Biology subjects

Teng, M.

Publications and source records attributed to Teng, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Using Polycistronic gRNAs and a tRNA Cleavage System in CRISPR-Based Genome Editing of Toxoplasma gondii

CRISPR/Cas9 is widely used for genetic manipulation in Toxoplasma gondii, but optimizing gRNA expression and multiplexed editing remains challenging. Here, we evaluated alternative promoters, gRNA scaffolds, and a tRNA-based polycistronic system to improve editing efficiency and reduce synthetic DNA costs. Four constructs targeting the SAG1 locus were used to compare the U6 and H1 promoters, as well as standard and modified gRNA scaffolds. U6-driven constructs supported robust gRNA-targeted Cas9 activity, whereas H1-driven constructs failed to produce detectable phenotypes. The alternative gRNA scaffold, modified by a number of base changes and insertions, increased knockout efficiency compared to the standard scaffold. To simplify dual gRNA-based methods, we establish a tRNA-based polycistronic system to simultaneously target two independent loci. Targeting the SAG1 and UPRT locus for proof-of-concept, all U6-driven polycistronic constructs conferred efficient SAG1 knockout and 5-fluorodeoxyuridine resistance, whereas H1-driven constructs exhibited position-dependent activity, with only the 3' gRNA being functional. These results demonstrate that U6-driven, tRNA-based polycistronic gRNA expression enables reliable multiplexed genome editing in T. gondii, with the minimal H1 promoter being insufficient for consistent multi-gRNA activity. This optimized system provides a scalable framework for dual-gRNA CRISPR strategies in this organism.

molecular biology↗

bioRxiv2026

SGFP-Grid Split GFP Graphene Grids

Affinity graphene grids provide a promising approach for selective protein capture in cryo-EM. Here, we introduce a split-GFP graphene grid platform(SGFP-G), in which graphene-conjugated GFP 1-10 selectively captures GFP11 tagged proteins from low concentration samples or cell lysates. This platform enables rapid assessment of target protein enrichment and particle distribution before vitrification via fluorescence imaging, while the grid design positions captured proteins away from the graphene surface and air-water interface. We also introduce a unique strategy to minimize nonspecific protein adsorption, thereby improving the selective enrichment of target proteins on this grid. Using GFP11-tagged apoferritin, we demonstrate fluorescence guided protein capture and obtain a 2.58 [A] cryoEM reconstruction, establishing SGFP-G as an affinity grid platform for high resolution structural studies with reduced sample requirements.

biochemistry↗

bioRxiv2026

Chemoproteomic Characterization of GPX4 Covalent Ligands and Targeted Degradation

Despite intensive efforts, the ferroptosis gatekeeper glutathione peroxidase 4 (GPX4) remains difficult to selectively target due to stringent structural constraints surrounding its catalytic selenocysteine, which impose tight requirements on warhead reactivity and geometry. Here, leveraging a chemoproteomic approach, we characterize a potent and selective covalent GPX4 inhibitor featuring a pyrimidinylmethyl isourea warhead and define the chemical features underlying its proteome-wide selectivity. This chemotype enables tunable electrophile reactivity through steric and electronic modulation of leaving group ability, suggesting potential broader utility for targeting other recalcitrant proteins. Building on this scaffold, we further develop two selective GPX4 degraders - one CRBN-dependent and the other CRBN-independent - enabling complementary modulation of GPX4 through both inhibition and degradation. Together, these molecules expand the GPX4 chemical toolbox for more nuanced interrogation of GPX4 biology.

cancer biology↗

bioRxiv2026

Vascular Destabilization and Pericyte Detachment are Mediated by hIAPP Aggregation in Transgenic Mice.

