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 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.

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.

Protein arginine methyltransferase CARM1 attenuates the paraspeckle-mediated nuclear retention of mRNAs containing IRAlus.

In many cells, mRNAs containing inverted repeated Alu elements (IRAlus) in their 3' untranslated regions (UTRs) are inefficiently exported to the cytoplasm. Such nuclear retention correlates with paraspeckle-associated protein complexes containing p54(nrb). However, nuclear retention of mRNAs containing IRAlus is variable, and how regulation of retention and export is achieved is poorly understood. Here we show one mechanism of such regulation via the arginine methyltransferase CARM1 (coactivator-associated arginine methyltransferase 1). We demonstrate that disruption of CARM1 enhances the nuclear retention of mRNAs containing IRAlus. CARM1 regulates this nuclear retention pathway at two levels: CARM1 methylates the coiled-coil domain of p54(nrb), resulting in reduced binding of p54(nrb) to mRNAs containing IRAlus, and also acts as a transcription regulator to suppress NEAT1 transcription, leading to reduced paraspeckle formation. These actions of CARM1 work together synergistically to regulate the export of transcripts containing IRAlus from paraspeckles under certain cellular stresses, such as poly(I:C) treatment. This work demonstrates how a post-translational modification of an RNA-binding protein affects protein-RNA interaction and also uncovers a mechanism of transcriptional regulation of the long noncoding RNA NEAT1.

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.

SnoVectors for nuclear expression of RNA.

Many long noncoding RNAs (lncRNAs) are constrained to the nucleus to exert their functions. However, commonly used vectors that were designed to express mRNAs have not been optimized for the study of nuclear RNAs. We reported recently that sno-lncRNAs are not capped or polyadenylated but rather are terminated on each end by snoRNAs and their associated proteins. These RNAs are processed from introns and are strictly confined to the nucleus. Here we have used these features to design expression vectors that can stably express virtually any sequence of interest and constrain its accumulation to the nucleus. Further, these RNAs appear to retain normal nuclear associations and function. SnoVectors should be useful in conditions where nuclear RNA function is studied or where export to the cytoplasm needs to be avoided.

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.

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.

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.

Research progress of long noncoding RNA in China.

RNA is essential for all kingdoms of life and exerts important functions beyond transferring genetic information from DNA to protein. With the advent of the state-of-the-art deep sequencing technology, a large portion of noncoding transcripts in eukaryotic genomes has been broadly identified. Among them, long noncoding RNAs (lncRNAs) have been emerged as a new class of RNA molecules that have regulatory potential in a variety of physiological and pathological processes. Here we summarize recent research progresses that have been made by scientists in China on lncRNAs, including their biogenesis, functional implication and the underlying mechanism of action at the current stage. © 2016 IUBMB Life, 68(11):887-893, 2016.

Enhanced degradation of azo dye in wastewater by pulsed discharge plasma coupled with MWCNTs-TiO2/γ-Al2O3 composite photocatalyst.

In order to improve the photocatalytic performance of TiO2 in pulsed discharge plasma systems, easily recycled multi-walled carbon nanotubes (MWCNTs)-TiO2 supported on γ-Al2O3 (MWCNTs-TiO2/γ-Al2O3) composite photocatalyst were prepared. The morphology and physicochemical properties of the prepared catalysts were investigated using XRD, SEM, FTIR and UV-vis spectroscopy. The photocatalytic activity was evaluated by degradation of azo dye acid orange II (AO7) in wastewater under pulsed discharge plasma. The results indicate that the MWCNTs-TiO2/γ-Al2O3 composite catalyst possesses enhanced photocatalytic activity facilitating the decomposition of AO7 compared with TiO2/γ-Al2O3 composite in pulsed discharge plasma systems. Under pulsed discharge plasma, almost 100% AO7 is degraded by the MWCNTs-TiO2/γ-Al2O3 composite after 60 min at optimal conditions. The degradation efficiency of AO7 is also affected by the dosage of the composite catalyst and pulsed discharge peak voltage. As the amount of MWCNTs-TiO2/γ-Al2O3 composite and pulsed discharge peak voltage increases, the degradation efficiency of AO7 increases. The photocatalyst was implemented for 6 cycles and the degradation efficiency of AO7 remains higher than 85% under pulsed discharge plasma. Results indicate that the catalyst displays easy separation and minimal deactivation after several uses. Possible decomposition mechanisms were also investigated. MWCNTs are capable of improving the photocatalytic activity of TiO2/γ-Al2O3 composite in pulsed discharge plasma systems primarily due to the photo-induced-electron absorption effect and the electron trap effect of MWCNTs. The results of this study establish the feasibility and potential implementation of MWCNTs-TiO2/γ-Al2O3 composites in pulsed discharge plasma systems for the degradation of dye wastewater.

