Biology subjects

Zhao, W.

Publications and source records attributed to Zhao, W..

At least 343 records · Page 19Linked to original sources

bioRxiv2019

CellGPS: Whole-body tracking of single cells by positron emission tomography

In vivo molecular imaging tools are critically important for determining the role played by cell trafficking in biological processes and cellular therapies. However, existing tools measure average cell behavior and not the kinetics and migration routes of individual cells inside the body. Furthermore, efflux and non-specific accumulation of contrast agents are confounding factors, leading to inaccurate estimation of cell distribution in vivo. In view of these challenges, we report the development of a \"cellular GPS\" capable of tracking single cells inside living subjects with exquisite sensitivity. We use mesoporous silica nanoparticles (MSN) to concentrate 68Ga radioisotope into live cells and inject these cells into live mice. From the pattern of annihilation photons detected by positron emission tomography (PET), we infer, in real time, the position of individual cells with respect to anatomical landmarks derived from X-ray computed tomography (CT). To demonstrate this technique, a single human breast cancer cell was tracked in a mouse model of experimental metastasis. The cell arrested in the lungs 2-3 seconds after tail-vein injection. Its average velocity was estimated at around 50 mm/s, consistent with blood flow rate. Other cells were tracked after injection through other routes, but no motion was detected within 10 min of acquisition. Single-cell tracking could be applied to determine the kinetics of cell trafficking and arrest during the earliest phase of the metastatic cascade, the trafficking of immune cells during cancer immunotherapy, or the distribution of cells after transplantation in regenerative medicine.

bioengineering↗

bioRxiv2019

N-Acetyl Cysteine Alleviates Coxsackievirus B-Induced Myocarditis by Suppressing caspase-1

Viral myocarditis caused by Coxsackievirus B (CVB) infection is a severe inflammatory disease of the myocardium, which may develop to cardiomyopathy and heart failure. No effective medicine is available to treat CVB infection. Here we evaluated the anti-CVB effect of N-acetyl cysteine (NAC), a widely used antioxidant. NAC significantly alleviated myocarditis and improved the overall condition of CVB type 3 (CVB3)-infected mice. Importantly, NAC treatment suppressed viral replication in both myocardium and cell culture. We show that NAC inhibited CVB3 replication when it was applied at the early stage of CVB3 infection. NACs antiviral mechanism, while independent of its antioxidant property, relies on its inhibition on caspase-1 activation, since the knockdown of caspase-1 blocked CVB3 replication. Moreover, NAC promotes procaspase-1 degradation via ubiquitin proteasome system, which may further contribute to the inhibited activity of caspase-1. NAC also inhibits the activity of viral proteases. Taken together, this study shows that NAC exerts potent anti-CVB effect by inhibiting caspase-1 and viral proteases. This study suggests that NAC can be a safe therapeutic option for CVB-induced myocarditis.

microbiology↗

bioRxiv2019

Importance of Matrix-dimensionality in Regulating the Bone Marrow Hematopoietic Cells Pool

In bone marrow, hematopoietic stem cells (HSCs) and multiple hematopoietic progenitor cells (HPCs) cooperate to differentiate and replenish blood and immune cells. It has long been recognized bone marrow niche parameters interact with hematopoietic stem and progenitor cells (HSPCs) and additional work is required to study niche physical signals controlling cell behavior. Here we presented that important biophysical signals, stiffness and dimensionality, regulating expansion of bone marrow HSPCs. Mice bone marrow derived progenitor cells were cultured in collagen I hydrogel in vitro. We found stiffer 3D matrix promoted the expansion of lineage negative (Lin-) progenitor cells and Lin-Sca-1+c-kit+ (LSK) HSPCs compared to softer hydrogel. Compared with cells cultured in 2D environment, 3D embedded construct had significant advantage on HSPCs expansion, accompanied by increases on myeloid and lymphoid lineage fractions. Bright changes on gene expression were subsequently discovered. According to these data, we concluded that culture matrix dimensionality is an important factor to regulate the behavior of subpopulations in hematopoietic cell pool, which should be considered in attempts to illuminate HSCs fate decision in vitro.\n\nStatement of SignificanceWe would like to submit the enclosed manuscript entitled \"Importance of Niche-dimensionality in Regulating the Bone Marrow Hematopoietic Cells Pool\", which we wish to be considered for publication in Biophysical Journal. Studies about the interaction between HSCs and factors provided by their microenvironment is largely focus on pure perspective of biology. But biophysical factors affecting HSC fate and behavior needs to be further explore. Herein we found ex vivo culture dimensionality affected HSPC expansion. Cell surface marker detection and mRNA expression analysis predicted the changes on myeloid and lymphoid lineage fractions. We hope niche physical signals which we identified will be considered to design HSC biomimetic niches in clinical applications. And we believe that our study will make it interesting to general readers. We deeply appreciate your consideration of our manuscript, and we look forward to receiving comments from the reviewers.