Aims/hypothesisHuman islet amyloid polypeptide (hIAPP) deposition is a common feature of type-2 diabetes (T2D). Previous studies have demonstrated hIAPP-mediated endothelial cell (EC) dysfunction and inflammation, but little is known about islet microvascular stability or pericyte function in hIAPP-containing islets. This study investigates how islet endothelial cells and pericytes are influenced by hIAPP aggregation. MethodsBulk RNAseq and qPCR were conducted on hIAPP or vehicle treated MS-1 cells and bead-purified human islet CD31+ cells from donors with or without T2D to determine how islet ECs respond to hIAPP exposure. Confocal imaging of living pancreatic slices obtained from hIAPP transgenic mice was conducted to evaluate the effect of hIAPP deposition on islet pericyte function and vasomotor responses. ResultshIAPP-treated MS-1 cells and ECs purified from T2D islets demonstrate downregulation of leading-edge genes associated with extracellular matrix and cell adhesion pathways. Pericytes from hIAPP-expressing mouse islets appear detached from underlying endothelial cells, which was associated with impaired vasomotor responses to constrictive or dilatory stimuli. Conclusions/interpretationhIAPP induces vascular destabilization by downregulating mRNA of key extracellular matrix and cell adhesion molecules in ECs, likely promoting the breakdown of EC-EC and EC-pericyte coupling. hIAPP disrupts EC-pericyte connections, and pericyte detachment ultimately impairs pericytes ability to modulate capillary diameter without impairing intracellular Ca2+ dynamics. Our data suggest that amyloid deposition compromises EC health and survival by altering islet microvascular morphology, stability, and function. This, in turn, may disrupt islet microvascular stability and exacerbate endocrine cell dysfunction in T2D. Research in contextO_ST_ABSWhat is already known about this subject?C_ST_ABS- hIAPP is cytotoxic to islet endothelial cells and beta cells, and contributes to islet failure in type-2 diabetes (T2D) - hIAPP transgenic mice demonstrate islet capillary dilation, loss of vascular structures, and increased pericyte density - Impaired pericyte anchorage and vascular fragmentation drive diabetes-related vasculopathies in other tissues, like the retina, kidney, and brain What is the key question?- How does the surviving microvasculature in islets respond to hIAPP deposition? What are the new findings?- Endothelial cells demonstrate transcriptional downregulation of key genes involved in cytoskeleton, ECM, and cell-adhesion maintenance, including Thbs1, Tln1, and Plec. - Amyloid deposits disrupt homeostatic interactions between endothelial cells and pericytes. - Amyloid-adjacent islet pericytes are detached from endothelial cells and display impaired ability to modulate capillary diameter. How might this impact on clinical practice in the foreseeable future?- Therapies targeting endothelial cell-pericyte interactions may restore islet microvascular stability and improve islet function, especially in the context of early T2D.

molecular biology↗

bioRxiv2025

Epstein-Barr Virus Encoded lncRNAs Control the Viral Lytic Switch

Epstein-Barr virus (EBV) uses a biphasic lifecycle, switching between latent and lytic phases to persistently infect most adults. Latency is observed in most tumor cells of the 200,000 EBV-associated cancers/year. EBV reactivation is increasingly implicated in autoimmune diseases, including multiple sclerosis. However, mechanisms that regulate EBV reactivation have remained incompletely understood. Here, we leveraged multi-omic approaches to reveal the existence of pro-latency and a pro-lytic viral long noncoding RNAs (lncRNAs) that counter-regulate the lytic switch. Known reactivation triggers rapidly induced expression of the pro-lytic lncRNA, which encodes an RNA G-quadruplex that mediated its interaction with CTCF. The pro-lytic lncRNA occupies viral origin of lytic replication enhancers and promotes their looping to the immediate early lytic promoter to trigger reactivation. The pro-latency lncRNA duplexes with the pro-lytic RNA to impede its interactions with CTCF. These studies lay a foundation for therapeutic approaches to manipulate the EBV lytic switch.

