RNA Elongation Research Articles

Recognition of Davidsoniella virescens on Fagus sylvatica Wood in Poland and Assessment of Its Pathogenicity.

Davidsoniella virescens is so far only known in North America. However, recently in southern Poland, blackish growth consisting of fungal mycelia and sporulation structures was found on the wood of Fagus sylvatica. As a result of isolation, 17 cultures of this fungus were obtained. All cultures produced an intense sweet odor. This fungus, both in situ and in vitro, abundantly produced perithecia with long necks and asexual stage. Particularly characteristic was the production of variable endoconidia in two types of phialophores differing mainly in the width of the collarette. The nucleotide sequences for five gene fragments of representative cultures were used in phylogenetic analyses: 18S; the internal transcribed spacer regions ITS1 and ITS2, including the 5.8S gene (ITS); 28S region of the ribosomal RNA (rRNA), β-tubulin 2 (TUB2) and translation elongation factor 1-α (TEF1). Based on morphological and phylogenetic analyses, the fungus on European beech in Poland was identified as Davidsoniella virescens. The optimal temperature for radial colony growth was 20 °C. However, the differences between colony diameter at 25 °C compared to that at the optimal temperature were not statistically significant. Six D. virescens isolates were used for pathogenicity assay. They were inoculated into wounds on stems of two-year-old seedlings of Fagus sylvatica and Acer saccharum (36 seedlings of each tree species). Final evaluation was performed 4 months after inoculation. No external symptoms were observed in any A. saccharum seedling, neither in the crown nor on the stem. However, 13.9% of F. sylvatica seedlings showed wilting symptoms throughout the entire crown within 3-6 weeks after inoculation. Moreover, after 4 months on the stems of 30.6% beech seedlings, necrotic lesions with a length of 1.3 to 7.2 cm were formed, without any symptoms of wilting. The most noticeable internal symptom was the discoloration of the wood, which was observed in all inoculated seedlings of both tree species. All D. virescens isolates caused greater wood discoloration in F. sylvatica than in A. saccharum. Most of the differences found in the extent of discoloration between host plants were statistically significant. The discoloration caused by all D. virescens isolates in F. sylvatica was significantly greater than in the control. However, none of the isolates tested on A. saccharum caused significantly greater wood discoloration compared to the control. Pathogenicity tests showed that the D. virescens isolates identified in southern Poland may pose a greater threat to native European beech than to foreign sugar maple.

Cryo-EM structures of Thogoto virus polymerase reveal unique RNA transcription and replication mechanisms among orthomyxoviruses

Influenza viruses and thogotoviruses account for most recognized orthomyxoviruses. Thogotoviruses, exemplified by Thogoto virus (THOV), are capable of infecting humans using ticks as vectors. THOV transcribes mRNA without the extraneous 5′ end sequences derived from cap-snatching in influenza virus mRNA. Here, we report cryo-EM structures to characterize THOV polymerase RNA synthesis initiation and elongation. The structures demonstrate that THOV RNA transcription and replication are able to start with short dinucleotide primers and that the polymerase cap-snatching machinery is likely non-functional. Triggered by RNA synthesis, asymmetric THOV polymerase dimers can form without the involvement of host factors. We confirm that, distinctive from influenza viruses, THOV-polymerase RNA synthesis is weakly dependent of the host factors ANP32A/B/E in human cells. This study demonstrates varied mechanisms in RNA synthesis and host factor utilization among orthomyxoviruses, providing insights into the mechanisms behind thogotoviruses’ broad-infectivity range.

PAF1c links S-phase progression to immune evasion and MYC function in pancreatic carcinoma

In pancreatic ductal adenocarcinoma (PDAC), endogenous MYC is required for S-phase progression and escape from immune surveillance. Here we show that MYC in PDAC cells is needed for the recruitment of the PAF1c transcription elongation complex to RNA polymerase and that depletion of CTR9, a PAF1c subunit, enables long-term survival of PDAC-bearing mice. PAF1c is largely dispensable for normal proliferation and regulation of MYC target genes. Instead, PAF1c limits DNA damage associated with S-phase progression by being essential for the expression of long genes involved in replication and DNA repair. Surprisingly, the survival benefit conferred by CTR9 depletion is not due to DNA damage, but to T-cell activation and restoration of immune surveillance. This is because CTR9 depletion releases RNA polymerase and elongation factors from the body of long genes and promotes the transcription of short genes, including MHC class I genes. The data argue that functionally distinct gene sets compete for elongation factors and directly link MYC-driven S-phase progression to tumor immune evasion.

