RNA Elongation Research Articles

Targeting the super elongation complex for oncogenic transcription driven tumor malignancies: Progress in structure, mechanisms and small molecular inhibitor discovery.

Oncogenic transcription activation is associated with tumor development and resistance derived from chemotherapy or target therapy. The super elongation complex (SEC) is an important complex regulating gene transcription and expression in metazoans closely related to physiological activities. In normal transcriptional regulation, SEC can trigger promoter escape, limit proteolytic degradation of transcription elongation factors and increase the synthesis of RNA polymerase II (POL II), and regulate many normal human genes to stimulate RNA elongation. Dysregulation of SEC accompanied by multiple transcription factors in cancer promotes rapid transcription of oncogenes and induce cancer development. In this review, we summarized recent progress in understanding the mechanisms of SEC in regulating normal transcription, and importantly its roles in cancer development. We also highlighted the discovery of SEC complex target related inhibitors and their potential applications in cancer treatment.

A panel of qPCR assays to detect and quantify soybean soil-borne pathogens.

Fusarium oxysporum,F. graminearum,F. acuminatum,F. equiseti,F. proliferatum,F. solani, and Rhizoctonia solani are soil-borne fungal pathogens that cause substantial yield loss in a widespread list of crops worldwide. The objective of this study was to develop a panel of TaqMan assays for the detection and quantification of these six widespread soil-borne fungal species using real-time polymerase chain reaction (qPCR). The primers and probes were designed based on the intergenic spacer ribosomal RNA and translation elongation factor 1-alpha gene (tef1). These assays, although not multiplexed, can be performed simultaneously as they have similar reaction conditions, allowing more efficiency when targeting multiple pathogens in a sample. The assays presented high efficiency (94.3%-108.9%) and sensitivity, with a limit of detection of 0.05 picograms (50 femtograms) of target DNA. Results from an assay targeting 19 non-target and closely related species confirmed the specificity of the developed assays. The assays were also evaluated to detect the target species in different matrices, such as soil and plant material. This panel of qPCR assays is an additional tool that can be used by plant pathologists, microbiologists, plant breeders, diagnostic clinics, and other researchers interested in these fungal species.

Natural Compounds as Non-Nucleoside Inhibitors of Zika Virus Polymerase through Integration of In Silico and In Vitro Approaches.

Although the past epidemic of Zika virus (ZIKV) resulted in severe neurological consequences for infected infants and adults, there are still no approved drugs to treat ZIKV infection. In this study, we applied computational approaches to screen an in-house database of 77 natural and semi-synthetic compounds against ZIKV NS5 RNA-dependent RNA-polymerase (NS5 RdRp), an essential protein for viral RNA elongation during the replication process. For this purpose, we integrated computational approaches such as binding-site conservation, chemical space analysis and molecular docking. As a result, we prioritized nine virtual hits for experimental evaluation. Enzymatic assays confirmed that pedalitin and quercetin inhibited ZIKV NS5 RdRp with IC50 values of 4.1 and 0.5 µM, respectively. Moreover, pedalitin also displayed antiviral activity on ZIKV infection with an EC50 of 19.28 µM cell-based assays, with low toxicity in Vero cells (CC50 = 83.66 µM) and selectivity index of 4.34. These results demonstrate the potential of the natural compounds pedalitin and quercetin as candidates for structural optimization studies towards the discovery of new anti-ZIKV drug candidates.

Discovery of non-nucleoside oxindole derivatives as potent inhibitors against dengue RNA-dependent RNA polymerase

