Biology subjects

Liao, Y.

Publications and source records attributed to Liao, Y..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening

Cas12a is a gene-editing tool that simplifies multiplexed gene targeting through its RNase activity, enabling maturation of individual crRNAs from a pre-crRNA-encoding RNA. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene-editing in cells from enAsCas12aKI mice. To test in vivo activity, we transduced haematopoietic stem cells from E-MycT/+;enAsCas12aKI/+animals with Trp53-targeting pre-crRNAs followed by transplantation into irradiated recipient animals. Tumour development was accelerated and TRP53 protein lost. We generated compact, genome-wide Cas12a knockout libraries targeting each gene with four guide RNAs encoded on two (Menuetto) or one (Scherzo) vector. Introducing these libraries into E-MycT/+;enAsCas12aKI/+lymphoma cells followed by treatment with an MCL-1 inhibitor (S63845) or TRP53-inducer (nutlin-3a) identified known and novel drug resistance genes. Finally, we demonstrate simultaneous gene knockouts (Trp53 or combined Bax/Bak) and activation (Cd19) in primary T cells and mouse dermal fibroblasts from crosses of our enAsCas12a and CRISPR activation models (dCas9a-SAM). Our enAsCas12a mouse model and accompanying libraries enhance genome engineering capabilities and complements current CRISPR technologies.

molecular biology↗

bioRxiv2024

Dual nuclease single-cell lineage tracing by Cas9 and Cas12a

Single-cell lineage tracing based on CRISPR/Cas9 gene editing enables the simultaneous linkage of cell states and lineage history at a high resolution. Despite its immense potential in resolving the cell fate determination and genealogy within an organism, existing implementations of this technology suffer from limitations in recording capabilities and considerable barcode dropout. Here, we introduce DuTracer, a versatile tool, which utilizes two orthogonal gene editing systems to record deep cell lineage history at single-cell resolution in an inducible manner. DuTracer shows the ability of enhanced lineage recording with minimized target dropouts and deepened tree depth. Application of DuTracer in mouse embryoid bodies and neuromesodermal organoids illustrates the lineage relationship of different cell types and proposes potential lineage-biased molecular drivers. Moreover, we have developed an entropy-based approach to quantify the lineage recording ability of DuTracer in cell differentiation models. Together, DuTracer facilitates the precise and regulatory interrogation of cellular lineages of complex biological processes.

developmental biology↗

bioRxiv2024

Adaptive loss of shortwave cone opsins during genomic evolution in cartilaginous fish

There is ample evidence of the loss of the shortwave visual protein gene in cartilaginous fish. However, the underlying logic and mechanisms by which organisms need to adapt to the environment behind the opsin loss are still unclear. Here, we report the assembly structure of the whole genome of Okamejei kenojei and Prionace glauca, identify TcMar difference between skates/rays and sharks/chimaeras, and analyze the distribution characteristics and intra group differentiation of opsin-related genes in cartilaginous fish. By establishing a zebrafish model with short wave opsin gene deletion, we find blue or violet light via short-wave sensitive opsins SWS1 or SWS2 can cause the photoreceptor layer thinning (the biomarker for age-related macular degeneration in human eye) through enactive the cell aging. The loss of sws is helpful for alleviating the short-wave light damage on the eye. Since the tapetum lucidum in the eye is found broad existence in various cartilaginous fish, a logical hypothesis was creatively proposed that the existence of tapetum lucidum on the retina of cartilaginous fish is interdependent with the loss of shortwave visual protein gene, providing a new perspective and explanatory path for further understanding of the evolution of visual genes in cartilaginous fish.

bioinformatics↗

bioRxiv2024

Unveiling Tissue Structure and Tumor Microenvironment from Spatially Resolved Transcriptomics by Hypergraph Learning