SARS-CoV-2-Virus structure and life cycle.

This book chapter presents a concise overview of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. It explores viral classification based on morphology and nucleic acid composition with a focus on DNA and RNA viruses, the SARS-CoV-2 structure including the structural as well as nonstructural proteins in detail, and the viral replication mechanisms. The chapter then delves into the characteristics and diversity of coronaviruses, particularly SARS-CoV-2, highlighting its similarities with other beta-coronaviruses. The replication and transcription complex, RNA elongation, and capping, as well as the role of accessory proteins in viral replication and modulation of the host immune response is discussed extensively.

Design and Execution of In Vitro Polymerase Assays for Measles Virus and Related Mononegaviruses.

Morbilliviruses such as measles virus (MeV) are responsible for major morbidity and mortality worldwide, despite the availability of an effective vaccine and global vaccination campaigns. MeV belongs to the mononegavirus order of viral pathogens that store their genetic information in non-segmented negative polarity RNA genomes. Genome replication and viral gene expression are carried out by a virus-encoded RNA-dependent RNA polymerase (RdRP) complex that has no immediate host cell analog. To better understand the organization and regulation of the viral RdRP and mechanistically characterize antiviral candidates, biochemical RdRP assays have been developed that employ purified recombinant polymerase complexes and synthetic RNA templates to monitor the initiation of RNA synthesis and RNA elongation in vitro. In this article, we will discuss strategies for the efficient expression and preparation of mononegavirus polymerase complexes, provide detailed protocols for the execution and optimization of RdRP assays, evaluate alternative options for the choice of template and detection system, and describe the application of the assay for the characterization of inhibitor candidates. Although MeV RdRP assays are the focus of this article, the general strategies and experimental approaches are readily transferable to related viruses in the mononegavirus order.

5′-cap RNA/SAM mimetic conjugates as bisubstrate inhibitors of viral RNA cap 2′-O-methyltransferases

Viral RNA cap 2′-O-methyltransferases are considered promising therapeutic targets for antiviral treatments, as they play a key role in the formation of viral RNA cap-1 structures to escape the host immune system. A better understanding of how they interact with their natural substrates (RNA and the methyl donor SAM) would enable the rational development of potent inhibitors. However, as few structures of 2′-O-MTases in complex with RNA have been described, little is known about substrate recognition by these MTases. For this, chemical tools mimicking the state in which the cap RNA substrate and SAM cofactor are bound in the enzyme’s catalytic pocket may prove useful. In this work, we designed and synthesized over 30 RNA conjugates that contain a short oligoribonucleotide (ORN with 4 or 6 nucleotides) with the first nucleotide 2′-O-attached to an adenosine by linkers of different lengths and containing S or N-heteroatoms, or a 1,2,3-triazole ring. These ORN conjugates bearing or not a cap structure at 5′-extremity mimic the methylation transition state with RNA substrate/SAM complex as bisubstrates of 2′-O-MTases. The ORN conjugates were synthesized either by the incorporation of a dinucleoside phosphoramidite during RNA elongation or by click chemistry performed on solid-phase post-RNA elongation. Their ability to inhibit the activity of the nsp16/nsp10 complex of SARS-CoV-2 and the NS5 protein of dengue and Zika viruses was assessed. Significant submicromolar IC50 values and Kd values in the µM range were found, suggesting a possible interaction of some ORN conjugates with these viral 2′-O-MTases.