biophysics↗

bioRxiv2019

Single-cell based elucidation of molecularly-distinct glioblastoma states and drug sensitivity

Glioblastoma heterogeneity and plasticity remain controversial, with proposed subtypes representing the average of highly heterogeneous admixtures of independent transcriptional states. Single-cell, protein-activity-based analysis allowed full quantification of >6,000 regulatory and signaling proteins, thus providing a previously unattainable single-cell characterization level. This helped identify four novel, molecularly distinct subtypes that successfully harmonize across multiple GBM datasets, including previously published bulk and single-cell profiles and single cell profiles from seven orthotopic PDX models, representative of prior subtype diversity. GBM is thus characterized by the plastic coexistence of single cells in two mutually-exclusive developmental lineages, with additional stratification provided by their proliferative potential. Consistently, all previous subtypes could be recapitulated by single-cell mixtures drawn from newly identified states. Critically, drug sensitivity was predicted and validated as highly state-dependent, both in single-cell assays from patient-derived explants and in PDX models, suggesting that successful treatment requires combinations of multiple drugs targeting these distinct tumor states.\n\nSignificanceWe propose a new, 4-subtype GBM classification, which harmonizes across bulk and single-cell datasets. Single-cell mixtures from these subtypes effectively recapitulate all prior classifications, suggesting that the latter are a byproduct of GBM heterogeneity. Finally, we predict single-cell level activity of three clinically-relevant drugs, and validate them in patient-derived explant.

systems biology↗

bioRxiv2019

Tomato yellow leaf curl virus V2 protein plays a critical role in the nuclear export of V1 protein and viral systemic infection

Geminiviruses are an important group of circular, single-stranded DNA viruses that cause devastating diseases in crops. Geminiviruses replicate their genomic DNA in the nucleus. The newly-synthesized viral DNA is subsequently transported to the cytoplasm, moved to adjacent cells through plasmodesmata with the help of viral movement proteins, and, ultimately, moved long-distance to establish systemic infection. Thus, the nucleocytoplasmic transportation is crucial for a successful infection by geminiviruses. For Tomato yellow leaf curl virus (TYLCV), the V1 protein is known to bind and shuttle viral genomic DNA, but the role of V2 protein in this process is still unclear. Here, we report that the nucleus-localized V1 protein dramatically decreases when co-expressed with V2 protein, and that V2-facilitated nuclear export of V1 protein depends on host exportin- and a specific V1-V2 interaction. Chemical inhibition of exportin- or a substitutions at cysteine 85 of V2 protein, which abolishes the V1-V2 interaction, blocks the promoted redistribution of V1 protein to the perinuclear region and the cytoplasm. When the V2C85S mutation is incorporated into a TYLCV infectious clone, the TYLCV-C85S causes delayed onset of very mild symptoms compared to wild-type TYLCV, indicating that the V1-V2 interaction and, thus, V2-mediated nuclear export of V1 protein is crucial for viral spread and systemic infection. Our data point to a critical role of the V2 protein in promoting the nuclear export of the V1 protein, likely by promoting V1-mediated nucleocytoplasmic transportation of TYLCV genomic DNA, and in turn, promoting viral systemic infection.\n\nAuthor summaryAs both replication and the transcription of geminiviruses occur in the nucleus, transportation of the viral genomic DNA into and out of the nucleus of the infected cells is essential for a successful infection cycle. However, the nuclear export of geminiviruses is still little known and even less is known about the process for monopartite geminiviruses. We use TYLCV, a typical monopartite begomovirus in the family Geminiviridae, to examine the nucleocytoplasmic transportation. In this study, we found TYLCV V2 is able to redistribute the nucleus-localized V1 protein to the perinuclear region. Moreover, the nuclear export of V1 protein is dependent on the V1-V2 interaction and host exportin-. Blocking the V1-V2 interaction impeded the V2-mediated V1 protein redistribution and decrease TYLCV infection efficiency with delayed and mild symptoms. This report shows us a new explanation for the role of V2 in the nuclear export of V1 protein and TYLCV viral systemic infection.