microbiology↗

bioRxiv2025

Functional landscape of circular RNAs in human cancer cells

Circular RNAs (circRNAs) constitute a novel class of noncoding RNAs showcasing distinct tissue- and cell-specific expression patterns. Despite the extensive profiling of circRNAs, their individual functions remain poorly understood. To fill this gap, we designed a genome-wide library of 65,300 shRNAs, targeting 9,663 clinically relevant circRNAs and 3,981 of their linear parental genes, and conducted functional screening in seven types of human cancer. We identified a total of 1,342 essential circRNAs (13.9% screened) that impact cell proliferation in at least one cell line, and in 96.5% of the cases, the linear counterparts are not essential. While a shared common subset emerges as functional regulators across all examined cell lines, the majority of circRNAs are functional in a cell type-specific manner. For a comprehensive presentation of the functional circRNA landscape in cancer, we introduce FunCirc, an online database encompassing functional circRNAs across cancer cell lines, coupled with circRNA expression profiles from diverse cancer and tissue types. Our work enhances the understanding of circRNA functions in cancer and provides the scientific community with a resource to further investigate their intricate roles.

cancer biology↗

bioRxiv2025

Circular RNA profiling and functional screening in breast cancer identify circNSD1 as a suppressor of tumor autophagy

Circular RNAs (circRNAs) have emerged as critical regulators of cell biology. However, their function in breast cancer remains elusive. Herein, through circRNA profiling of 38 breast tumors and 10 benign breast tissues, we identified 509 of differentially expressed circRNAs. Integration with transcriptome-wide functional screening of approximately 10,000 circRNAs pinpointed circNSD1(6) as a top ranked tumor suppressor in breast cancer. circNSD1(6) is downregulated in tumors and suppression of the circular form, but not the linear form, promotes breast cancer proliferation and tumor growth. Mechanistically, circNSD1(6) interacts with autophagy receptor SQSTM1/p62 and inhibits the oligomerization of p62, thereby suppressing p62 body formation and p62-dependent autophagy. Furthermore, circNSD1(6) ameliorates p62-mediated Keap1 sequestration, hence suppresses the Nrf2 pathway and oxidative stress tolerance. Our study provides a landscape view of the transcription and function of circRNA in breast cancer, and uncovers the crucial role of circNSD1(6) in autophagy and antioxidant stress.

cancer biology↗

bioRxiv2024

The DNA loop release factor WAPL suppresses Epstein-Barr virus latent membrane protein expression to maintain the highly restricted latency I program

Epstein-Barr virus (EBV) uses latency programs to colonize the memory B-cell reservoir, and each program is associated with human malignancies. However, knowledge remains incomplete of epigenetic mechanisms that maintain the highly restricted latency I program, present in memory and Burkitt lymphoma cells, in which EBNA1 is the only EBV-encoded protein expressed. Given increasing appreciation that higher order chromatin architecture is an important determinant of viral and host gene expression, we investigated roles of Wings Apart-Like Protein Homolog (WAPL), a host factor that unloads cohesins to control DNA loop size and that was discovered as an EBNA2-associated protein. WAPL knockout (KO) in Burkitt cells de-repressed LMP1 and LMP2A expression but not other EBV oncogenes to yield a viral program reminiscent of EBV latency II, which is rarely observed in B-cells. WAPL KO also increased LMP1/2A levels in latency III lymphoblastoid cells. WAPL KO altered EBV genome architecture, triggering formation of DNA loops between the LMP promoter region and the EBV origins of lytic replication (oriLyt). Hi-C analysis further demonstrated that WAPL KO reprograms EBV genomic DNA looping. LMP1 and LMP2A de-repression correlated with decreased histone repressive marks at their promoters. We propose that EBV coopts WAPL to negatively regulate latent membrane protein expression to maintain Burkitt latency I. Author SummaryEBV is a highly prevalent herpesvirus etiologically linked to multiple lymphomas, gastric and nasopharyngeal carcinomas, and multiple sclerosis. EBV persists in the human host in B-cells that express a series of latency programs, each of which is observed in a distinct type of human lymphoma. The most restricted form of EBV latency, called latency I, is observed in memory cells and in most Burkitt lymphomas. In this state, EBNA1 is the only EBV-encoded protein expressed to facilitate infected cell immunoevasion. However, epigenetic mechanisms that repress expression of the other eight EBV-encoded latency proteins remain to be fully elucidated. We hypothesized that the host factor WAPL might have a role in restriction of EBV genes, as it is a major regulator of long-range DNA interactions by negatively regulating cohesin proteins that stabilize DNA loops, and WAPL was found in a yeast 2-hybrid screen for EBNA2-interacting host factors. Using CRISPR together with Hi-ChIP and Hi-C DNA architecture analyses, we uncovered WAPL roles in suppressing expression of LMP1 and LMP2A, which mimic signaling by CD40 and B-cell immunoglobulin receptors, respectively. These proteins are expressed together with EBNA1 in the latency II program. We demonstrate that WAPL KO changes EBV genomic architecture, including allowing the formation of DNA loops between the oriLyt enhancers and the LMP promoter regions. Collectively, our study suggests that WAPL reinforces Burkitt latency I by preventing the formation of DNA loops that may instead support the latency II program.