A series of thiazole linked Oxindole-5-Sulfonamide (OSA) derivatives were designed as inhibitors of RNA-dependent RNA polymerase (RdRp) activity of Dengue virus. These were synthesized and then evaluated for their efficacy in ex-vivo virus replication assay using human cell lines. Among 20 primary compounds in the series, OSA-15 was identified as a hit. A series of analogues were synthesized by replacing the difluoro benzyl group of OSA-15 with different substituted benzyl groups. The efficacy of OSA-15derivatives was less than that of the parent compound, except OSA-15-17, which has shown improved efficacy than OSA-15. The further optimization was carried out by adding dimethyl (DM) groups to both the sulfonamide and oxindole NH’s to produce OSA-15-DM and OSA-15-17-DM. These two compounds were showing no detectable cytotoxicity and the latter was more efficacious. Further, both these compounds were tested for inhibition in all the serotypes of the Dengue virus using an ex-vivo assay. The EC50 of OSA-15-17-DM was observed in a low micromolar range between 2.5 and 5.0 µg/ml. Computation docking and molecular dynamics simulation studies confirmed the binding of identified hits to DENV RdRp. OSA15-17-DM blocks the RNA entrance and elongation site for their biological activity with high binding affinity. Overall, the identified oxindole derivatives are novel compounds that can inhibit Dengue replication, working as non-nucleoside inhibitors (NNI) to explore as anti-viral RdRp activity.

Novel Species and Records of Dictyosporiaceae from Freshwater Habitats in China and Thailand.

China and Thailand are rich in fungal diversity with abundant freshwater resources that are favorable for numerous fungal encounters. Resulting from this, the majority of the Dictyosporiaceae species reported were from these two countries. During the investigation on the diversity of lignicolous freshwater fungi in the Greater Mekong Subregion, eleven collections of cheirosporous species on submerged wood were collected from lentic and lotic habitats in China and Thailand. Phylogenetic analysis that combined nuclear small-subunit ribosomal RNA (SSU), internal transcribed spacer region (ITS), nuclear large subunit ribosomal RNA (LSU) and translation elongation factor 1α (tef 1-α) loci revealed six new species: Dictyocheirospora chiangmaiensis, D. multiappendiculata, D. suae, Digitodesmium aquaticum, Vikalpa grandispora and V. sphaerica. In addition, four known species were also identified and reported based on morphological and phylogenetic evidence. The detailed descriptions and illustrations of these taxa are provided with an updated phylogenetic tree of Dictyosporiaceae.

First report of Leaf Spot Caused by Paramyrothecium foliicola on Tomato (Solanum lycopersicum) in China.

Tomato (Solanum lycopersicum) is a staple vegetable across the world. In October 2019, leaf spots were observed on tomato (cv. Tianmi) in a greenhouse in JiZhou District Tianjin, China(117°10 'E; 39°55 'N). Symptoms initially appeared as small brown spots, which gradually expanded and turned into circular, oval or irregular spots (some spots with distinct concentric zones). In severe cases, some spots coalesced and eventually covered the whole leaf. Disease incidence ranged between 12 and 18%. Twenty symptomatic leaves from five plants were collected and cut into small pieces, surface disinfested in 2% NaClO for 60 s, rinsed three times in sterile water, and subsequently plated on potato dextrose agar (PDA). Plates were incubated at 25°C in the dark for 7 days. A total of 102 isolates were obtained and 92 isolates had the same morphology. Colonies were initially white with abundant aerial mycelia and formed sporodochia with conidial masses in olivaceous green concentric rings. All isolates formed single-celled, hyaline, and rod-shaped conidia were 4.91 to 7.43 (avg. 6.53±0.72) × 1.41 to 2.45 （avg. 2.11±0.30）μm with rounded ends (n=50). Conidiophores were highly branched. These characteristics resembled a Paramyrothecium-like fungus (Lombard et al. 2016). The genomic DNA of three representative single-spored isolates TJJXPF1-3 were extracted and the internal transcribed spacer (ITS) region, β-tubulin (tub2), large subunit ribosomal RNA (LSU), calmodulin (cmdA) and translation elongation factor 1-alpha (tef1) genes were amplified and sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), LR0R/LR5 (Rehner and Samuels 1995; Vilgalys and Hester 1990), CAL-228F/CAL2Rd (Carbone and Kohn 1999; Groenewald et al. 2013) and EF1-728F/EF2 (O'Donnell et al. 1998), respectively. All sequences were deposited in GenBank (ITS: MW463444, OM368178, OM368179; tub2: MW269542，OM714930，OM714931; LSU: OM349050, OM397398, OM390582; cmdA: MW280443, OM350474, OM350476; tef1: MW560083, OM350475, OM350477). BLASTN analysis showed 99.3-100% similarity with reference isolate QB1 of P. foliicola (MK335967, MT415353, MT415362, MT415356 and MT415359). Multilocus phylogenetic analysis showed that TJJXPF1-3 best grouped with the P. foliicola clade, which was identified by morphological characteristics and phylogenetic analysis. To fulfill Koch's postulates, pathogenicity tests were conducted by spray-inoculation with a conidial suspension of isolate TJJXPF1 prepared with distilled water (1×105 conidia/mL) on five 45-day old tomato plants. Three healthy plants were sprayed with sterile water as control. All treatments were incubated in an artificial climate chamber (25°C, 80% RH, 12h light/12h dark ). After two weeks, leaf spots were observed on all inoculated plants, which were similar to those in the greenhouse of JiZhou District, while control plants remained asymptomatic. Additionally, the pathogens were reisolated from symptomatic leaves and three representative isolates TJJXPF4-6 were identified as P. foliicola. The pathogenicity tests were repeated thrice. To our knowledge, this is the first report of leaf spot caused by P. foliicola on tomato in China. This disease could be a serious threat to tomato production in the future. Our findings will help to differentiate this disease from other leaf spot-like diseases and develop disease control strategies.