Spatial omics technologies have revolutionized life sciences by enabling the simultaneous acquisition of biomolecular and spatial information. Identifying spatial patterns is crucial for understanding organ development and tumor microenvironments. However, the emergence of diverse spatial omics resolutions in these technologies has made it challenging to accurately characterize spatial domains at finer resolutions. To address this, we propose HyperSTAR, a hypergraph-based method designed to precisely identify spatial domains across varying resolutions by leveraging higher-order relationships among spatially adjacent tissue programs. Specifically, a gene expression-guided hyperedge decomposition module is introduced to refine the hypergraph structure to accurately delineate spatial domains boundaries. Additionally, a hypergraph attention convolutional neural network is designed to adaptively learn the importance of each hyperedge, enhancing the models ability to capture complex higher-order relationships within spatially neighboring multi-spots and/or single cells. HyperSTAR outperforms existing graph neural network models in tasks such as uncovering tissue substructures, inferring spatiotemporal patterns, and denoising spatially resolved gene expressions. It effectively handles diverse spatial omics data types and scales seamlessly to large datasets. The method successfully reveals spatial heterogeneity in breast cancer sections, with findings validated through functional and survival analyses of independent clinical data. HyperSTAR represents a significant advancement in spatial omics analysis, representing a robust tool for exploring complex spatial patterns across varying resolutions and data types. Its ability to capture intricate higher-order relationships among spatially neighboring spots/cells makes it an invaluable tool for advancing research in life sciences, particularly in cancer and developmental biology.

bioinformatics↗

bioRxiv2024

Spatiotemporal control of subcellular O-GlcNAc signaling using Opto-OGT

The posttranslational modification of intracellular proteins through O-linked {beta}-N-acetylglucosamine (O-GlcNAc) is a conserved regulatory mechanism in multicellular organisms. Catalyzed by O-GlcNAc transferase (OGT), this dynamic modification plays an essential role in signal transduction, gene expression, organelle function, and systemic physiology. Here we present Opto-OGT, an optogenetic probe that allows for precise spatiotemporal control of OGT activity through light stimulation. By fusing a photosensitive cryptochrome protein to OGT, Opto-OGT can be robustly and reversibly activated with high temporal resolution by blue light and exhibits minimal background activity without illumination. Transient activation of Opto-OGT results in mTORC activation and AMPK suppression which recapitulate nutrient-sensing signaling. Furthermore, Opto-OGT can be customized to be localized at specific subcellular sites. By targeting OGT to the plasma membrane, we demonstrate downregulation of site-specific AKT phosphorylation and signaling outputs in response to insulin stimulation. Thus, Opto-OGT is a powerful tool to define the role of O-GlcNAcylation in cell signaling and physiology.

cell biology↗

bioRxiv2024

Mutant TP53 switches therapeutic vulnerability during gastric cancer progression within Interleukin-6 family cytokines

Although aberrant activation of the KRAS and PI3K pathway alongside TP53 mutations account for frequent aberrations in human gastric cancers, neither the sequence nor the individual contributions of these mutations have been clarified. Here, we establish an allelic series of mice to afford conditional expression in glandular epithelium of KrasG12D; Pik3caH1047R or Trp53R172H and/or ablation of Pten or Trp53. We find that KrasG12D;Pik3caH1047Ris sufficient to induce adenomas, and that lesions progress to carcinoma when also harboring Pten-deletions. Additional challenge with either Trp53 loss- or gain-of-function alleles further accelerated tumor progression and triggered metastatic disease. While tumor-intrinsic STAT3 signaling in response to gp130 family cytokines remained as a gatekeeper for all stages of tumor development, metastatic progression required a mutant Trp53-induced interleukin (IL)-11 to IL-6 dependency switch. Consistent with poorer survival of patients with high IL6 expression, we identify IL6/STAT3 signaling as a therapeutic vulnerability for TP53-mutant gastric cancer.

cancer biology↗

bioRxiv2024

Dynamic self-association of archaeal tubulin-like protein CetZ1 drives Haloferax volcanii morphogenesis