pathology↗

bioRxiv2019

Serum-free culture system for spontaneous human mesenchymal stem cell spheroids formation

Human mesenchymal stem cells (hMSCs) are widely used in clinical research because of their multipotential, immunomodulatory, and reparative properties. Previous studies determined that hMSC spheroids from three-dimensional (3D) culture possess higher therapeutic efficacy than conventional hMSCs from monolayer (2D) culture. To date, various 3D culture methods have been developed to form hMSC spheroids, but most of them used culture medium containing fetal bovine serum (FBS), which is not suitable for further clinical use. Here, we demonstrate that dissociated single MSCs seeded in induced pluripotent stems medium (MiPS), adhere loosely to the dish and spontaneously migrate to form spheroids during day 3 to day 6. Through component deletion screening and complementation experiments, the knockout serum replacement (KSR) was identified as necessary and sufficient for hMSC spheroid formation. Transcriptome analysis showed that the overall expression profiles were highly similar between 2D culture with FBS and KSR derived spheroids. Interestingly, genes related to inflammatory response, immune response, and angiogenesis were up-regulated in spheroids at day 6, and qPCR results further validated the increased expression level of related genes, including STC1, CCL7, HGF, IL24, and TGFB3. When spheroids were re-plated in normal FBS medium, cells formed a typical spindle-shaped morphology, and FACS results showed that the recovered cells retained MSC-specific surface markers, such as CD73, CD90, and CD105. In summary, we developed a practical and convenient method to generate hMSC spheroids for clinical research and therapy.

cell biology↗

bioRxiv2019

Metagenomic Next-generation Sequencing of Cerebrospinal Fluid for the Diagnosis of Central Nervous System Infections: A Multicentre Prospective Study

BackgroundInfectious encephalitis and meningitis are often treated empirically without identification of the causative pathogen. Metagenomic next-generation sequencing (mNGS) is a high throughput technology that enables the detection of pathogens independent of prior clinical or laboratory information.\n\nMethodsThe present study was a multicentre prospective evaluation of mNGS of cerebrospinal fluid (CSF) for the diagnosis of suspected central nervous system infections.\n\nResultsA total of 276 patients were enrolled in this study between Jan 1, 2017 and Jan 1, 2018. Identification of an etiologic pathogen in CSF by mNGS was achieved in 101 patients (36.6%). mNGS detected 11 bacterial species, 7 viral species, 2 fungal species, and 2 parasitic species. The five leading positive detections were varicella-zoster virus (17), Mycobacterium tuberculosis (14), herpes simplex virus 1 (12), Epstein-Barr virus (12), and Cryptococcus neoformans (7). False positives occurred in 12 (4.3%) patients with bacterial infections known to be widespread in hospital environments. False negatives occurred in 16 (5.8%) patients and included bacterial, viral and fungal aetiologies.\n\nConclusionsmNGS of CSF is a powerful diagnostic method to identify the pathogen for many central nervous system infections.

genomics↗

bioRxiv2019

Qualitative analyses of polishing and pre-coating FIB milled crystals for MicroED

Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction limits the thickness of crystals that can be investigated by MicroED, mainly due to absorption. Recent studies have demonstrated that focused ion beam (FIB) can thin even very large crystals into ideal sized lamellae however it is not clear how to best apply FIB-milling for MicroED. Here, The effects of polishing the lamellae, whereby the last few nanometers are milled away using a low-current gallium beam, are explored in both platinum pre-coated and uncoated samples. Our results suggest that pre-coating samples with a thin layer of platinum followed by polishing the crystal surfaces prior to data collection consistently led to superior results as indicated by higher signal/noise ratio, higher resolution and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED.