Caseinolytic Mitochondrial Matrix Peptidase Proteolytic Subunit (CLPP) As a Novel Mediator of, and Target in High Risk Multiple Myeloma

Background: Up to 30% of newly diagnosed myeloma (NDMM) patients have high-risk disease characterized by increased proliferation, resistance to apoptosis, focal and bone marrow-independent growth, and intraclonal heterogeneity. High-risk can be defined by fluorescence in situ hybridization (FISH) features including 17p deletion, translocation (t)(4;14), t(14;16), or t(14;20), and gain of 1q. However, some standard risk patients also exhibit high-risk characteristics, indicating that identifying novel targetable mediators of this phenotype is of interest. Methods: We conducted single cell RNA-Sequencing (scRNA-Seq) of CD138 + monoclonal/neoplastic and polyclonal/normal plasma cells (PCs) from 72 individuals across the plasma cell dyscrasia spectrum and 3 healthy controls to identify differentially expressed genes associated with myeloma pathobiology. Also, we leveraged publicly available gene expression profiling (GEP) databases, and performed pre-clinical studies both in vitro and in vivo. Results: CLPP was expressed with increased frequency in NDMM and relapsed/refractory myeloma patients compared to the precursors monoclonal gammopathy of undetermined significance and smoldering myeloma (GSE6477; p=4.03x10 -14). Notably, CLPP is the proteolytic component, along with CLPX, of the CLP complex which cleaves peptides and proteins in an ATP-dependent manner and is associated with the inner mitochondrial membrane. The GEP data were confirmed in our scRNA-Seq database, which showed CLPP over-expression occurred preferentially in each patient's monoclonal versus their own polyclonal PCs. Moreover, analysis of the Broad Institute Cancer Cell Line Encyclopedia revealed MM cells had the highest mean CLPP expression of any tumor cell type. In the NDMM CoMMpass database, high CLPP levels were associated with markers of increased disease burden (beta-2-microglobulin (p<0.001), calcium (p=0.0027), circulating plasma cells (p<0.001), marrow plasmacytosis (p<0.001), and lactate dehydrogenase (p=0.0025)). Moreover, these patients showed a significantly shorter progression-free and overall survival (both p<0.0001), and did not cluster in any of the Arkansas GEP70-defined myeloma subtypes. In MM.1S and H929 myeloma cells, specific CLPP knockdown with an inducible shRNA decreased cell proliferation and activated apoptosis as shown by increased Annexin V positivity. Similarly, pharmacologic CLPP inhibition produced a time- and concentration-dependent induction of apoptosis in a panel of cell lines representing myeloma molecular subtypes and drug resistant phenotypes. Ectopic CLPP overexpression promoted myeloma organoid growth, while its suppression had the opposite effect. Functional analysis revealed that CLPP knockdown or inhibition impaired cell proteolytic activity, mitochondrial transmembrane potential, and reduced adhesion to stromal cells while inducing reactive oxygen species and autophagy. To further pursue the underlying molecular mechanisms, we first examined the net downstream effects of CLPP knockdown and inhibition by mass spectrometry followed by mapping to the KEGG database. Recurrently up-regulated pathways included the mitochondrial unfolded protein response (UPR mt), lysosome transport, and autophagy utilizing and metabolic processes. In contrast, down-regulated pathways included oxidative phosphorylation, c-Myc signaling, RNA polymerase I-directed transcription, DNA replication and elongation, and the G2M checkpoint. Next, our metabolomics analysis revealed a significant attenuation of the Warburg effect in myeloma cells upon CLPP inhibition consistent with reduced glycolysis and pentose phosphate pathway activity. Notably, mitochondrial ATP production was significantly reduced, as confirmed by a seahorse XF Real-time ATP Rate assay. Finally, in vivo experiments demonstrated that pharmacologic CLPP inhibition impaired myeloma growth in an MM.1S-based xenograft model ( Figure). Conclusions: These pre-clinical data support the hypothesis that CLPP represents a novel mediator of high-risk behavior in multiple myeloma, demonstrate that its targeting results in mitochondrial dysfunction that attenuates myeloma growth and survival, and support development of pharmacologic CLPP inhibitors to target myeloma.