Tubulin superfamily (TSF) proteins include the well-known eukaryotic tubulin and bacterial FtsZ families, and lesser-known archaeal CetZ family. In eukaryotes and bacteria, GTP-dependent polymerization and self-association of tubulin and FtsZ protofilaments are integral to the formation of cytoskeletal structures with essential roles in cell division, growth, and morphology. Archaeal CetZs are implicated in the control of cell shape and motility through unknown mechanisms. Here, we reveal a sequence of subcellular localization patterns of CetZ1, the prototypical member of the CetZ family, during stages of Haloferax volcanii rod cell development, in which it plays an essential role. Like tubulin and FtsZ, we found that CetZ1 formed GTP-dependent polymers in vitro, which appear to associate laterally as irregular polymer bundles. Mutations targeting regions predicted to mediate self-association and dynamic turnover of CetZ1, including the longitudinal (GTPase T7 and T4 loops) and lateral assembly interfaces, perturbed or altered rod shape development and subcellular assembly and dynamics, and caused corresponding effects on polymerization in vitro. Remarkably, a conspicuous amphipathic protrusion in the large microtubule (M-) loop, a characteristic of the CetZ1 subfamily, also strongly influenced function and assembly. Our findings reveal the importance of dynamic CetZ1 self-association in cellular morphogenesis involving multiple regions of the TSF fold, including tubulin- and FtsZ-like structural characteristics and CetZ1-specific features. Furthermore, they support a mechanism involving CetZ1 dynamic guidance of cell envelope-associated structures that reshape the cell during morphogenesis.

molecular biology↗

bioRxiv2024

Illuminating the Dark Cancer Phosphoproteome Through a Machine-Learned Co-Regulation Map of 26,280 Phosphosites

Mass spectrometry-based phosphoproteomics offers a comprehensive view of protein phosphorylation, but limited knowledge about the regulation and function of most phosphosites restricts our ability to extract meaningful biological insights from phosphoproteomics data. To address this, we combine machine learning and phosphoproteomic data from 1,195 tumor specimens spanning 11 cancer types to construct CoPheeMap, a network mapping the co-regulation of 26,280 phosphosites. Integrating network features from CoPheeMap into a machine learning model, CoPheeKSA, we achieve superior performance in predicting kinase-substrate associations. CoPheeKSA reveals 24,015 associations between 9,399 phosphosites and 104 serine/threonine kinases, including many unannotated phosphosites and under-studied kinases. We validate the accuracy of these predictions using experimentally determined kinase-substrate specificities. By applying CoPheeMap and CoPheeKSA to phosphosites with high computationally predicted functional significance and cancer-associated phosphosites, we demonstrate the effectiveness of these tools in systematically illuminating phosphosites of interest, revealing dysregulated signaling processes in human cancer, and identifying under-studied kinases as putative therapeutic targets.

cancer biology↗

bioRxiv2024

Coronavirus Nucleocapsid Protein Enhances the binding of p-PKCα to RACK1: Implications for Inhibition of Nucleocytoplasmic Trafficking and Suppression of the Innate Immune Response

