-gate mutant of pol VICE391

pol VICE391 (RumAʹ2B) is a low-fidelity polymerase that promotes considerably higher levels of spontaneous “SOS-induced” mutagenesis than the related E. coli pol V (UmuDʹ2C). The molecular basis for the enhanced mutagenesis was previously unknown. Using single molecule fluorescence microscopy to visualize pol V enzymes, we discovered that the elevated levels of mutagenesis are likely due, in part, to prolonged binding of RumB to genomic DNA leading to increased levels of DNA synthesis compared to UmuC.We have generated a steric gate pol VICE391 variant (pol VICE391_Y13A) that readily misincorporates ribonucleotides into the E. coli genome and have used the enzyme to investigate the molecular mechanisms of Ribonucleotide Excision Repair (RER) under conditions of increased ribonucleotide-induced stress. To do so, we compared the extent of spontaneous mutagenesis promoted by pol V and pol VICE391 to that of their respective steric gate variants. Levels of mutagenesis promoted by the steric gate variants that are lower than that of the wild-type enzyme are indicative of active RER that removes misincorporated ribonucleotides, but also misincorporated deoxyribonucleotides from the genome.Using such an approach, we confirmed that RNase HII plays a pivotal role in RER. In the absence of RNase HII, Nucleotide Excision Repair (NER) proteins help remove misincorporated ribonucleotides. However, significant RER occurs in the absence of RNase HII and NER. Most of the RNase HII and NER-independent RER occurs on the lagging strand during genome duplication. We suggest that this is most likely due to efficient RNase HI-dependent RER which recognizes the polyribonucleotide tracts generated by pol VICE391_Y13A. These activities are critical for the maintenance of genomic integrity when RNase HII is overwhelmed, or inactivated, as ΔrnhB or ΔrnhB ΔuvrA strains expressing pol VICE391_Y13A exhibit genome and plasmid instability in the absence of RNase HI.

Endoribonucleolytic Cleavage of m6A-Containing RNAs by RNase P/MRP Complex.

N6-methyladenosine (m6A) is the most abundant internal modification in RNAs and plays regulatory roles in a variety of biological and physiological processes. Despite its important roles, the molecular mechanism underlying m6A-mediated gene regulation is poorly understood. Here, we show that m6A-containing RNAs are subject to endoribonucleolytic cleavage via YTHDF2 (m6A reader protein), HRSP12 (adaptor protein), and RNase P/MRP (endoribonucleases). We demonstrate that HRSP12 functions as an adaptor to bridge YTHDF2 and RNase P/MRP, eliciting rapid degradation of YTHDF2-bound RNAs. Transcriptome-wide analyses show that m6A RNAs that are preferentially targeted for endoribonucleolytic cleavage have an HRSP12-binding site and a RNase P/MRP-directed cleavage site upstream and downstream of the YTHDF2-binding site, respectively. We also find that a subset of m6A-containing circular RNAs associates with YTHDF2 in an HRSP12-dependent manner and is selectively downregulated by RNase P/MRP. Thus, our data expand the known functions of RNase P/MRP to endoribonucleolytic cleavage of m6A RNAs.

The dual role of ribonucleases in tumor-host relationship.

Ribonucleases are enzymes that destroy RNA, play an important role in protein synthesis, epigenetic regulation of genetic activity, cell proliferation and apoptosis. Ribonucleases are important antimicrobial, antiviral and immune defense factors. Despite the same biochemical properties, they exhibit unequal, sometimes opposite biological effects. While mostly ribonucleases inhibit cell proliferation, induce apoptosis and inhibit the growth of tumors, some ribonucleases stimulate vascular growth, proliferation and tumor development. RNase inhibitors have an opposite effect. The correct use of these features of RNases can provide additional opportunities for the development of a strategy of targeted influence on tumor growth.

