RNA editing and immune control: from mechanism to therapy.

Adenosine-to-inosine RNA editing, catalyzed by the enzymes ADAR1 and ADAR2, stands as a pervasive RNA modification. A primary function of ADAR1-mediated RNA editing lies in labeling endogenous double-stranded RNAs (dsRNAs) as 'self', thereby averting their potential to activate innate immune responses. Recent findings have highlighted additional roles of ADAR1, independent of RNA editing, that are crucial for immune control. Here, we focus on recent progress in understanding ADAR1's RNA editing-dependent and -independent roles in immune control. We describe how ADAR1 regulates various dsRNA innate immune receptors through distinct mechanisms. Furthermore, we discuss the implications of ADAR1 and RNA editing in diseases, including autoimmune diseases and cancers.

ADAR1p150 prevents MDA5 and PKR activation via distinct mechanisms to avert fatal autoinflammation.

Effective immunity requires the innate immune system to distinguish foreign nucleic acids from cellular ones. Cellular double-stranded RNAs (dsRNAs) are edited by the RNA-editing enzyme ADAR1 to evade being recognized as viral dsRNA by cytoplasmic dsRNA sensors, including MDA5 and PKR. The loss of ADAR1-mediated RNA editing of cellular dsRNA activates MDA5. Additional RNA-editing-independent functions of ADAR1 have been proposed, but a specific mechanism has not been delineated. We now demonstrate that the loss of ADAR1-mediated RNA editing specifically activates MDA5, whereas loss of the cytoplasmic ADAR1p150 isoform or its dsRNA-binding activity enabled PKR activation. Deleting both MDA5 and PKR resulted in complete rescue of the embryonic lethality of Adar1p150-/- mice to adulthood, contrasting with the limited or no rescue by removing MDA5 or PKR alone. Our findings demonstrate that MDA5 and PKR are the primary in vivo effectors of fatal autoinflammation following the loss of ADAR1p150.

Protocol for rapidly inducing genome-wide RNA Pol II hyperphosphorylation by selectively disrupting INTAC phosphatase activity.

While several inhibitors targeting RNA polymerase II (Pol II) kinases have been applied for inhibiting RNA Pol II phosphorylation, there are few approaches for inducing RNA Pol II hyperphosphorylation. Here, we present a protocol for constructing the INTS8 degradation tag (dTAG) system combined with ectopic expression of N-terminally truncated INTS8 (INTS8-ΔN) in DLD-1 cells. We describe steps for INTS8-dTAG cell line construction, validation of knockin and degradation, and INTS8-ΔN rescue. We then detail validation of RNA Pol II phosphorylation upregulation. For complete details on the use and execution of this protocol, please refer to Hu et al. (2023).1.

A case of duplicated inferior vena cava with bilateral iliac vein compression.

Duplicated inferior vena cava with bilateral iliac vein compression is extremely rare. We report a case of an 87-year-old man presented with bilateral lower extremity swelling, who was noted to have duplicated inferior vena cava, as revealed by computed tomography angiography (CTA). This revealed bilateral iliac vein compression caused by surrounding structures. Anticoagulant treatment combined with stent implantation completely alleviated this chronic debilitating condition during the follow-up of 2 months with no recurrence.

INTAC endonuclease and phosphatase modules differentially regulate transcription by RNA polymerase II.

Gene expression in metazoans is controlled by promoter-proximal pausing of RNA polymerase II, which can undergo productive elongation or promoter-proximal termination. Integrator-PP2A (INTAC) plays a crucial role in determining the fate of paused polymerases, but the underlying mechanisms remain unclear. Here, we establish a rapid degradation system to dissect the functions of INTAC RNA endonuclease and phosphatase modules. We find that both catalytic modules function at most if not all active promoters and enhancers, yet differentially affect polymerase fate. The endonuclease module induces promoter-proximal termination, with its disruption leading to accumulation of elongation-incompetent polymerases and downregulation of highly expressed genes, while elongation-competent polymerases accumulate at lowly expressed genes and non-coding elements, leading to their upregulation. The phosphatase module primarily prevents the release of paused polymerases and limits transcriptional activation, especially for highly paused genes. Thus, both INTAC catalytic modules have unexpectedly general yet distinct roles in dynamic transcriptional control.

ADAR1p150 Prevents MDA5 and PKR Activation via Distinct Mechanisms to Avert Fatal Autoinflammation.