biochemistry↗

bioRxiv2019

Systematically understanding the key roles of microglia in AD development

Alzheimers disease (AD) is the leading cause of age-related dementia, affecting over 5 million people in the United States. Unfortunately, current therapies are largely palliative and several potential drug candidates have failed in late-stage clinical trials. Studies suggest that microglia-mediated neuroinflammation might be responsible for the failures of various therapies. Microglia contribute to A{beta} clearance in the early stage of neurodegeneration and may contribute to AD development at the late stage by releasing pro-inflammatory cytokines. However, the activation profile and phenotypic changes of microglia during the development of AD are poorly understood. To systematically understand the key role of microglia in AD progression and predict the optimal therapeutic strategy in silico, we developed a 3D multi-scale model of AD (MSMAD) by integrating multi-level experimental data, to manipulate the neurodegeneration in a simulated system. Based on our analysis, we revealed how TREM2-related signal transduction leads to an imbalance in the activation of different microglia phenotypes, thereby promoting AD development. Our MSMAD model also provides an optimal treatment strategy for improving the outcome of AD treatment.

systems biology↗

bioRxiv2019

Role of Rare and Low Frequency Variants in Gene-Alcohol Interactions on Plasma Lipid Levels

BackgroundAlcohol intake influences plasma lipid levels and such effects may be modulated by genetic variants. ObjectiveWe aimed to characterize the role of aggregated rare and low-frequency variants in gene by alcohol consumption interactions associated with fasting plasma lipid levels. DesignIn the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, fasting plasma triglycerides (TG), and high- and low-density lipoprotein cholesterol (HDL-c and LDL-c) were measured in 34,153 European Americans from five discovery studies and 32,275 individuals from six replication studies. Rare and low-frequency protein coding variants (minor allele frequency [≤] 5%) measured by an exome array were aggregated by genes and evaluated by a gene-environment interaction (GxE) test and a joint test of genetic main and GxE interaction effects. Two dichotomous self-reported alcohol consumption variables, current drinker, defined as any recurrent drinking behavior, and regular drinker, defined as the subset of current drinkers who consume at least two drinks per week, were considered. ResultsWe discovered and replicated 21 gene-lipid associations at 13 known lipid loci through the joint test. Eight loci (PCSK9, LPA, LPL, LIPG, ANGPTL4, APOB, APOC3 and CD300LG) remained significant after conditioning on the common index single nucleotide polymorphism (SNP) identified by previous genome-wide association studies, suggesting an independent role for rare and low-frequency variants at these loci. One significant gene-alcohol interaction on TG was discovered at a Bonferroni corrected significance level (p-value <5x10-5) and replicated (p-value <0.013 for the interaction test) in SMC5. ConclusionsIn conclusion, this study applied new gene-based statistical approaches to uncover the role of rare and low-frequency variants in gene-alcohol consumption interactions on lipid levels.

epidemiology↗

bioRxiv2019

Comparison of three different eye-tracking tasks for distinguishing autistic from typically developing children and autistic symptom severity

Altered patterns of visual social attention preference detected using eye-tracking and a variety of different paradigms are increasingly proposed as sensitive biomarkers for autism spectrum disorder. However, few eye tracking studies have compared the relative efficacy of different paradigms to discriminate between autistic compared with typically developing children and their sensitivity to specific symptoms. To target this issue, the current study used three common eye tracking protocols contrasting social versus non-social stimuli in young (2-7 years old) Chinese autistic (n = 35) and typically developing (n = 34) children matched for age and gender. Protocols included dancing people vs. dynamic geometrical images, biological motion (dynamic light point walking human or cat) vs. non-biological motion (scrambled controls) and child playing with toy vs. toy alone. Although all three paradigms differentiated autistic and typically developing children, the dancing people versus dynamic geometry pattern paradigm was the most effective, with autistic children showing marked reductions in visual preference for dancing people and correspondingly increased one for geometric patterns. Furthermore, this altered visual preference in autistic children was correlated with the ADOS social affect score and had the highest discrimination accuracy. Our results therefore indicate that decreased visual preference for dynamic social stimuli may be the most effective visual attention-based paradigm for use as a biomarker for autism in Chinese children. Clinical trial ID: NCT03286621 (clinicaltrials.gov); Clinical trial name: Development of Eye-tracking Based Markers for Autism in Young Children. Lay summaryEye-tracking measures may be useful in aiding diagnosis and treatment of autism, although it is unclear which specific tasks are optimal. Here we compare the ability of three different social eye-gaze tasks to discriminate between autistic and typically developing young Chinese children and their sensitivity to specific autistic symptoms. Our results show that a dynamic task comparing visual preference for social (individuals dancing) versus geometric patterns is the most effective both for diagnosing autism and sensitivity to its social affect symptoms.

clinical trials↗

bioRxiv2019

Oxytocin modulation of self-other distinction is replicable and influenced by oxytocin receptor (OXTR) genotype