microbiology↗

bioRxiv2024

A novel protein Moat prevents ectopic epithelial folding by limiting Bazooka/Par3-dependent adherens junctions

Cortical myosin contraction and cell adhesion work together to promote tissue shape changes, but how they are modulated to achieve diverse morphogenetic outcomes remains unclear. Epithelial folding occurs via apical constriction, mediated by apical accumulation of contractile myosin engaged with adherens junctions, as in Drosophila ventral furrow formation. While levels of contractile myosin correlate with apical constriction, whether levels of adherens junctions modulate apical constriction is unknown. We identified a novel Drosophila gene moat that maintains low levels of Bazooka/Par3-dependent adherens junctions and thereby restricts apical constriction to ventral furrow cells with high-level contractile myosin. In moat mutants, abnormally high levels of Bazooka/Par3-dependent adherens junctions promote ectopic apical constriction in cells with low-level contractile myosin, insufficient for apical constriction in wild type. Such ectopic apical constriction expands infolding behavior from ventral furrow to ectodermal anterior midgut, which normally forms a later circular invagination. In moat mutant ventral furrow, a perturbed apical constriction gradient delays infolding. Our results indicate that levels of adherens junctions can modulate the outcome of apical constriction, providing an additional mechanism to define morphogenetic boundaries. Summary StatementCharacterization of a novel gene moat demonstrates ectopic expansion of apical constriction due to abnormally high levels of Bazooka/Par3-dependent adherens junctions without defects in early patterning gene expression.

developmental biology↗

bioRxiv2023

Exploration of the Tunability of BRD4 Degradation by DCAF16 Trans-labelling Covalent Glues

Small molecules that can induce protein degradation by inducing proximity between a desired target and an E3 ligase have the potential to greatly expand the number of proteins that can be manipulated pharmacologically. Current strategies for targeted protein degradation are mostly limited in their target scope to proteins with preexisting ligands. Alternate modalities such as molecular glues, as exemplified by the glutarimide class of ligands for the CUL4CRBN ligase, have been mostly discovered serendipitously. We recently reported a trans-labelling covalent glue mechanism which we named Template-assisted covalent modification, where an electrophile decorated small molecule binder of BRD4 was effectively delivered to a cysteine residue on an E3 ligase DCAF16 as a consequence of a BRD4-DCAF16 protein-protein interaction. Herein, we report our medicinal chemistry efforts to evaluate how various electrophilic modifications to the BRD4 binder, JQ1, affect DCAF16 trans-labeling and subsequent BRD4 degradation efficiency. We discovered a decent correlation between the ability of the electrophilic small molecule to induce ternary complex formation between BRD4 and DCAF16 with its ability to induce BRD4 degradation. Moreover, we show that a more solvent-exposed warhead presentation is optimal for DCAF16 recruitment and subsequent BRD4 degradation. Unlike the sensitivity of CUL4CRBN glue degraders to chemical modifications, the diversity of covalent attachments in this class of BRD4 glue degraders suggests a high tolerance and tunability for the BRD4-DCAF16 interaction. This offers a potential new avenue for a rational design of covalent glue degraders by introducing covalent warheads to known binders.