Effective immunity requires the innate immune system to distinguish foreign (non-self) nucleic acids from cellular (self) nucleic acids. Cellular double-stranded RNAs (dsRNAs) are edited by the RNA editing enzyme ADAR1 to prevent their dsRNA structure pattern being recognized as viral dsRNA by cytoplasmic dsRNA sensors including MDA5, PKR and ZBP1. A loss of ADAR1-mediated RNA editing of cellular dsRNA activates MDA5. However, additional RNA editing-independent functions of ADAR1 have been proposed, but a specific mechanism has not been delineated. We now demonstrate that the loss of ADAR1-mediated RNA editing specifically activates MDA5, while loss of the cytoplasmic ADAR1p150 isoform or its dsRNA binding activity enabled PKR activation. Deleting both MDA5 and PKR resulted in complete rescue of the embryonic lethality of Adar1p150 -/- mice to adulthood, contrasting with the limited or no rescue by removing MDA5, PKR or ZBP1 alone, demonstrating that this is a species conserved function of ADAR1p150. Our findings demonstrate that MDA5 and PKR are the primary in vivo effectors of fatal autoinflammation following the loss of ADAR1p150.

Coordinated regulation of RNA polymerase II pausing and elongation progression by PAF1.

Pleiotropic transcription regulator RNA polymerase II (Pol II)-associated factor 1 (PAF1) governs multiple transcriptional steps and the deposition of several epigenetic marks. However, it remains unclear how ultimate transcriptional outcome is determined by PAF1 and whether it relates to PAF1-controlled epigenetic marks. We use rapid degradation systems and reveal direct PAF1 functions in governing pausing partially by recruiting Integrator-PP2A (INTAC), in addition to ensuring elongation. Following acute PAF1 degradation, most destabilized polymerase undergoes effective release, which presumably relies on skewed balance between INTAC and P-TEFb, resulting in hyperphosphorylated substrates including SPT5. Impaired Pol II progression during elongation, along with altered pause release frequency, determines the final transcriptional outputs. Moreover, PAF1 degradation causes a cumulative decline in histone modifications. These epigenetic alterations in chromatin likely further influence the production of transcripts from PAF1 target genes.

SPT5 stabilizes RNA polymerase II, orchestrates transcription cycles, and maintains the enhancer landscape.

Transcription progression is governed by multitasking regulators including SPT5, an evolutionarily conserved factor implicated in virtually all transcriptional steps from enhancer activation to termination. Here we utilize a rapid degradation system and reveal crucial functions of SPT5 in maintaining cellular and chromatin RNA polymerase II (Pol II) levels. Rapid SPT5 depletion causes a pronounced reduction of paused Pol II at promoters and enhancers, distinct from negative elongation factor (NELF) degradation resulting in short-distance paused Pol II redistribution. Most genes exhibit downregulation, but not upregulation, accompanied by greatly impaired transcription activation, altered chromatin landscape at enhancers, and severe Pol II processivity defects at gene bodies. Phosphorylation of an SPT5 linker at serine 666 potentiates pause release and is antagonized by Integrator-PP2A (INTAC) targeting SPT5 and Pol II, while phosphorylation of the SPT5 C-terminal region links to 3' end termination. Our findings position SPT5 as an essential positive regulator of global transcription.

Identification of Integrator-PP2A complex (INTAC), an RNA polymerase II phosphatase.

The 14-subunit metazoan-specific Integrator contains an endonuclease that cleaves nascent RNA transcripts. Here, we identified a complex containing Integrator and protein phosphatase 2A core enzyme (PP2A-AC), termed INTAC. The 3.5-angstrom-resolution structure reveals that nine human Integrator subunits and PP2A-AC assemble into a cruciform-shaped central scaffold formed by the backbone and shoulder modules, with the phosphatase and endonuclease modules flanking the opposite sides. As a noncanonical PP2A holoenzyme, the INTAC complex dephosphorylates the carboxy-terminal repeat domain of RNA polymerase II at serine-2, -5, and -7 and thus regulates transcription. Our study extends the function of PP2A to transcriptional regulation and reveals how dual enzymatic activities-RNA cleavage and RNA polymerase II dephosphorylation-are structurally and functionally integrated into the INTAC complex.

Simultaneous removal of chromium(VI) and tetracycline hydrochloride from simulated wastewater by nanoscale zero-valent iron/copper-activated persulfate.