Intranasal oxytocin (OXT) has been associated with effects on diverse social-emotional domains in humans, however progress in the field is currently hampered by poor replicability. Limited statistical power and individual differences in biological factors, such as oxytocin receptor (OXTR) genetics, may have contributed to these variable findings. To this end, we present a pharmaco-genetic study aiming at (1) replicating previous findings suggesting that intranasal oxytocin (24 IU) blurs self-other distinction in a large sample of n = 170 male subjects, (2) determining whether variations in common receptor polymorphisms (rs237887, rs2268491, rs2254298, rs53576, rs2268498) influence sensitivity to oxytocins behavioral effects. Employing a validated oxytocin-sensitive trait judgment paradigm, we confirmed that it blurred self-other distinction in terms of decision time and subsequent memory. However, oxytocin only influenced decision time in rs53576 G carriers, whereas effects on memory performance were most pronounced in rs2268498 TT homozygotes. In summary, the current study replicates our previous findings showing that oxytocin blurs self-other distinction and suggests that sensitivity to its effects in this domain are receptor genotype dependent.

neuroscience↗

bioRxiv2019

Human extinction learning is accelerated by an angiotensin antagonist via ventromedial prefrontal cortex and its connections with basolateral amygdala

Recent translational research suggests a role of the renin-angiotensin (RA) system in threat extinction and underlying neuroplasticity; however, whether and how pharmacological modulation of the RA system influences physiological and neural manifestations of threat during extinction learning in humans is unclear. Here we report that pre-extinction administration of losartan, an angiotensin II type 1 receptor antagonist, accelerated attenuation of physiological threat expression. During early extinction, losartan enhanced threat-signal specific ventromedial prefrontal cortex (vmPFC) activation and its coupling with the basolateral amygdala. Multivoxel pattern analysis revealed that losartan reduced whole brain, particularly vmPFC, threat expression and voxel-wise mediation analyses further confirmed that losartan-accelerated extinction crucially involved vmPFC processing. Overall the results provide initial evidence for a critical role of the RA system in extinction learning in humans and suggest that adjunct losartan administration may facilitate the efficacy of extinction-based therapies. ClinicalTrials.gov, Identifier: NCT03396523

clinical trials↗

bioRxiv2019

Functional Near-Infrared Spectroscopy (fNIRS) informed neurofeedback: regional-specific modulation of lateral orbitofrontal activation and cognitive flexibility

Cognitive flexibility and reward processing critically rely on the orbitofrontal cortex. Dysregulations in these domains and orbitofrontal activation have been reported in major psychiatric disorders. Haemodynamic brain imaging informed neurofeedback allows regional-specific control over brain activation and thus may represent an innovative intervention to regulate orbitofrontal dysfunctions. Against this background the present proof-of-concept study evaluated the feasibility and behavioral relevance of functional Near-Infrared Spectroscopy (fNIRS) assisted neurofeedback training of the lateral orbitofrontal cortex (lOFC). In a randomized sham-controlled between-subject design 60 healthy participants underwent four subsequent runs of training to enhance lOFC activation. Training-induced changes in the lOFC, attentional set shifting performance and reward experience served as primary outcomes. Feedback from the target channel significantly increased regional-specific lOFC activation over the four training runs in comparison with sham feedback. The experimental group demonstrated a trend for faster responses during set shifting relative to the sham group. Within the experimental group stronger training-induced lOFC increases were associated with higher reward experience. The present results demonstrate that fNIRS-informed neurofeedback allows regional-specific regulation of lOFC activation and may have the potential to modulate associated behavioral domains. As such fNIRS-informed neurofeedback may represent a promising strategy to regulate OFC dysfunctions in psychiatric disorders.

neuroscience↗

bioRxiv2018

A circuit of protein-protein regulatory interactions enables polarity establishment in a bacterium

Asymmetric cell division generates specialized daughter cells that play a variety of roles including tissue morphogenesis in eukaryotes and pathogenesis in bacteria. In the gram-negative bacterium Caulobacter crescentus, asymmetric localization of two biochemically distinct signaling hubs at opposite cell poles provides the foundation for asymmetric cell division. Through a set of genetic, synthetic biology and biochemical approaches we have characterized the regulatory interactions between three scaffolding proteins. These studies have revealed that the scaffold protein PodJ functions as a central mediator for organizing the new cell signaling hub, including promoting bipolarization of the central developmental scaffold protein PopZ. In addition, we identified that the old pole scaffold SpmX serves as a negative regulator of PodJ subcellular accumulation. These two scaffold-scaffold regulatory interactions serve as the core of an integrated cell polarization circuit that is layered on top of the cell-cycle circuitry to coordinate cell differentiation and asymmetric cell division.