The hallmark of coronavirus infection lies in its ability to evade host immune defenses, a process intricately linked to the nuclear entry of transcription factors crucial for initiating the expression of antiviral genes. Central to this evasion strategy is the manipulation of the nucleocytoplasmic trafficking system, which serves as an effective target for the virus to modulate the expression of immune response-related genes. In this investigation, we discovered that infection with the infectious bronchitis virus (IBV) dynamically impedes the nuclear translocation of several transcription factors such as IRF3, STAT1, STAT2, NF-{kappa}B p65, and the p38 mitogen-activated protein kinase (MAPK), leading to compromised transcriptional induction of key antiviral genes such as IFN{beta}, IFITM3, and IL-8. Further examination revealed that during the infection process, components of the nuclear pore complex (NPC), particularly FG-Nups (such as NUP62, NUP153, NUP42, and TPR), undergo cytosolic dispersion from the nuclear envelope; NUP62 undergoes phosphorylation, and NUP42 exhibits a mobility shift in size. These observations suggest a disruption in nucleocytoplasmic trafficking. Screening efforts identified the IBV nucleocapsid protein (N) as the agent responsible for the cytoplasmic distribution of FG-Nups, subsequently hindering the nuclear entry of transcription factors and suppressing the expression of antiviral genes. Interactome analysis further revealed that the IBV N protein interacts with the scaffold protein RACK1, facilitating the recruitment of activated protein kinase C alpha (p-PKC) to RACK1 and relocating the RACK1-PKC complex to the cytoplasm. These observations are conserved across pan-coronaviruses N proteins. Concurrently, the presence of both RACK1 and PKC/{beta} proved essential for the phosphorylation and cytoplasmic dispersion of NUP62, the suppression of antiviral cytokine expression, and efficient virus replication. These findings unveil a novel, highly effective, and evolutionarily conserved mechanism. Author summaryCoronaviruses employ diverse strategies to suppress the host innate immune defense. In this study, we uncovered a novel and highly effective strategy utilized by pan-coronaviruses to inhibit the innate immune response. Specifically, we found that the coronavirus N protein facilitates the binding of p-PKC to RACK1, leading to the phosphorylation of NUP62 and the cytoplasmic redistribution of multiple FG-Nups. This phenomenon is accompanied by the disruption of nuclear translocation of several innate immune response-related transcription factors and suppression of antiviral/pro-inflammatory genes expression. Our research represents the first elucidation of how the N protein targets and impairs NPC function through the promotion of RACK1-PKC interaction and NUP62 phosphorylation/disassembly. This discovery unveils a novel mechanism employed by pan-coronaviruses to counteract the host immune response.

microbiology↗

bioRxiv2024

PHOSPHATE-INDUCED 1 and its paralogs positively regulate cellulose biosynthesis in Arabidopsis.

Cellulose is not only the most abundant biopolymer on Earth but also an essential multi-purpose resource, primarily synthesized by cellulose synthase complexes (CSCs) located on plasma membrane in vascular plants. Although insights into the functions of CSCs have been provided by the identification of cellulose synthase catalytic subunits, our understanding of the regulation of CSC activity remains limited. In this study, combining molecular and genetic methods, we demonstrated that both PHI-1 and its paralogs residing in the plasma membrane are involved in the cellulose biosynthesis regulation as a positive regulatory component of CSCs by feeding developmental and environmental cues into their activities in Arabidopsis (Arabidopsis thaliana). Our finding reveals a mechanism for how plants regulate cellulose synthesis in response to developmental and environmental signals, and thus improves the likelihood that cellulose biosynthesis could be feasibly adapted for sustainable purposes.

plant biology↗

bioRxiv2024

Germinal Center Cytokines Driven Epigenetic Control of Epstein-Barr Virus Latency Gene Expression

Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues. Author SummaryA longstanding question has remained how Epstein-Barr virus (EBV) epigenetically switches between latency programs as it navigates the B-cell compartment. EBV uses its latency III program to stimulate newly infected B cell growth and then trafficking into secondary lymphoid tissue germinal centers (GC). In latency III, the viral C promoter stimulates expression of six Epstein-Barr nuclear antigens (EBNA) that in turn induce two latent membrane proteins (LMP). However, knowledge has remained incomplete about how GC microenvironmental cues trigger switching to latency II, where only one EBNA and two LMP are expressed, a program observed in Hodgkin lymphoma. Building on prior evidence that GC cytokines are a major cue, we systematically tested effects of cytokines secreted by GC-resident T follicular helper and follicular dendritic cells on EBV latency gene expression and on epigenetic remodeling of their promoters. This highlighted that a range of GC cytokines repress latency III EBNA, while only several support LMP1 expression, major events in the transition between the latency III and II programs. We identified key downstream roles of JAK/STAT signaling in relaying cytokine signals to the EBV epigenome, including obligatory STAT3 and 5 roles in rewiring of C and LMP promoter histone epigenetic marks.