RNA elongation contributes to gene expression regulation during early endothelial-to-mesenchymal transition

Abstract Background Endothelial dysfunction is characterized by endothelial-to-mesenchymal transition (EndMT), a process in which endothelial cells (EC) transform into mesenchymal cells and lose part of their endothelial identity. EndMT-positive cells are present in vulnerable atherosclerotic plaques. EndMT is triggered by stress stimuli such as inflammation or hypoxia that require an adaptive change in gene expression mediated at the level of RNA elongation. RNA elongation is controlled by the super elongation complex (SEC), which is built around scaffolding proteins of the AF family (AFF1-AFF4). The SEC controls the pausing of RNA Polymerase II (RNAPII). We hypothesized that RNA elongation plays a regulatory role in EndMT. Methods and Results Single cell sequencing data revealed that the SEC scaffold proteins AFF1 and AFF4 are ubiquitously expressed in human carotid artery plaques. AFF1 and AFF4 are higher expressed in EC compared to AFF2 and AFF3. In an in vitro EndMT model, AFF1 and AFF4 were upregulated compared to controls (AFF1: 1.9-fold; AFF4: 2.6-fold). SEC inhibition using chemical compounds reduced the protein levels of AFF1 (-67.4%) and AFF4 (-46.5%). EndMT induction was reduced by SEC inhibition, as measured by mesenchymal marker gene expression (SM22: -88.8%; CNN1: -94.0%). The rapid induction of gene expression depends on the release of paused RNAPII present at the promoter site. ChIP-sequencing revealed a reduced occupancy of RNAPII at promoter sites in EndMT as early as 2 h compared to controls, suggesting a rapid pause release of RNAPII during EndMT. Indeed, transcriptome-wide RNA elongation rates were increased by 20.2% as early as 10 min after induction of EndMT, as determined by analysis of newly synthesized 4sU-labelled RNA. SEC inhibition reduced transcriptome-wide RNA elongation rates (-26.8%). These data suggest a regulatory contribution of RNA elongation in early EndMT. Among the genes that were immediately and prominently upregulated after the induction of EndMT was the SPARC mRNA. RNA initiation and elongation of SPARC mRNA was increased 10 min after EndMT induction, which was reduced by SEC inhibition. 72 hours after EndMT induction, inhibition of SEC resulted in a reduction in SPARC mRNA (-72.2%), protein concentration (-97.2%) and reduced SPARC delivery to the supernatant (-43.9%). Knockdown of SPARC enhanced EndMT induction (SM22: 1.6-fold; CNN1: 3.6-fold), which was attenuated by treatment with recombinant SPARC. Conclusions We observed increased RNA elongation rates during EndMT associated with decreased pausing of RNA Polymerase II. The RNA elongation mediated specific gene expression such as the upregulation of SPARC. Taken together, our results indicate that RNA elongation plays a regulatory role in EndMT.

DIFFERENT PROTEIN GROUPS INVOLVED IN TRANSCRIPTION REGULATION IN GENES OF DEVELOPMENT AND HOUSEKEEPING IN <i>DROSOPHILA</i>

Antibodies to histone modifications and an insulator protein involved in the processes of transcription initiation and elongation are mapped in Drosophila polytene chromosomes. The CHRIZ protein (chromatin insulator) and H3K36me3 histone modification (RNA elongation) are detected only in the localization of housekeeping genes (interbands and gray bands of polytene chromosomes) and never in the regions of development genes (black bands and large puffs arising from them). Antibodies to H3S10P histone modification, which is associated with the initial elongation of the RNA strand during transcription, are found exclusively in small puffs, but not in housekeeping gene localization sites or large ecdysone-induced puffs, where housekeeping genes are localized. Antibodies to H4R3me2 histone modification (a co-repressor of the ecdysone receptor) are detected only in large α-induced puffs.