pharmacology and toxicology↗

bioRxiv2023

Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders

Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics1-3. Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs4-6. The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans-labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). BRD4BD2, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 [A] cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4BD2 revealed that the loop conformation around BRD4His437, rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.

biochemistry↗

bioRxiv2022

A CRISPR screen identifies redox vulnerabilities for KEAP1/NRF2 mutant non-small cell lung cancer

The redox regulator NRF2 is hyperactivated in a large percentage of non-small cell lung cancer (NSCLC) cases, which is associated with chemotherapy and radiation resistance. To identify redox vulnerabilities for KEAP1/NRF2 mutant NSCLC, we conducted a CRISPR-Cas9-based negative selection screen for antioxidant enzyme genes whose loss sensitized cells to sub-lethal concentrations of the superoxide (O2*-)-generating drug {beta}-Lapachone. While our screen identified expected hits in the pentose phosphate pathway, the thioredoxin-dependent antioxidant system, and glutathione reductase, we also identified the mitochondrial superoxide dismutase 2 (SOD2) as one of the top hits. Surprisingly, {beta}-Lapachone did not generate mitochondrial O2*- but rather SOD2 loss enhanced the efficacy of {beta}-Lapachone due to loss of iron-sulfur protein function, loss of mitochondrial ATP maintenance and deficient NADPH production. Importantly, inhibition of mitochondrial electron transport activity sensitized to {beta}-Lapachone, demonstrating these effects may be translated to increase ROS sensitivity therapeutically.

cancer biology↗

bioRxiv2021

Summarizing internal dynamics boosts differential analysis and functional interpretation of super enhancers

Super enhancers (SEs) are broad enhancer domains usually containing multiple constituent enhancers that hold elevated activities in gene regulation. Disruption in one or more constituent enhancers causes aberrant SE activities that lead to gene dysregulation in diseases. To quantify SE aberrations, differential analysis is performed to compare SE activities between cell conditions. The state-of-art strategy in estimating differential SEs relies on overall activities and neglect the changes in length and structure of SEs. Here, we propose a novel computational method to identify differential SEs by weighting the combinatorial effects of constituent-enhancer activities and locations (i.e., internal dynamics). In addition to overall activity changes, our method identified four novel classes of differential SEs with distinct enhancer structural alterations. We demonstrate that these structure alterations hold distinct regulatory impact, such as regulating different number of genes and modulating gene expression with different strengths, highlighting the differentiated regulatory roles of these unexplored SE features. When compared to the existing method, our method showed improved identification of differential SEs that were linked to better discernment of cell-type-specific SE activity and functional interpretation. We implement an R package, DASE, to facilitate the use of our method.

bioinformatics↗

bioRxiv2020

The mutational landscape of human somatic and germline cells

During the course of a lifetime normal human cells accumulate mutations. Here, using multiple samples from the same individuals we compared the mutational landscape in 29 anatomical structures from soma and the germline. Two ubiquitous mutational signatures, SBS1 and SBS5/40, accounted for the majority of acquired mutations in most cell types but their absolute and relative contributions varied substantially. SBS18, potentially reflecting oxidative damage, and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The mutation rate was lowest in spermatogonia, the stem cell from which sperm are generated and from which most genetic variation in the human population is thought to originate. This was due to low rates of ubiquitous mutation processes and may be partially attributable to a low cell division rate of basal spermatogonia. The results provide important insights into how mutational processes affect the soma and germline.