Targeting RNA elongation in endothelial-to-mesenchymal transition

Abstract Background and purpose EndMT describes a process in which endothelial cells lose their endothelial features such as the maintenance of the vascular barrier function and trans-differentiate into mesenchymal like cells. ECs which have underwent EndMT are enriched in vulnerable plaques in humans. Several stress stimuli such as hypoxia or inflammation demand immediate induction of adaptive gene expression to improve cell survival. In contrast to the usual induction of gene expression which requires several hours, release of paused RNA Polymerase II (RNAPII) from promoter allows rapid gene induction via RNA elongation. This rapid response mechanism is mediated by the super elongation complex (SEC), which is built around the scaffold proteins AFF1 and AFF4. Here, we hypothesized that RNA elongation regulates EndMT and therefore investigated on the contribution of the SEC scaffold proteins during the process of EndMT. Methods and results After induction of EndMT in human primary endothelial cells (EC) the elongation factors AFF1 and AFF4 were induced (AFF1: 1.9-fold and AFF4: 2.7-fold, P<0.05). Overexpression of AFF4 resulted in increased expression of the mesenchymal marker genes SM22 (1.8-fold, P<0.05) and VCAN (2.0-fold, P<0.05). Accordingly, treatment of EC with two small molecules inhibiting the SEC reduced the expression of several mesenchymal markers SM22 (−87.6%, P<0.05) and Calponin (−93.8%, P<0.05) during EndMT. After induction of EndMT, a large number of newly synthesized RNA transcripts were upregulated, indicating regulation of gene expression on the level of RNA elongation during EndMT. Sequencing of newly transcribed RNA (4sU-sequencing) after treatment with the SEC inhibitors revealed a lower transcript density downstream of promoter sites, suggesting a decreased RNA elongation rate in ECs. SEC inhibitors decreased the expression of the majority of EndMT-induced transcripts (63.6%). Among these rapid induced genes we found the mRNA of RE1 (+10,8-fold). We validated the regulation of RE1 by qPCR (2.8-fold, P<0.05) and single-cell sequencing. Chromatin immunoprecipitation (ChIP) sequencing revealed a reduced occupation of RNAPII in proximity of promotor sites of RE1 upon induction of EndMT. These data suggest a SEC-mediated regulation on the level of RNA elongation of RE1 during EndMT. Conclusion SEC scaffold proteins AFF1 and AFF4 are upregulated during EndMT. Targeting the SEC during EndMT reduces the expression of common mesenchymal markers, consistent with an overall higher occupancy of paused RNAPII at promoter-proximal sites. We identify RE1 as a rapidly induced gene during EndMT that is mediated by the SEC scaffold proteins AFF1 and AFF4. Our findings suggest that RNA elongation is an inducer of EndMT. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)

Distinctive features of the respiratory syncytial virus priming loop compared to other non-segmented negative strand RNA viruses.