In this paper, metallic copper (Cu) was supported on nanoscale zero-valent iron (nZVI) to form a nanoscale bimetallic composite (nZVI-Cu), which was used to activate persulfate (PS) to simultaneously remove the compound contaminants Cr(VI) and tetracycline hydrochloride (TCH) in simulated wastewater. nZVI, nZVI-Cu, and nZVI-Cu-activated PS (nZVI-Cu/PS) were characterized by SEM, TEM, XRD, and XPS. The effects of the bimetallic composite on Cr(VI) and TCH removal were compared in the nZVI, nZVI-activated PS (nZVI/PS), nZVI-Cu, and nZVI-Cu/PS systems. The results showed that nZVI and Cu can form a nanobimetallic system, which can create galvanic cells; thus, the galvanic corrosion of nZVI and the transfer of electrons are accelerated. For a single contaminant, the removal efficiency of Cr(VI) and TCH is the highest when nZVI is loaded with 3 wt% and 1 wt% Cu, respectively. The ratio of nZVI-Cu with 3 wt% Cu to PS is 7:1, and the removal efficiency of Cr(VI) and TCH compound contaminants is ~ 100% after 60 min under acidic conditions, which indicates that the Cr(VI) reduction and TCH oxidation were complete in the nZVI-Cu/PS system. The mechanisms of simultaneous removal of Cr(VI) and TCH in the nZVI-Cu/PS system are proposed. The removal of Cr is because of the adsorption-reduction effects of the nZVI-Cu bimetallic material. The degradation of TCH is mainly due to the action of oxidative free radicals generated by Fe2+-activated PS. The free radical capture experiments showed that SO- 4· plays a major role in the process of TCH degradation.

Fish consumption could reduce the risk of oral cancer in Europeans: A meta-analysis.

OBJECTIVE: More and more researches focused on studying the relationship of fish consumption with the risk of oral cancer, among which discrepancies have risen. The current study aimed to evaluate the possible relationship about fish consumption and oral cancer risk. METHODS: The PubMed, Web of Science and Chinese National Knowledge Infrastructure were searched up to May 31th, 2019. Pooled odds ratios (OR) and 95% confidence intervals (CI) was calculated using a random-effect model by Stata 12.0 software. RESULTS: A total of 15 publications involving 5211 cases and 70,005 participants were used in the current study. Overall, a significantly reduced association about fish consumption on oral cancer risk was found in all included subjects (OR = 0.74, 95% CI = 0.64-0.85). Subgroup analysis by geographic location suggested that highest category of fish consumption compared with lowest category had an inverse association on oral cancer risk in Europeans only (OR = 0.67, 95% CI = 0.55-0.82), instead of other populations. No publication bias was detected. CONCLUSIONS: In summary, our results indicated that fish consumption may contribute to the lower development of oral cancer in European populations, instead of other populations.

Disruption of nuclear speckles reduces chromatin interactions in active compartments.

BACKGROUND: Nuclei of eukaryotes contain various higher-order chromatin architectures and nuclear bodies (NBs), which are critical for proper nuclear functions. Recent studies showed that active chromatin regions are associated with nuclear speckles (NSs), a type of NBs involved in RNA processing. However, the functional roles of NSs in 3D genome organization remain unclear. RESULTS: Using mouse hepatocytes as the model, we knocked down SRRM2, a core protein component scaffolding NSs, and performed Hi-C experiments to examine genome-wide chromatin interactions. We found that Srrm2 depletion disrupted the NSs and changed the expression of 1282 genes. The intra-chromosomal interactions were decreased in type A (active) compartments and increased in type B (repressive) compartments. Furthermore, upon Srrm2 knockdown, the insulation of TADs was decreased specifically in active compartments, and the most significant reduction occurred in A1 sub-compartments. Interestingly, the change of intra-TAD chromatin interactions upon Srrm2 depletion was not associated with the alteration of gene expression. CONCLUSIONS: We show that disruption of NSs by Srrm2 knockdown causes a global decrease in chromatin interactions in active compartments, indicating critical functions of NSs in the organization of the 3D genome.

Genome-wide screening of NEAT1 regulators reveals cross-regulation between paraspeckles and mitochondria.