Abstract P1134: RNA Elongation Mediates Rapid Induction Of Gene Expression During Endothelial-to-mesenchymal Transition

Endothelial-to-mesenchymal transition (EndMT) is triggered by stress stimuli and characterized by the conversion of endothelial cells (EC) into mesenchymal cells. Stress stimuli such as hypoxia or inflammation depend on rapid gene expression mediated by RNA elongation. RNA elongation is controlled by the super elongation complex (SEC) through transient pausing of RNA Polymerase II (RNAPII). We aimed to analyze the regulatory role of RNA elongation on rapid gene expression during the onset of EndMT. In an in vitro EndMT model, SEC scaffolds AFF1 and AFF4 were upregulated compared to controls. SEC inhibition using chemical compounds reduced the protein levels of AFF1 and AFF4. EndMT was reduced by SEC inhibition, whereas overexpression of AFF4 increased EndMT as measured by mesenchymal marker gene expression. Rapid gene expression depends on the release of paused RNAPII at promoter sites. ChIP-sequencing revealed a reduction of RNAPII occupancy at promoter sites in EndMT compared to controls, suggesting the release of RNAPII during EndMT. Transcriptome-wide RNA elongation rates were increased as early as 10 min after induction of EndMT, as determined by analysis of nascent 4sU-labeled RNA. SEC inhibition reduced global RNA elongation rates, suggesting a regulatory contribution of RNA elongation in EndMT. Among the genes that were immediately upregulated after the induction of EndMT was the SPARC mRNA. RNA initiation and elongation of SPARC mRNA were increased 10 min after EndMT induction, which was reduced by SEC inhibition. At 72 h after EndMT induction, inhibition of SEC decreased SPARC mRNA, protein concentration and SPARC delivery to the supernatant. Silencing of SPARC reduced the angiogenic potential of EC and enhanced EndMT induction, which was attenuated by treatment with recombinant SPARC. Treatment with recombinant SPARC also improved endothelial impedance under EndMT conditions. Spatial transcriptomics revealed increased SPARC expression in brachiocephalic artery plaques from LDLR-/- mice. In conclusion, we observed increased RNA elongation rates associated with reduced RNAPII pausing during EndMT that mediate rapid gene expression of target genes such as SPARC. Our results suggest that RNA elongation plays a regulatory role in EndMT.

First Report of Root Rot caused by Coniella fragariae on Paeoniae radix Rubra in China.

Paeoniae radix Rubra is a traditional Chinese herbal medicine, which has the effect of clearing heat and cooling blood, activating blood and removing stasis. It has become popular in the Chinese market in recent years due to its extremely high medicinal value and showy flower color. In May 2021, typical symptoms of root rot were observed in a field (35°7'12″ N, 103°58'48″ E) in Dingxi, Gansu province, China. Approximately 10% of the plants in the field had typical root rot symptoms, and the root of each affected plant is at least 5% severe. The roots of the naturally infected plants in the field discolored and decayed with black brown spots on the surface of the root bark, the root bark detached from the phloem，and some leaves were chlorosis, shrunken and smaller, and the branches were dead and underdeveloped. In the transverse section, the xylem was black diffusion and abnormal odor. Three diseased plants with typical symptoms were chosen at random and brought back to the lab. Small pieces cut from the margins of lesions were surface disinfested with 75% ethanol for 15 s, and 0.5% NaClO solution for 30 s, rinsed three times in sterile distilled water, dried on sterile filter paper, plaed onto potato dextrose agar (PDA), and incubated at 25 ± 1℃ for 7 days in the dark. The pure cultures were obtained by single-spore isolation. All isolates produced wavy on the surface, radial from the inside out, initially white or milky white to orange colonies with abundant black brown oily conidiomata pycnidia on PDA at 25 ± 1℃ after 15 days in the dark. The conidiomata pycnidia is spherical to irregularly spherical, 231.5 to 512.4 μm, initially transparent with age turning brown, with a dark brown internal conidial mass inside, and with a 13.1 to 45.4 μ m wide ostiole central. Young conidia (n=100) developed from conidiogenous cells, which were simple, tapering, hyaline, smooth, and 12.3 to 18.0 × 2.5 to 4.6 μm, 1.0 to 1.5 µm wide at apex. Mature conidia (n=100) were ellipsoid, apices tapering, subobtusely rounded, brown, and 6.5 to 11.0 × 4.1 to 7.5 µm. The morphological characteristics of the isolates were consistent with previous descriptions of the genus Coniella (Crous et al., 2014). A representation isolate CS-1 was deposited in the Institute of Plant Protection, Gansu Academy of Agricultural Sciences and used for further studies. To confirm the identity of the causal fungus, the internal transcribed spacer (ITS), 28S large subunit of nuclear ribosomal RNA (LSU) and partial translation elongation factor 1-alpha (TEF1-α) gene of the representative isolate CS-1 were amplified and sequenced using primers ITS1/ITS4 (White et al., 1990), LROR/LR7 (Chethan et al., 2017) and EF1-728F/EF1-986R (Carbone and Kohn, 1999), respectively, and deposited on GenBank with accession numbers OP824764 (ITS), OP824767 (LSU)/span>and OP903926 (TEF1-α). Blastn analysis of all sequences resulted in E-value of 0.0 (ITS and LSU) and nearly 0.0 (TEF1-α), with Query cover values of 90% to 99% identity with C. fragariae, confirming the hypothesis based on morphological features examination. To conduct a pathogenicity test, three root segments of healthy plants were wounded using sterilized needles and inoculated by pipetting 10 μL of conidial suspension (1×107 conidia/mL) onto each wound, and controls were inoculated with 10 μL sterile distilled water. These root segments were kept in a moist chamber at 25°C in the dark. The experiment was repeated three times. After 14 days, root rot symptoms were observed on all of the inoculated root segments and identical to those observed in the field, whereas control root segments did not develop symptoms. The pathogen was re-isolated from the lesions of inoculated root segments, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of C. fragariae causing root rot on P. radix Rubra in China. This identification can aid in the selection of appropriate management measures for this disease.