De novo initiation by viral RNA-dependent RNA polymerases often requires a polymerase priming residue, located within a priming loop, to stabilize the initiating NTPs. Polymerase structures from three different non-segmented negative strand RNA virus (nsNSV) families revealed putative priming loops in different conformations, and an aromatic priming residue has been identified in the rhabdovirus polymerase. In a previous study of the respiratory syncytial virus (RSV) polymerase, we found that Tyr1276, the L protein aromatic amino acid residue that most closely aligns with the rhabdovirus priming residue, is not required for RNA synthesis but two nearby residues, Pro1261 and Trp1262, were required. In this study, we examined the roles of Pro1261 and Trp1262 in RNA synthesis initiation. Biochemical studies showed that substitution of Pro1261 inhibited RNA synthesis initiation without inhibiting back-priming, indicating a defect in initiation. Biochemical and minigenome experiments showed that the initiation defect incurred by a P1261A substitution could be rescued by factors that would be expected to increase the stability of the initiation complex, specifically increased NTP concentration, manganese, and a more efficient promoter sequence. These findings indicate that Pro1261 of the RSV L protein plays a role in initiation, most likely in stabilizing the initiation complex. However, we found that substitution of the corresponding proline residue in a filovirus polymerase had no effect on RNA synthesis initiation or elongation. These results indicate that despite similarities between the nsNSV polymerases, there are differences in the features required for RNA synthesis initiation.

Characterization of SARS-CoV-2 replication complex elongation and proofreading activity

The replication complex (RC) of SARS-CoV-2 was recently shown to be one of the fastest RNA-dependent RNA polymerases of any known coronavirus. With this rapid elongation, the RC is more prone to incorporate mismatches during elongation, resulting in a highly variable genomic sequence. Such mutations render the design of viral protein targets difficult, as drugs optimized for a given viral protein sequence can quickly become inefficient as the genomic sequence evolves. Here, we use biochemical experiments to characterize features of RNA template recognition and elongation fidelity of the SARS-CoV-2 RdRp, and the role of the exonuclease, nsp14. Our study highlights the 2′OH group of the RNA ribose as a critical component for RdRp template recognition and elongation. We show that RdRp fidelity is reduced in the presence of the 3′ deoxy-terminator nucleotide 3′dATP, which promotes the incorporation of mismatched nucleotides (leading to U:C, U:G, U:U, C:U, and A:C base pairs). We find that the nsp10–nsp14 heterodimer is unable to degrade RNA products lacking free 2′OH or 3′OH ribose groups. Our results suggest the potential use of 3′ deoxy-terminator nucleotides in RNA-derived oligonucleotide inhibitors as antivirals against SARS-CoV-2.

RNA polymerase I (Pol I) lobe-binding subunit Rpa12.2 promotes RNA cleavage and proofreading

Elongating nuclear RNA polymerases (Pols) frequently pause, backtrack, and are then reactivated by endonucleolytic cleavage. Pol backtracking and RNA cleavage are also crucial for proofreading, which contributes to transcription fidelity. RNA polymerase I (Pol I) of the yeast Saccharomyces cerevisiae synthesizes exclusively 35S rRNA, the precursor transcript of mature ribosomal 5.8S, 18S, and 25S rRNA. Pol I contains the specific heterodimeric subunits Rpa34.5/49 and subunit Rpa12.2, which have been implicated in RNA cleavage and elongation activity, respectively. These subunits are associated with the Pol I lobe structure and encompass different structural domains, but the contribution of these domains to RNA elongation is unclear. Here, we used Pol I mutants or reconstituted Pol I enzymes to study the effects of these subunits and/or their distinct domains on RNA cleavage, backtracking, and transcription fidelity in defined in vitro systems. Our findings suggest that the presence of the intact C-terminal domain of Rpa12.2 is sufficient to support the cleavage reaction, but that the N-terminal domains of Rpa12.2 and the heterodimer facilitate backtracking and RNA cleavage. Since both N-terminal and C-terminal domains of Rpa12.2 were also required to faithfully incorporate NTPs in the growing RNA chain, efficient backtracking and RNA cleavage might be a prerequisite for transcription fidelity. We propose that RNA Pols containing efficient RNA cleavage activity are able to add and remove nucleotides until the matching nucleotide supports RNA chain elongation, whereas cleavage-deficient enzymes can escape this proofreading process by incorporating incorrect nucleotides.