The long noncoding RNA NEAT1 (nuclear enriched abundant transcript 1) nucleates the formation of paraspeckles, which constitute a type of nuclear body with multiple roles in gene expression. Here we identify NEAT1 regulators using an endogenous NEAT1 promoter-driven enhanced green fluorescent protein reporter in human cells coupled with genome-wide RNAi screens. The screens unexpectedly yield gene candidates involved in mitochondrial functions as essential regulators of NEAT1 expression and paraspeckle formation. Depletion of mitochondrial proteins and treatment of mitochondrial stressors both lead to aberrant NEAT1 expression via ATF2 as well as altered morphology and numbers of paraspeckles. These changes result in enhanced retention of mRNAs of nuclear-encoded mitochondrial proteins (mito-mRNAs) in paraspeckles. Correspondingly, NEAT1 depletion has profound effects on mitochondrial dynamics and function by altering the sequestration of mito-mRNAs in paraspeckles. Overall, our data provide a rich resource for understanding NEAT1 and paraspeckle regulation, and reveal a cross-regulation between paraspeckles and mitochondria.

Improvement of wheat seed vitality by dielectric barrier discharge plasma treatment.

Influences of discharge voltage on wheat seed vitality were investigated in a dielectric barrier discharge (DBD) plasma system at atmospheric pressure and temperature. Six different treatments were designed, and their discharge voltages were 0.0, 9.0, 11.0, 13.0, 15.0, and 17.0 kV, respectively. Fifty seeds were exposed to the DBD plasma atmosphere with an air flow rate of 1.5 L min-1 for 4 min in each treatment, and then the DBD plasma-treated seeds were prepared for germination in several Petri dishes. Each treatment was repeated three times. Germination indexes, growth indexes, surface topography, water uptake, permeability, and α-amylase activity were measured. DBD plasma treatment at appropriate energy levels had positive effects on wheat seed germination and seedling growth. The germination potential, germination index, and vigor index significantly increased by 31.4%, 13.9%, and 54.6% after DBD treatment at 11.0 kV, respectively, in comparison to the control. Shoot length, root length, dry weight, and fresh weight also significantly increased after the DBD plasma treatment. The seed coat was softened and cracks were observed, systematization of the protein was strengthened, and amount of free starch grain increased after the DBD plasma treatment. Water uptake, relative electroconductivity, soluble protein, and α-amylase activity of the wheat seed were also significantly improved after the DBD plasma treatment. Roles of active species and ultraviolet radiation generated in the DBD plasma process in wheat seed germination and seedling growth are proposed. Bioelectromagnetics. 39:120-131, 2018. © 2017 Wiley Periodicals, Inc.

Evaluation of activated carbon fiber supported nanoscale zero-valent iron for chromium (VI) removal from groundwater in a permeable reactive column.

An activated carbon fiber supported nanoscale zero-valent iron (ACF-nZVI) composite for Cr(VI) removal from groundwater was synthesized according to the liquid phase reduction method. The techniques of N2 adsorption/desorption, FESEM, EDX, XRD and XPS were used to characterize the ACF-nZVI composite and the interaction between the ACF-nZVI composite and Cr(VI) ions. Batch experiments were conducted to evaluate the effects of several factors, including the amount of nZVI on activated carbon fiber (ACF), pH value, initial Cr(VI) concentration, and co-existing ions on Cr(VI) removal. The results indicate that presence of ACF can inhibit the aggregation of nanoscale zero-valent iron (nZVI) particles and increase its reactivity, and the Cr(VI) removal efficiency increases with increasing amounts of nZVI on ACF and a decrease in the initial Cr(VI) concentration. In acidic conditions, almost 100% of Cr(VI) in solution can be removed after 60 min of reaction, and the removal efficiency decreases with increasing initial pH values. The Cr(VI) removal is also dependent on the co-existing ions. Reusability experiments on ACF-nZVI demonstrate that the ACF-nZVI composite can keep a high reactivity after five successive reduction cycles. The removal mechanisms are proposed as a two-step interaction including the physical adsorption of Cr(VI) on the surface or inner layers of the ACF-nZVI composite and the subsequent reduction of Cr(VI) to Cr(III) by nZVI.

Enhanced removal of humic acid from micro-polluted source water in a surface discharge plasma system coupled with activated carbon.