M phase phosphorylation of the epigenetic regulator UHRF1 regulates its physical association with the deubiquitylase USP7 and stability.

UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) plays an important role in DNA CpG methylation, heterochromatin function and gene expression. Overexpression of UHRF1 has been suggested to contribute to tumorigenesis. However, regulation of UHRF1 is largely unknown. Here we show that the deubiquitylase USP7 interacts with UHRF1. Using interaction-defective and catalytic mutants of USP7 for complementation experiments, we demonstrate that both physical interaction and catalytic activity of USP7 are necessary for UHRF1 ubiquitylation and stability regulation. Mass spectrometry analysis identified phosphorylation of serine (S) 652 within the USP7-interacting domain of UHRF1, which was further confirmed by a UHRF1 S652 phosphor (S652ph)-specific antibody. Importantly, the S652ph antibody identifies phosphorylated UHRF1 in mitotic cells and consistently S652 can be phosphorylated by the M phase-specific kinase CDK1-cyclin B in vitro. UHRF1 S652 phosphorylation significantly reduces UHRF1 interaction with USP7 in vitro and in vivo, which is correlated with a decreased UHRF1 stability in the M phase of the cell cycle. In contrast, UHRF1 carrying the S652A mutation, which renders UHRF1 resistant to phosphorylation at S652, is more stable. Importantly, cells carrying the S652A mutant grow more slowly suggesting that maintaining an appropriate level of UHRF1 is important for cell proliferation regulation. Taken together, our findings uncovered a cell cycle-specific signaling event that relieves UHRF1 from its interaction with USP7, thus exposing UHRF1 to proteasome-mediated degradation. These findings identify a molecular mechanism by which cellular UHRF1 level is regulated, which may impact cell proliferation.

Salting-out assisted liquid-liquid extraction coupled to dispersive liquid-liquid microextraction for the determination of chlorophenols in wine by high-performance liquid chromatography.

A novel procedure of sample preparation combined with high-performance liquid chromatography with diode array detection is introduced for the analysis of highly chlorinated phenols (trichlorophenols, tetrachlorophenols, and pentachlorophenol) in wine. The main features of the proposed method are (i) low-toxicity diethyl carbonate as extraction solvent to selectively extract the analytes without matrix effect, (ii) the combination of salting-out assisted liquid-liquid extraction and dispersive liquid-liquid microextraction to achieve an enrichment factor of 334-361, and (iii) the extract is analyzed by high-performance liquid chromatography to avoid derivatization. Under the optimum conditions, correlation coefficients (r) were >0.997 for calibration curves in the range 1-80 ng/mL, detection limits and quantification limits ranged from 0.19 to 0.67 and 0.63 to 2.23 ng/mL, respectively, and relative standard deviation was <8%. The method was applied for the determination of chlorophenols in real wines, with recovery rates in the range 82-104%.

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.

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.

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.

Resolution of 2-nitroalcohols by Burkholderia cepacia lipase-catalyzed enantioselective acylation.

Racemic 2-nitro-1-phenylethanol was resolved by via enantioselective transesterification catalyzed by Burkholderia cepacia lipase. The reaction afforded excellent E values (E > 200) and enantioselectivity (up to >99% enantiomeric excesses [ee]) of both remaining substrates and acetylated product. Moreover, the lipase displayed high enantioselectivity in the resolution of additional 2-nitroalcohols (E up to >200). This method provides an efficient alternative for obtaining enantiopure 2-nitroalcohols.

Large chromatin domains in pluripotent and differentiated cells.

Pluripotent stem cells are able to proliferate unlimitedly and to generate all somatic cell types, thus holding a great promise in medical applications. Epigenetic modifications are believed to play crucial roles in regulating pluripotency and differentiation. Recent genome-wide studies on mammalian systems have revealed several types of large chromatin domains which are associated with higher-order organization of the genome. The elucidation of genomic distribution and dynamics of these domains have shed light on the mechanisms underling pluripotency and lineage commitment.

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.