Structures of dengue virus RNA replicase complexes

Dengue is a mosquito-borne viral infection caused by dengue virus (DENV), a member of the flaviviruses. The DENV genome is a 5'-capped positive-sense RNA with a unique 5'-stem-loop structure (SLA), which is essential for RNA replication and 5' capping. The virus-encoded proteins NS5 and NS3 are responsible for viral genome replication, but the structural basis by which they cooperatively conduct the required tasks has remained unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of SLA-bound NS5 (PC), NS3-bound PC (PC-NS3), and an RNA-elongating NS5-NS3 complex (EC). While SLA bridges the NS5 methyltransferase and RNA-dependent RNA polymerase domains in PC, the NS3 helicase domain displaces it in elongation complex (EC). The SLA- and NS3-binding sites overlap with that of human STAT2. These structures illuminate the key steps in DENV genome replication, namely, SLA-dependent replication initiation, processive RNA elongation, and 5' capping of the nascent genomic RNA, thereby providing foundations to combat flaviviruses.

Expression of RNA polymerase I catalytic core is influenced by RPA12.

RNA Polymerase I (Pol I) has recently been recognized as a cancer therapeutic target. The activity of this enzyme is essential for ribosome biogenesis and is universally activated in cancers. The enzymatic activity of this multi-subunit complex resides in its catalytic core composed of RPA194, RPA135, and RPA12, a subunit with functions in RNA cleavage, transcription initiation and elongation. Here we explore whether RPA12 influences the regulation of RPA194 in human cancer cells. We use a specific small-molecule Pol I inhibitor BMH-21 that inhibits transcription initiation, elongation and ultimately activates the degradation of Pol I catalytic subunit RPA194. We show that silencing RPA12 causes alterations in the expression and localization of Pol I subunits RPA194 and RPA135. Furthermore, we find that despite these alterations not only does the Pol I core complex between RPA194 and RPA135 remain intact upon RPA12 knockdown, but the transcription of Pol I and its engagement with chromatin remain unaffected. The BMH-21-mediated degradation of RPA194 was independent of RPA12 suggesting that RPA12 affects the basal expression, but not the drug-inducible turnover of RPA194. These studies add to knowledge defining regulatory factors for the expression of this Pol I catalytic subunit.