microbiology↗

bioRxiv2023

Inactivation of p53 drives breast cancer brain metastasis by altering fatty acid metabolism

Brain metastasis (BM) is a dire prognosis across cancer types. It is largely unknown why some tumors metastasize to the brain whereas others do not. We analyzed genomic and transcriptional data from clinical samples of breast cancer BM (BCBM) and found that nearly all of them carried p53-inactivating genetic alterations through mutations, copy-number loss, or both. Importantly, p53 pathway activity was already perturbed in primary tumors giving rise to BCBM, often by loss of the entire 17p chromosome-arm. This association was recapitulated across other carcinomas. Experimentally, p53 knockout was sufficient to drastically increase BCBM formation and growth in vivo, providing a causal link between p53 inactivation and brain tropism. Mechanistically, p53-deficient BC cells exhibited altered lipid metabolism, particularly increased fatty acid (FA) synthesis and uptake, which are characteristic of brain-metastasizing cancer cells. FA metabolism was further enhanced by astrocytes in a p53-dependent manner, as astrocyte-conditioned medium increased FASN, SCD1, and CD36 expression and activity, and enhanced the survival, proliferation and migration of p53-deficient cancer cells. Consequently, these cells were more sensitive than p53-competent cells to FA synthesis inhibitors, in isogenic cell cultures, in BCBM-derived spheroids, and across dozens of BC cell lines. Lastly, a significant association was observed between p53 inactivation, astrocyte infiltration, and SCD1 expression in clinical human BCBM samples. In summary, our study identifies p53 inactivation as a driver of BCBM and potentially of BM in general; suggests a p53-dependent effect of astrocytes on BC cell behavior; and reveals FA metabolism as an underlying, therapeutically-targetable molecular mechanism.

cancer biology↗

bioRxiv2023

High-resolution ultrastructure and morphology of marine ammonia-oxidizing archaea Nitrosopumilus maritimus SCM1 revealed by Cryo-ET and STEM-EDS

Nitrososphaerota, formerly known as Thaumarchaeota, constitute a diverse and widespread group of ammonia-oxidizing archaea (AOA) inhabiting ubiquitously in marine and terrestrial environments, playing a pivotal role in global nitrogen cycling. Despite their significance in Earths ecosystems, the cellular organization of AOA remains largely unexplored, leading to a significant question unanswered of how the machinery of these organisms underpins metabolic functions. In this study, we combined chromatic-aberration-corrected cryo-electron tomography (cryo-ET), scanning transmission electron microscopy (STEM), and energy dispersive X-ray spectroscopy (EDS) to unveil the cellular organization and elemental composition of Nitrosopumilus maritimus SCM1, a representative member of marine Nitrososphaerota. Our tomograms show the native ultrastructural morphology of SCM1 and one to several dense storage granules in the cytoplasm. STEM-EDS analysis identifies two types of storage granules in which one type is possibly composed of polyphosphate, while the other type is polyhydroxyalkanoate. Collectively, these findings provide visual evidence for the resilience of AOA in the vast oligotrophic marine environment.

microbiology↗

bioRxiv2023

Bio-inspired mineralization collagen induce fibrocartilage regeneration after tendon-bone injury by activating Gli1+Dkk3+ progenitor cells