Surface discharge plasma (SDP) combined with activated carbon (AC) was employed to eliminate dissolved organic matter from micro-polluted source water, with humic acid (HA) as the model pollutant. Synergistic effect on HA removal was observed in the SDP-AC system; HA removal efficiency reached 60.9% within 5-min treatment in the SDP-AC system with 5.0 g AC addition, whereas 16.7 and 17.4% of HA were removed in sole SDP system and AC adsorption, respectively. Scanning electron microscope and Boehm titration analysis showed that chemical reactions between active species and functional groups of AC occurred. The existence of isopropanol or benzoquinone exhibited inhibitive effects on HA removal in the SDP system, while these inhibitive effects were weakened in the SDP-AC system. The influences of AC on ozone equivalent concentration and H2O2 concentration were evaluated, and there were approximately 39 and 20% decline in ozone equivalent concentration and H2O2 concentration within 6-min treatment in the SDP-AC system, respectively, compared with those in the sole SDP system. Dissolved organic carbon, specific ultraviolet absorbance, and UV absorption ratios analysis demonstrated that the SDP treatment destroyed the chromophoric groups, double bonds, and aromatic structure of HA molecules, and these destructive actions were strengthened by AC.

Source apportionment of perfluoroalkyl substances in surface sediments from lakes in Jiangsu Province, China: Comparison of three receptor models.

Receptor models have been proved as useful tools to identify source categories and quantitatively calculate the contributions of extracted sources. In this study, sixty surface sediment samples were collected from fourteen lakes in Jiangsu Province, China. The total concentrations of C4-C14-perfluoroalkyl carboxylic acids and perfluorooctane sulfonic acid (∑12PFASs) in sediments ranged from 0.264 to 4.44ng/gdw (dry weight), with an average of 1.76ng/gdw. Three commonly-applied receptor models, namely principal component analysis-multiple linear regression (PCA-MLR), positive matrix factorization (PMF) and Unmix models, were employed to apportion PFAS sources in sediments. Overall, these three models all could well track the ∑12PFASs concentrations as well as the concentrations explained in sediments. These three models identified consistently four PFAS sources: the textile treatment sources, the fluoropolymer processing aid/fluororesin coating sources, the textile treatment/metal plating sources and the precious metal sources, contributing 28.1%, 37.0%, 29.7% and 5.3% by PCA-MLR model, 30.60%, 39.3%, 22.4% and 7.7% by PMF model, and 20.6%, 52.4%, 20.2% and 6.8% by Unmix model to the ∑12PFASs, respectively. Comparative statistics of multiple analytical methods could minimize individual-method weaknesses and provide convergent results to enhance the persuasiveness of the conclusions. The findings could give us a better knowledge of PFAS sources in aquatic environments.

Synthesis and photocatalytic performance of reduced graphene oxide-TiO2 nanocomposites for orange II degradation under UV light irradiation.

To enhance the photocatalytic activity of TiO2, reduced graphene oxide-TiO2 (RGO-TiO2) composites with sandwich-like structure were synthesized using a simple solvothermal method. The morphology, crystalline information, and structural property of the photocatalyst were characterized by field emission scanning electron microscopy, transmission electron microscopy, X-ray diffraction, and Fourier transmission infrared spectroscopy. The photocatalytic performances of the RGO-TiO2 composites were evaluated by the degradation of orange II (AO7) in water under UV light irradiation. The results showed that the RGO-TiO2 composites exhibited much higher photocatalytic activity than TiO2 and that the removal efficiency of AO7 could reach above 95% only after 20 min of UV light irradiation under the optimum condition. The improved photocatalytic activity might be attributed to the improved charge transfer and significant separation of the photoinduced electrons and holes in the presence of a two-dimensional graphene network. The results of recycling experiments show that RGO-TiO2 composites have a high photostability, which is expected in the practical application. Radical trapping experiments indicated that ·OH plays a crucial role in the process of AO7 degradation.

Hydrolysis mechanism of carbendazim hydrolase from the strain Microbacterium sp. djl-6F.

The carbendazim (MBC) hydrolyzing enzyme gene was cloned and heterologously expressed in Escherichia coli BL21 (DE3) from a newly isolated MBC-degrading bacterium strain Microbacterium sp. strain djl-6F. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis revealed that purified MheI-6F protein catalyzes direct hydrolysis of MBC into 2-aminobenzimidazole (2-AB) with a high turnover rate and moderate affinity (Km of 6.69µmol/L and kcat of 160.88/min) without the need for any cofactors. The optimal catalytic condition of MheI-6F was identified as 45°C, pH7.0. The enzymatic activity of MheI-6F was found to be diminished by metal ions, and strongly inhibited by sodium dodecyl sulfate (SDS). Through generating amino acid mutations in MheI-6F, Cys16 and Cys222 were identified as the catalytic groups that are essential for the hydrolysis of MBC. This is the first report on the biodegradation of MBC at the enzymatice level.