PNPase is involved in the coordination of mRNA degradation and expression in stationary phase cells of Escherichia coli

BackgroundExoribonucleases are crucial for RNA degradation in Escherichia coli but the roles of RNase R and PNPase and their potential overlap in stationary phase are not well characterized. Here, we used a genome-wide approach to determine how RNase R and PNPase affect the mRNA half-lives in the stationary phase. The genome-wide mRNA half-lives were determined by a dynamic analysis of transcriptomes after transcription arrest. We have combined the analysis of mRNA half-lives with the steady-state concentrations (transcriptome) to provide an integrated overview of the in vivo activity of these exoribonucleases at the genome-scale.ResultsThe values of mRNA half-lives demonstrated that the mRNAs are very stable in the stationary phase and that the deletion of RNase R or PNPase caused only a limited mRNA stabilization. Intriguingly the absence of PNPase provoked also the destabilization of many mRNAs. These changes in mRNA half-lives in the PNPase deletion strain were associated with a massive reorganization of mRNA levels and also variation in several ncRNA concentrations. Finally, the in vivo activity of the degradation machinery was found frequently saturated by mRNAs in the PNPase mutant unlike in the RNase R mutant, suggesting that the degradation activity is limited by the deletion of PNPase but not by the deletion of RNase R.ConclusionsThis work had identified PNPase as a central player associated with mRNA degradation in stationary phase.

RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B

ABSTRACTEndoribonuclease Y (RNase Y) is a crucial regulator of virulence in Gram-positive bacteria. In the human pathogen Streptococcus pyogenes, RNase Y is required for the expression of the major secreted virulence factor streptococcal pyrogenic exotoxin B (SpeB), but the mechanism involved in this regulation remains elusive. Here, we demonstrate that the 5′ untranslated region of speB mRNA is processed by several RNases including RNase Y. In particular, we identify two RNase Y cleavage sites located downstream of a guanosine (G) residue. To assess whether this nucleotide is required for RNase Y activity in vivo, we mutated it and demonstrate that the presence of this G residue is essential for the processing of the speB mRNA 5′ UTR by RNase Y. Although RNase Y directly targets and processes speB, we show that RNase Y-mediated regulation of speB expression occurs primarily at the transcriptional level and independently of the processing in the speB mRNA 5′ UTR. To conclude, we demonstrate for the first time that RNase Y processing of an mRNA target requires the presence of a G. We also provide new insights on the speB 5′ UTR and on the role of RNase Y in speB regulation.

HepG2 Attenuation Induced by RNase A Modulates Gene Profiling and Immunophenotypic Characterization of Some Immune Cells Operating in Cancer Vaccine

Hepatocellular carcinoma (HCC) ranks as the third leading cause of cancer death. Attempts to induce an effective immune response against cancer by immunotherapeutic intervention, including activation of dendritic cells (DCs), were established. The present study was undertaken to investigate the attenuation of HepG2 cells using ribonuclease enzyme A (RNase A) as a possible biological factor to sensitize allogenic DCs and lymphocytes isolated from Egyptian HCC patients. Attenuation of HepG2 cells resulted in a significant increase in activated DC and T-lymphocyte markers, upregulation of CD44 gene expression and increased lactate dehydrogenase as well as interleukin-12 levels. In contrast, a significant decrease in mature DCs, B-cells, T-helper, cytotoxic T-cells, and NK-cells, as well as LMP-2 gene expression was recorded. In conclusion, the attenuation of HepG2 cells with RNase A and subsequent pulsation to allogenic DCs and lymphocytes caused a differential immune response. Further studies are recommended to explain the role of RNase A in modulating antigen expression on the tumor cell surface.

RNase E cleavage shapes the transcriptome of Rhodobacter sphaeroides and strongly impacts phototrophic growth.

Bacteria adapt to changing environmental conditions by rapid changes in their transcriptome. This is achieved not only by adjusting rates of transcription but also by processing and degradation of RNAs. We applied TIER-Seq (transiently inactivating an endoribonuclease followed by RNA-Seq) for the transcriptome-wide identification of RNase E cleavage sites and of 5' RNA ends, which are enriched when RNase E activity is reduced in Rhodobacter sphaeroides. These results reveal the importance of RNase E for the maturation and turnover of mRNAs, rRNAs, and sRNAs in this guanine-cytosine-rich α-proteobacterium, some of the latter have well-described functions in the oxidative stress response. In agreement with this, a role of RNase E in the oxidative stress response is demonstrated. A remarkably strong phenotype of a mutant with reduced RNase E activity was observed regarding the formation of photosynthetic complexes and phototrophic growth, whereas there was no effect on chemotrophic growth.