Oridonin inhibits SARS-CoV-2 replication by targeting viral proteinase and polymerase

COVID-19 has become a global public health crisis since its outbreak in China in December 2019. Currently there are few clinically effective drugs to combat SARS-CoV-2 infection. The main protein (Mpro), papain-like protease (PLpro) and RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 are involved in the viral replication, and might be prospective targets for anti-coronavirus drug development. Here, we investigated the antiviral activity of oridonin, a natural small-molecule compound, against SARS-CoV-2 infection in vitro. The time-of-addition analysis showed that oridonin efficiently inhibited SARS-CoV-2 infection by interfering with the genome replication at the post-entry stage. Mechanistically, the inhibition of viral replication by oridonin depends on the oxidation activity of α, β-unsaturated carbonyl. Further experiments showed that oridonin not only effectively inhibited SARS-CoV-2 Mpro activity, but also had some inhibitory effects on PLpro-mediated deubiquitinating and viral polymerase-catalyzed RNA elongation activities at high concentrations. In particular, oridonin could inhibit the bat SARS-like CoV and the newly emerged SARS-CoV-2 omicron variants (BA.1 and BA.2), which highlights its potential as a pan-coronavirus antiviral agent. Overall, our data provide strong evidence that oridonin is an efficient antiviral agent against SARS-CoV-2 infection.

Polypeptide antibiotic actinomycin D induces Mcl-1 uncanonical downregulation in lung cancer cell apoptosis

AimsActinomycin (Act) D, a polypeptide antibiotic, is used clinically to inhibit the growth of malignant tumors. Act D binds to DNA at the transcription initiation complex to prevent the elongation of RNA. Act D causes DNA damage, growth inhibition, and cell death. Myeloid cell leukemia (Mcl-1) is an anti-apoptotic Bcl-2 family member protein, and the present study explored the effects and molecular mechanism of Act D-induced Mcl-1 downregulation. Main methodsHuman adenocarcinoma A549 cells were used to check the cytotoxic signaling pathways of Act D, particularly in apoptotic mechanism, in a cell-based study approach. Specific blockers targeting the apoptotic factors were examined for their possible roles. Key findingsWe found that Act D caused cell growth inhibition and apoptosis. Propidium iodide-based flow cytometric analysis and immunostaining confirmed cell apoptosis. Treatment with Act D caused DNA damage, followed by p53-independent cell death. Western blotting showed a significant decrease in Mcl-1 expression, mitochondrial transmembrane potential loss, and caspase-9/caspase-3 cascade activation. The proteasome inhibitor MG132 reversed Act D-induced Mcl-1 downregulation. However, pharmacological inhibition of glycogen synthase kinase-3, p53 expression, ER stress, autophagy, and vesicle acidification, which are Mcl-1-regulating signaling pathways, did not rescue these effects. Notably, Cullin-Ring E3 ligase partially mediated Mcl-1 downregulation. Administration of transforming growth factor-β induced mesenchymal cell differentiation, but Act D still decreased Mcl-1 and caused cell apoptosis. SignificanceAll of these data show a potential pro-apoptotic effect for Act D by facilitating Mcl-1 uncanonical downregulation.

Structure of the Newcastle Disease Virus L protein in complex with tetrameric phosphoprotein

Newcastle disease virus (NDV) belongs to Paramyxoviridae, which contains lethal human and animal pathogens. NDV RNA genome is replicated and transcribed by a multifunctional 250 kDa RNA-dependent RNA polymerase (L protein). To date, high-resolution structure of NDV L protein complexed with P protein remains to be elucidated, limiting our understanding of the molecular mechanisms of Paramyxoviridae replication/transcription. Here, we used cryo-EM and enzymatic assays to investigate the structure-function relationship of L-P complex. We found that C-terminal of CD-MTase-CTD module of the atomic-resolution L-P complex conformationally rearranges, and the priming/intrusion loops are likely in RNA elongation conformations different from previous structures. The P protein adopts a unique tetrameric organization and interacts with L protein. Our findings indicate that NDV L-P complex represents elongation state distinct from previous structures. Our work greatly advances the understanding of Paramyxoviridae RNA synthesis, revealing how initiation/elongation alternates, providing clues for identifying therapeutic targets against Paramyxoviridae.