The establishment of CDK9/RNA PolII/H3K4me3/DNA methylation feedback promotes HOTAIR expression by RNA elongation enhancement in cancer

Long non-coding RNA HOX Transcript Antisense RNA (HOTAIR) is overexpressed in multiple cancers with diverse genetic profiles. Importantly, since HOTAIR heavily contributes to cancer progression by promoting tumor growth and metastasis, HOTAIR becomes a potential target for cancer therapy. However, the underlying mechanism leading to HOTAIR deregulation is largely unexplored. Here, we performed a pan-cancer analysis using more than 4,200 samples and found that intragenic exon CpG island (Ex-CGI) was hypermethylated and was positively correlated to HOTAIR expression. Also, we revealed that Ex-CGI methylation promotes HOTAIR expression through enhancing the transcription elongation process. Furthermore, we linked up the aberrant intragenic tri-methylation on H3 at lysine 4 (H3K4me3) and Ex-CGI DNA methylation in promoting transcription elongation of HOTAIR. Targeting the oncogenic CDK7-CDK9-H3K4me3 axis downregulated HOTAIR expression and inhibited cell growth in many cancers. To our knowledge, this is the first time that a positive feedback loop that involved CDK9-mediated phosphorylation of RNA Polymerase II Serine 2 (RNA PolII Ser2), H3K4me3, and intragenic DNA methylation, which induced robust transcriptional elongation and heavily contributed to the upregulation of oncogenic lncRNA in cancer has been demonstrated. Targeting the oncogenic CDK7-CDK9-H3K4me3 axis could be a novel therapy in many cancers through inhibiting the HOTAIR expression.

First report of Plectosphaerella cucumerina causing root rot on fennel in China.

Fennel (Foeniculum vulgare Mill.) is herbaceous plant commonly cultivated for culinary and medicinal uses in China (Shi et al. 2016 ). In May 2019, disease of fennel was observed in Yumen City, Gansu Province, China (N 40°28'/E 97°05'). The incidence across the fields (about 0.23 hectare) was about 4.5%. The outer leaves of diseased fennel wilted, the rhizome changed color from brown to dark brown，necrosis and rot symptoms developed on the root. Finally, the whole plant wilted and died. When pulling up, it was easy to break the root. To identify the pathogen, 15 samples of diseased plants were collected and symptomatic rhizome tissues were surface disinfected with 0.1% HgCl solution for 30 s, rinsed in sterilized water 3 times, placed on potato dextrose agar (PDA), and incubated at 25℃ in the dark. About 7 days, hyphal tips from the tissue edge were transferred to a new PDA for purification. Three isolates obtained were named as hxa, hxb and hxc. To confirm their pathogenicity, two-month old fennel seedings planted in pots, three seedings per pot, with sterilized nutrient soil were inoculated by pouring 50 ml of conidial suspension (107 conidium/mL) produced from the three isolates. Plants inoculated with sterilized water only were included as controls. Six pots of inoculated plants were maintained in climatic cabinet / chamber (> 85% RH, 25°C). The pathogenicity tests were conducted twice. After 7 days, the plants inoculated with conidial suspension of hxa developed brown necrosis and wilt symptoms resembling those originally observed in the field, whereas the controls and the plants inoculated with the other two isolates had no symptoms. Furthermore, hxa was reisolated from rhizome of these diseased plants. The results indicated that isolate hxa was the pathogen causing root rot of fennel. The colonies of hxa on PDA were white-to-cream, slimy, mycelium appressed, aerial mycelium absent. Mycelium was hyaline, septate and formed hyphal coils. Conidiophores were solitary, hyaline, sometimes crooked or sinuous, widest at the base, gradually tapering to the apex. Conidia were smooth, hyaline, aseptate, elliptical and ovoid, measuring 5.97 to 9.51 × 2.13 to 3.58 um (avg. 7.58×2.78, n=100). These morphological characters of the fungal isolates were identical to those of Plectosphaerella cucumerina (Carlucci et al. 2012). For molecular identification, genomic DNA was extracted from the mycelium, and the rDNA internal transcribed spacer (ITS) region, portions of the 28S ribosomal RNA (LSU), calmodulin (CaM) and translation elongation factor 1α (Ef-1α) gene were amplified using primer pairs ITS1/ITS4, LROR/LR5, CMD5/CMD6 and 688f/1251R (White et al., 1990; Hopple et al., 1999; Hong et al., 2005; Alves et al., 2008), respectively. The sequences of these genes were deposited in GenBank (accessions: ITS as MW426266, LSU as MW433724, CaM as MW448071 and EF-1a as MW459981) and used in analysis to generate a phylogenetic tree. These sequences showed 100, 100, 96 and 97.32% homology to the sequences of P. cucumerina (GenBank accession no. EU594566, MH867359, KY416911 and KY964491), respectively. According to morphological characteristics and phylogenetic analysis, isolate hxa was identified as P. cucumerina. To our knowledge, this is the first report of P. cucumerina causing root rot of fennel in China as well as worldwide. This finding may help to take effective control measures of root rot on fennel.