A fibrocartilaginous connection between the tendon and bone, plays a critical role in transferring force from muscle to bone to enable joint movement. However, due to the high mechanical stress it experiences, the enthesis is vulnerable to injury and incapable of regenerating. The spatial relationship and functional basis of the principal components of the fibrocartilage - mineral and collagen - have not been clearly elucidated, which is a significant remaining gap in reconstructing complex architectures for promoting interface tissue regeneration. Here, using three-dimensional electron tomography imaging and high-resolution two-dimensional electron microscopy, we discover that mineral particles form a continuous cross-fibrillar phase within the fibrocartilage region. By developing a "floating mineralization" system, we fabricate a three-layer hydrogel that mimics the hierarchical nano- to micro-scale structure of tendon-bone interface (TBI). The middle layer is noteworthy for its resemblance to the nanostructure of fibrocartilage and its superior ability to induce mineralized fibrochondrogenesis in vitro. Based on motor function analysis, imaging diagnosis, histological staining, immunofluorescence staining, and biomechanics performance, we demonstrate that in situ transplantation of the gradient hydrogel achieved tendon-fibrocartilage-bone synchronous regeneration and result in 68% maximum mechanical recovery at 8-week postoperation. Single-cell RNA sequencing analysis reveals that a unique atlas of in situ stem/progenitor cells is generated during the TBI healing in vivo. Notably, the bio-inspired hydrogel microenvironment drived endogenous Gli1+Dkk3+ progenitor cells, playing a key role in TBI regeneration. Therefore, we have successfully decoded and reconstructed the nanostructure of fibrocartilage, which has great potential in TBI regeneration.

bioengineering↗

bioRxiv2023

Multi-omics analysis reveals signatures of selection and loci associated with complex traits in pigs

Selection signatures that contribute to phenotypic diversity, especially morphogenesis in pigs, remain to be further elucidated. To reveal the regulatory role of genetic variations in phenotypic differences between Eastern and Western pig breeds, we performed a systematic analysis based on seven high-quality de novo assembled genomes, 1,081 resequencing data representing 78 domestic breeds, 162 methylomes, and 162 transcriptomes of skeletal muscle from Tongcheng (Eastern) and Landrace (Western) pigs at 27 developmental stages. Selective sweep uncovers different genetic architectures behind divergent selection directions for the Eastern and Western breeds. Notably, two loci showed functional alterations by almost fixed missense mutations. By integrating time-course transcriptome and methylome, we revealed differences in developmental timing during myogenesis between Eastern and Western breeds. Genetic variants under artificial selection have critical regulatory effects on progression patterns of heterochronic genes like GHSR and BDH1, by the interaction of local DNA methylation status, particularly during embryonic development. Altogether, our work not only provides valuable resources for understanding pig complex traits, but also contributes to human biomedical research.

genomics↗

bioRxiv2023

Genomic insights into post-domestication expansion and selection of body size in ponies

Horses domestication revolutionized human civilization by changing transportation, farming, and warfare patterns. Despite extensive studies on modern domestic horse origins, the intricate demographic history and genetic signatures of pony size demand further exploration. Here, we present a high-quality genome of the Chinese Debao pony and extensively analyzed 385 individuals from 49 horse breeds. We reveal the conservation of ancient components in East Asian horses and close relationships between Asian horses and specific European pony lineages. Genetic analysis uncovers Asian paternal origin for European pony breeds, and these pony-sized horses share a close genetic affinity due to the presence of a potential ancestral ghost pony population. Additionally, we identify promising cis-regulatory elements influencing horse withers height by regulating genes like RFLNA and FOXO1. Overall, our study provides insightful perspectives into the development history and genetic determinants underlying body size in ponies and offers broader implications for horse population management and improvement. TeaserDecoding pony genetics: exploring origins and size determinants sheds light on their historical and biological impacts.

genomics↗

bioRxiv2023

Dividing out quantification uncertainty allows efficient assessment of differential transcript expression