AT-752 targets multiple sites and activities on the Dengue virus replication enzyme NS5

AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2′-methyl-2′-fluoro guanosine 5′-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2′-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 Å resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2′-O but not N7-methylation activity. AT-9010 is discriminated ∼10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibition through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC50 ≈ 0.50 μM), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses.

Effects of Histone H2B Ubiquitylations and H3K79me3 on Transcription Elongation.

Post-translational modifications of histone proteins often mediate gene regulation by altering the global and local stability of the nucleosome, the basic gene-packing unit of eukaryotes. We employed semisynthetic approaches to introduce histone H2B ubiquitylations at K34 (H2BK34ub) and K120 (H2BK120ub) and H3K79 trimethylation (H3K79me3). With these modified histones, we investigated their effects on the kinetics of transcription elongation by RNA polymerase II (Pol II) using single-molecule FRET. Pol II pauses at several locations within the nucleosome for a few seconds to minutes, which governs the overall transcription efficiency. We found that H2B ubiquitylations suppress pauses and shorten the pause durations near the nucleosome entry while H3K79me3 shortens the pause durations and increases the rate of RNA elongation near the center of the nucleosome. We also found that H2BK34ub facilitates partial rewrapping of the nucleosome upon Pol II passage. These observations suggest that H2B ubiquitylations promote transcription elongation and help maintain the chromatin structure by inducing and stabilizing nucleosome intermediates and that H3K79me3 facilitates Pol II progression possibly by destabilizing the local structure of the nucleosome. Our results provide the mechanisms of how these modifications coupled by a network of regulatory proteins facilitate transcription in two different regions of the nucleosome and help maintain the chromatin structure during active transcription.

Structure, activity and function of the lysine methyltransferase SETD5.

SET domain-containing 5 (SETD5) is an uncharacterized member of the protein lysine methyltransferase family and is best known for its transcription machinery by methylating histone H3 on lysine 36 (H3K36). These well-characterized functions of SETD5 are transcription regulation, euchromatin formation, and RNA elongation and splicing. SETD5 is frequently mutated and hyperactive in both human neurodevelopmental disorders and cancer, and could be down-regulated by degradation through the ubiquitin-proteasome pathway, but the biochemical mechanisms underlying such dysregulation are rarely understood. Herein, we provide an update on the particularities of SETD5 enzymatic activity and substrate specificity concerning its biological importance, as well as its molecular and cellular impact on normal physiology and disease, with potential therapeutic options.

Quasi Markov state models elucidates the role of bacterial RNA polymerase loading gate and trigger loop dynamics in antibiotics inhibition.

The rise of antibiotic resistance urges the development of new antibiotics. Bacterial RNA Polymerase (RNAP) transcribes DNA to RNA and is an effective target for antibiotics. RNAP has a crab-claw-like shape with two pincers, clamp and β-lobe domains, forming a loading gate. During the initiation of transcription,the opening of the loading gate is necessary to load the template DNA. Myx is an antibiotic that binds to the switch 2 region, under the clamp, to prevent clamp closing/opening and inhibit transcription initiation. To study the inhibition mechanism of Myx in atomic resolution, we built quasi-Markov State Models (qMSM) based on extensive molecular dynamics (MD) simulations. qMSM can obtain the slow timescale dynamics of complex conformational change using shorter MD simulations compared to traditional MSM. qMSM identifies four states of clamp opening: closed, two partially closed, and open states. We have shown that the opening of the Myx binding site only occurs in a partially closed state, which allows binding of Myx. This suggests that Myx selectively binds to a partially closed state and follows the conformational selection mechanism. Notably, we find that β-lobe opening is sufficient for loading of DNA during transcription initiation. This highlights the critical role of β-lobe to initiate transcription and β-lobe's potential as a target for future antibiotics development. We also utilize qMSMto study the folding of the trigger loop (TL) of RNAP. During RNA elongation, TL closes/folds to stabilize the NTP substrate in the active site for catalysis. CBR is antibiotics that inhibit the folding of TL. Studying the molecular mechanism of TL folding will be useful to understand the inhibition mechanism of antibiotics, CBR, and future drug design targeting TL.