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication.

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3'UTR-U30 (5'-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3').

THOC2 and THOC5 Regulate Stemness and Radioresistance in Triple-Negative Breast Cancer.

Triple‐negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Radioresistance and stemness are substantial obstacles to TNBC treatment. The THO complex (THOC) is a subunit of the TRanscription–EXport complex that functions in the coupling of transcription to nascent RNA splicing, elongation, and export. However, its role in regulating TNBC therapeutic resistance is not reported yet. In this study, the authors demonstrate that cancer stem cells are enriched in radioresistant TNBC cells and describe the role of the THOC in regulating TNBC radioresistance and stemness. The authors find that THOC2 and THOC5 are upregulated in radioresistant TNBC cells and associated with a poor prognosis in TNBC patients. Further investigation reveals that THOC2 promotes the stem‐like properties and radioresistance of TNBC cells in a THOC5‐dependent manner by facilitating the release of sex‐determining region Y (SRY)‐box transcription factor 2 (SOX2) and homeobox transcription factor (NANOG) transcripts from the nucleus. Silencing THOC2 or THOC5 expression decreases the protein expression of SOX2 and NANOG, depletes the stem‐like properties, and causes radiosensitization in these TNBC cells. Moreover, THOC2 or THOC5 depletion blocks the xenograft tumorigenesis and growth of radioresistant TNBC in vivo. These findings uncover the novel correlations of THOC with TNBC stemness and therapeutic resistance, proposing alternative therapeutic strategies against relapsed TNBC.

A novel mechanism of enhanced transcription activity and fidelity for influenza A viral RNA-dependent RNA polymerase.

During RNA elongation, the influenza A viral (IAV) RNA-dependent RNA polymerase (RdRp) residues in the active site interact with the triphosphate moiety of nucleoside triphosphate (NTP) for catalysis. The molecular mechanisms by which they control the rate and fidelity of NTP incorporation remain elusive. Here, we demonstrated through enzymology, virology and computational approaches that the R239 and K235 in the PB1 subunit of RdRp are critical to controlling the activity and fidelity of transcription. Contrary to common beliefs that high-fidelity RdRp variants exert a slower incorporation rate, we discovered a first-of-its-kind, single lysine-to-arginine mutation on K235 exhibited enhanced fidelity and activity compared with wild-type. In particular, we employed a single-turnover NTP incorporation assay for the first time on IAV RdRp to show that K235R mutant RdRp possessed a 1.9-fold increase in the transcription activity of the cognate NTP and a 4.6-fold increase in fidelity compared to wild-type. Our all-atom molecular dynamics simulations further elucidated that the higher activity is attributed to the shorter distance between K235R and the triphosphate moiety of NTP compared with wild-type. These results provide novel insights into NTP incorporation and fidelity control mechanisms, which lay the foundation for the rational design of IAV vaccine and antiviral targets.

Allosteric transcription stimulation by RNA polymerase II super elongation complex

The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and the subcomplex ELL2-EAF1, which stimulates RNA polymerase II (RNA Pol II) elongation. Here, we report the cryoelectron microscopy (cryo-EM) structure of ELL2-EAF1 bound to a RNA Pol II elongation complex at 2.8Å resolution. The ELL2-EAF1 dimerization module directly binds the RNA Pol II lobe domain, explaining how SEC delivers P-TEFb to RNA Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that the stimulation of RNA elongation requires the dimerization module and the ELL2 linker that tethers the module to the RNA Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a closed conformation of the RNA Pol II active center cleft.