microbiology↗

bioRxiv2018

Membrane curvature sensing of the lipid-anchored K-Ras small GTPases

Cell morphologies, defined by plasma membrane (PM) local curvature, change during mitogen-dependent function and pathology, such as growth, division and proliferation. The lipid-anchored Ras small GTPases are essential upstream regulators of the mitogen-activated protein kinases (MAPKs) cascades and play key roles in many pathological conditions, especially cancer. Ras signaling is mostly compartmentalized to the cell PM through the formation of nanometer-sized domains, termed as nanoclusters, and undergo selective lipid sorting for efficient effector recruitment and activation. Thus, Ras function might be sensitive to changing PM curvature, potentially regulating mechanosensing of mitogen signaling. We employed nanofabrication and super-resolution imaging and found that Ras functions respond to PM curvature modulations in an isoform specific manner: nanoclustering and signaling of the most oncogenically prevalent isoform K-Ras favor less curved PM, while those of another isoform H-Ras favor more curved PM. We then examined whether Ras membrane curvature sensing is mediated by lipid sorting. We found that anionic phospholipids sense changing PM curvature in distinct manners: phosphatidylserine (PS) localization shows preference for less curved membrane but phosphoinositol 4,5-bisphosphate (PIP2) localization favors more curved PM. Depletion of endogenous PS abolishes K-Ras PM curvature sensing. Exogenous PS addback and synthetic bilayer binding assays further show that only mixed-chain PS species, but not other PS species, mediate K-Ras curvature sensing. Taken together, the Ras proteolipid nano-assemblies on the PM act as relay stations to convert mechanical stimulations to mitogenic signaling circuits, thus a novel mechanism for cancer cell mechanotransduction

biochemistry↗

bioRxiv2018

Bayesian Shrinkage Estimation of High Dimensional Causal Mediation Effects in Omics Studies

Causal mediation analysis aims to examine the role of a mediator or a group of mediators that lie in the pathway between an exposure and an outcome. Recent biomedical studies often involve a large number of potential mediators based on high-throughput technologies. Most of the current analytic methods focus on settings with one or a moderate number of potential mediators. With the expanding growth of omics data, joint analysis of molecular-level genomics data with epidemiological data through mediation analysis is becoming more common. However, such joint analysis requires methods that can simultaneously accommodate high-dimensional mediators and that are currently lacking. To address this problem, we develop a Bayesian inference method using continuous shrinkage priors to extend previous causal mediation analysis techniques to a high-dimensional setting. Simulations demonstrate that our method improves the power of global mediation analysis compared to simpler alternatives and has decent performance to identify true non-null mediators. We also construct tests for natural indirect effects using a permutation procedure. The Bayesian method helps us to understand the structure of the composite null hypotheses. We applied our method to Multi-Ethnic Study of Atherosclerosis (MESA) and identified DNA methylation regions that may actively mediate the effect of socioeconomic status (SES) on cardiometabolic outcome.

epidemiology↗

bioRxiv2018

A global map of RNA binding protein occupancy guides functional dissection of post-transcriptional regulation of the T cell transcriptome

RNA binding proteins (RBPs) mediate constitutive RNA metabolism and gene specific regulatory interactions. To identify RNA cis-regulatory elements, we developed GCLiPP, a biochemical technique for detecting RBP occupancy transcriptome-wide. GCLiPP sequence tags corresponded with known RBP binding sites, specifically correlating to abundant cytosolic RBPs. To demonstrate the utility of our occupancy profiles, we performed functional dissection of 3' UTRs with CRISPR/Cas9 genome editing. Two RBP occupied sites in the CD69 3' UTR destabilized the transcript of this key regulator of lymphocyte tissue egress. Comparing human Jurkat T cells and mouse primary T cells uncovered hundreds of biochemically shared peaks of GCLiPP signal across homologous regions of human and mouse 3' UTRs, including a cis-regulatory element that governs the stability of the mRNA that encodes the proto-oncogene PIM3 in both species. Our GCLiPP datasets provide a rich resource for investigation of post-transcriptional regulation in the immune system.

genomics↗