genomics↗

bioRxiv2020

Defining a Global Map of Functional Group Based 3D Ligand-binding Motifs

Uncovering conserved 3D protein-ligand binding patterns at the basis of functional groups (FGs) shared by a variety of small molecules can greatly expand our knowledge of protein-ligand interactions. Despite that conserved binding patterns for a few commonly used FGs have been reported in the literature, large-scale identification and evaluation of FG-based 3D binding motifs are still lacking. Here, we developed AFTME, an alignment-free method for automatic mapping of 3D motifs to different FGs of a specific ligand through two-dimensional clustering. Applying our method to 233 nature-existing ligands, we defined 481 FG-binding motifs that are highly conserved across different ligand-binding pockets. Systematic analysis further reveals four main classes of binding motifs corresponding to distinct sets of FGs. Combinations of FG-binding motifs facilitate proteins to bind a wide spectrum of ligands with various binding affinities. Finally, we showed that these general binding patterns are also applicable to target-drug interactions, providing new insights into structure-based drug design.

bioinformatics↗

bioRxiv2020

Histone loaders CAF1 and HIRA restrict Epstein-Barr virus B-cell lytic reactivation

Epstein-Barr virus (EBV) infects 95% of adults worldwide and causes infectious mononucleosis. EBV is associated with endemic Burkitt lymphoma, Hodgkin lymphoma, post-transplant lymphomas, nasopharyngeal and gastric carcinomas. In these cancers and in most infected B-cells, EBV maintains a state of latency, where nearly 80 lytic cycle antigens are epigenetically suppressed. To gain insights into host epigenetic factors necessary for EBV latency, we recently performed a human genome-wide CRISPR screen that identified the chromatin assembly factor CAF1 as a putative Burkitt latency maintenance factor. CAF1 loads histones H3 and H4 onto newly synthesized host DNA, though its roles in EBV genome chromatin assembly are uncharacterized. Here, we identified that CAF1 depletion triggered lytic reactivation and transforming virion secretion from Burkitt cells, despite strongly also inducing interferon stimulated genes. CAF1 perturbation diminished occupancy of histones 3.1, 3.3 and repressive H3K9me3 and H3K27me3 marks at multiple viral genome lytic cycle regulatory elements. Suggestive of an early role in establishment of latency, EBV strongly upregulated CAF1 expression in newly infected primary human B-cells prior to the first mitosis, and histone 3.1 and 3.3 were loaded on the EBV genome by this timepoint. Knockout of CAF1 subunit CHAF1B impaired establishment of latency in newly EBV-infected Burkitt cells. A non-redundant latency maintenance role was also identified for the DNA synthesis-independent histone 3.3 loader HIRA. Since EBV latency also requires histone chaperones ATRX and DAXX, EBV coopts multiple host histone pathways to maintain latency, and these are potential targets for lytic induction therapeutic approaches. IMPORTANCEEpstein-Barr virus (EBV) was discovered as the first human tumor virus in endemic Burkitt lymphoma, the most common childhood cancer in sub-Saharan Africa. In Burkitt lymphoma and in 200,000 EBV-associated cancers per year, epigenetic mechanisms maintain viral latency, where lytic cycle factors are silenced. This property complicated EBVs discovery and facilitates tumor immunoevasion. DNA methylation and chromatin-based mechanisms contribute to lytic gene silencing. Here, we identify histone chaperones CAF1 and HIRA, which have key roles in host DNA replication-dependent and replication independent pathways, respectively, are each important for EBV latency. EBV strongly upregulates CAF1 in newly infected B-cells, where viral genomes acquire histone 3.1 and 3.3 variants prior to the first mitosis. Since histone chaperones ATRX and DAXX also function in maintenance of EBV latency, our results suggest that EBV coopts multiple histone pathways to reprogram viral genomes and highlights targets for lytic induction therapeutic strategies.