Real-time single-cell characterization of the eukaryotic transcription cycle reveals correlations between RNA initiation, elongation, and cleavage.

The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel application of Bayesian inference techniques to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters as well as single-cell correlations between these parameters. These measurements, combined with theoretical modeling, suggest a substantial variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution.

Structural visualization of transcription activated by a multidrug-sensing MerR family regulator

Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved –35 and –10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of transcriptional regulators activate suboptimal 19–20 bp spacer promoters in response to myriad cellular signals, ranging from heavy metals to drug-like compounds. The regulation of transcription by MerR family regulators is not fully understood. Here we report one crystal structure of a multidrug-sensing MerR family regulator EcmrR and nine cryo-electron microscopy structures that capture the EcmrR-dependent transcription process from promoter opening to initial transcription to RNA elongation. These structures reveal that EcmrR is a dual ligand-binding factor that reshapes the suboptimal 19-bp spacer DNA to enable optimal promoter recognition, sustains promoter remodeling to stabilize initial transcribing complexes, and finally dissociates from the promoter to reverse DNA remodeling and facilitate the transition to elongation. Our findings yield a comprehensive model for transcription regulation by MerR family factors and provide insights into the transition from transcription initiation to elongation.

Long-Range Supercoiling-Mediated RNA Polymerase Cooperation in Transcription.

It is widely believed that DNA supercoiling plays an important role in the regulation of transcriptional dynamics. Recent studies show that it could affect transcription not only through the buildup and relaxation of torsional strain on DNA strands but also via effective long-range supercoiling-mediated interactions between RNA polymerase (RNAP) molecules. Here, we present a theoretical study that quantitatively analyzes the effect of long-range RNAP cooperation in transcription dynamics. Our minimal chemical-kinetic model assumes that one or two RNAP molecules can simultaneously participate in the transcription, and it takes into account their binding to and dissociation from DNA. It also explicitly accounts for competition between the supercoiling buildup that reduces the RNA elongation speed and gyrase binding that rescues the RNA synthesis. The full analytical solution of the model accompanied by Monte Carlo computer simulations predicts that the system should exhibit transcriptional bursting dynamics, in agreement with experimental observations. The analysis also revealed that when there are two polymerases participating in the elongation rather than one, the transcription process becomes much more efficient since the level of stochastic noise decreases while more RNA transcripts are produced. Our theoretical investigation clarifies molecular aspects of the supercoiling-mediated RNAP cooperativity during transcription.

Diversity of Fusarium species causing invasive and disseminated infections

IntroductionInvasive fusariosis (IF) is considered an emerging fungal disease and an important problem worldwide that increasingly affects immunocompromised individuals. There is currently concern about establishing the genetic diversity and phylogenetic relationship of the species Fusarium causing invasive fusariosis. Materials and MethodsThe aim of this study was to characterize the molecular profile and morphological characteristics of Fusarium species isolated from 21 patients with invasive fusariosis. Multilocus sequence typing was performed for molecular identification of the following genes: the second largest subunit of the RNA polymerase gene (RPB2) and elongation factor 1 alpha (EF-1α). The morphological features of different species were carefully described and revised by experienced mycologists. ResultsMorphological and molecular analyses revealed that the F. solani species complex (FSSC) and F. oxysporum species complex (FOSC) were the most common species isolated from patients with invasive fusariosis; FSSC-2 h (5), FSSC-1 (2) and FOSC-183 (2) were the most frequent haplotypes. The macroscopic characterization revealed great variation in the tonalities of the FSSC colonies and particularities amongst the species in relation to the macroconidia structures, while the FOSC was more homogeneous and presented shades from white to lilac. ConclusionsOur study characterized the diversity, haplotypes, and morphological aspects of Fusarium species and the haplotypes prevalent in patients with invasive fusariosis. FSSC and FSSC-2 h were the predominant species and haplotype, respectively. Although we have described interesting morphological aspects in Fusarium species, particularly haplotypes, their identification cannot rely on phenotypical aspects. Molecular biology techniques are necessary and should be introduced for routine use in mycology laboratories.