Differential expression analysis of RNA-seq is one of the most commonly performed bioinformatics analyses. Transcript-level quantifications are inherently more uncertain than gene-level read counts because of ambiguous assignment of sequence reads to transcripts. While sequence reads can usually be assigned unambiguously to a gene, reads are very often compatible with multiple transcripts for that gene, particularly for genes with many isoforms. Software tools designed for gene-level differential expression do not perform optimally on transcript counts because the read-to-transcript ambiguity (RTA) disrupts the mean-variance relationship normally observed for gene level RNA-seq data and interferes with the efficiency of the empirical Bayes dispersion estimation procedures. The pseudoaligners kallisto and Salmon provide bootstrap samples from which quantification uncertainty can be assessed. We show that the overdispersion arising from RTA can be elegantly estimated by fitting a quasi-Poisson model to the bootstrap counts for each transcript. The technical overdispersion arising from RTA can then be divided out of the transcript counts, leading to scaled counts that can be input for analysis by established gene-level software tools with full statistical efficiency. Comprehensive simulations and test data show that an edgeR analysis of the scaled counts is more powerful and efficient than previous differential transcript expression pipelines while providing correct control of the false discovery rate. Simulations explore a wide range of scenarios including the effects of paired vs single-end reads, different read lengths and different numbers of replicates.

bioinformatics↗

bioRxiv2023

Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

The endoribonuclease (EndoU) nsp15 of coronaviruses plays an important role in evasion of host innate immune responses by reducing the abundance of viral double-stranded RNA, whereas less is known about potential host cellular targets of nsp15. In this study, we show that cellular protein synthesis is inhibited upon over-expression of nsp15 from four genera of coronaviruses and this is accompanied by nuclear retention of the poly(A) binding protein cytoplasmic 1 (PABPC1). We also show that the EndoU activity of nsp15 is indispensable for both, inhibition of protein synthesis and PABPC1 nuclear relocation. FISH analysis using oligo-dT probes, revealed an overlap between the localization of cellular mRNA and that of overexpressed nsp15 in some cells, suggesting that, when expressed alone, nsp15 may target host mRNA. When investigating the association of nsp15 on protein shut off in the context of a viral infection, we observed that the{gamma} -coronavirus infectious bronchitis virus (IBV), induced host translation shutoff in an p-eIF2-independent manner and mainly retained PABPC1 in the cytoplasm, whereas the nsp15 EndoU-deficient IBV accumulated viral dsRNA and caused p-PKR-p-eIF2-dependent host protein translation shutoff, accompanied with PABPC1 nuclear relocation or stress granule (SG) localization. This phenomenon suggests that during infection with wild type IBV, although the cellular translation is inhibited, initiation of viral mRNA translation leads to PABPC1 binding to viral mRNA, thereby preventing its nuclear entry; during infection with nsp15 EndoU-deficient IBV however, the eIF2-dependent host protein translation shutoff prevents both host and viral mRNA translation initiation, releasing PABPC1 from binding to cytosolic and viral mRNA, thereby relocating it to the nucleus or to SG. Altogether, this study reveals unique yet conserved mechanisms of host protein shutoff that add to our understanding of how coronaviruses regulate host protein expression through a mechanism that involves catalytically active nsp15 EndoU, and describes how nsp15 may target both, viral and host mRNA. Author summaryIt has been reported that coronavirus infection suppresses host protein translation, - and {beta}- coronavirus nsp1 is responsible for inhibition of host gene expression. However, for{gamma} - and{delta} -coronavirus, there is no nsp1 and the underlying mechanisms by which virus regulates host translation are not well characterized. Here, we show that coronavirus endoribonuclease nsp15 is responsible for the inhibition of host translation by targeting to host factors, meanwhile it helps virus bypass the PKR-eIF2 mediated host translation shutoff, which is harmful for virus gene expression, by reducing the accumulation of viral dsRNA. This novel finding gives insight how does nsp15 targets to both host factors and viral RNA, to facilitate virus replication. Moreover, the novel function of nsp15 is found to be conserved among coronaviruses, revealing the essential role of this endoribonuclease in hijacking host translation machinery for virus replication.

microbiology↗