microbiology↗

bioRxiv2019

Genotype-dependent and non-gradient patterns of RSV gene expression

Respiratory syncytial virus (RSV) is a nonsegmented negative-strand (NNS) RNA virus and a leading cause of severe lower respiratory tract illness in infants and the elderly. Transcription of the ten RSV genes proceeds sequentially from the 3 promoter and requires conserved gene start (GS) and gene end (GE) signals. Previous studies using the prototypical GA1 genotype Long and A2 strains have indicated a gradient of gene transcription. However, recent reports show data that appear inconsistent with a gradient. To better understand RSV transcriptional regulation, mRNA abundances from five RSV genes were measured by quantitative real-time PCR (qPCR) in three cell lines and cotton rats infected with virus isolates belonging to four different genotypes (GA1, ON, GB1, BA). Relative mRNA levels reached steady-state between four and 24 hours post-infection. Steady-state patterns were genotype-specific and non-gradient, where mRNA levels from the G (attachment) gene exceeded those from the more promoter-proximal N (nucleocapsid) gene across isolates. Transcript stabilities could not account for the non-gradient patterns observed, indicating that relative mRNA levels more strongly reflect transcription than decay. While the GS signal sequences were highly conserved, their alignment with N protein in the helical ribonucleocapsid, i.e., N-phase, was variable, suggesting polymerase recognition of GS signal conformation affects transcription initiation. The effect of GS N-phase on transcription efficiency was tested using dicistronic minigenomes. Ratios of minigenome gene expression showed a switch-like dependence on N-phase with a period of seven nucleotides. Our results indicate that RSV gene expression is in part sculpted by polymerases that initiate transcription with a probability dependent on GS signal N-phase.\n\nAuthor SummaryRSV is a major viral pathogen that causes significant morbidity and mortality, especially in young children. Shortly after RSV enters a host cell, transcription from its nonsegmented negative-strand (NNS) RNA genome starts at the 3 promoter and proceeds sequentially. Transcriptional attenuation is thought to occur at each gene junction, resulting in a gradient of gene expression. However, recent studies showing non-gradient levels of RSV mRNA suggest that transcriptional regulation may have additional mechanisms. We show using RSV isolates belonging to four different genotypes that gene expression is genotype-dependent and one gene (the G or attachment gene) is consistently more highly expressed than an upstream neighbor. We hypothesize that variable alignment of highly conserved gene start (GS) signals with nucleoprotein (i.e., variable GS N-phase) can affect transcription and give rise to non-gradient patterns of gene expression. We show using dicistronic RSV minigenomes wherein the reporter genes differ only in the N-phase of one GS signal that GS N-phase affects gene expression. Our results suggest the existence of a novel mechanism of transcriptional regulation that might play a role in other NNS RNA viruses.

molecular biology↗

bioRxiv2018

A practical guide to methods controlling false discoveries in computational biology

BackgroundIn high-throughput studies, hundreds to millions of hypotheses are typically tested. Statistical methods that control the false discovery rate (FDR) have emerged as popular and powerful tools for error rate control. While classic FDR methods use only p-values as input, more modern FDR methods have been shown to increase power by incorporating complementary information as \"informative covariates\" to prioritize, weight, and group hypotheses. However, there is currently no consensus on how the modern methods compare to one another. We investigated the accuracy, applicability, and ease of use of two classic and six modern FDR-controlling methods by performing a systematic benchmark comparison using simulation studies as well as six case studies in computational biology\n\nResultsMethods that incorporate informative covariates were modestly more powerful than classic approaches, and did not underperform classic approaches, even when the covariate was completely uninformative. The majority of methods were successful at controlling the FDR, with the exception of two modern methods under certain settings. Furthermore, we found the improvement of the modern FDR methods over the classic methods increased with the informativeness of the covariate, total number of hypothesis tests, and proportion of truly non-null hypotheses.\n\nConclusionsModern FDR methods that use an informative covariate provide advantages over classic FDR-controlling procedures, with the relative gain dependent on the application and informativeness of available covariates. We present our findings as a practical guide and provide recommendations to aid researchers in their choice of methods to correct for false discoveries.

bioinformatics↗