Biology subjects

Wei, G.

Publications and source records attributed to Wei, G..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Structural and Mechanistic Bases for Resistance of the M66I Capsid Variant to Lenacapavir

Lenacapavir (LEN) is the first in class viral capsid protein (CA) targeting antiretroviral for treating multi-drug-resistant HIV-1 infection. Clinical trials and cell culture experiments have identified resistance associated mutations (RAMs) in the vicinity of the hydrophobic CA pocket targeted by LEN. The M66I substitution conferred by far the highest level of resistance to the inhibitor compared to other RAMs. Here we investigated structural and mechanistic bases for how the M66I change affects LEN binding to CA and viral replication. The high-resolution X-ray structure of the CA(M66I) hexamer revealed that the {beta}-branched side chain of Ile66 induces steric hindrance specifically to LEN thereby markedly reducing the inhibitor binding affinity. By contrast, the M66I substitution did not affect binding of Phe-Gly (FG)-motif-containing cellular cofactors CPSF6, NUP153, or SEC24C, which engage the same hydrophobic pocket of CA. In cell culture the M66I variant did not acquire compensatory mutations or replicate in the presence of LEN. Analysis of viral replication intermediates revealed that HIV-1(M66I CA) predominantly formed correctly matured viral cores, which were more stable than their wildtype counterparts. The mutant cores stably bound to the nuclear envelope but failed to penetrate inside the nucleus. Furthermore, the M66I substitution markedly altered HIV-1 integration targeting. Taken together, our findings elucidate mechanistic insights for how the M66I change confers remarkable resistance to LEN and affects HIV-1 replication. Moreover, our structural findings provide powerful means for future medicinal chemistry efforts to rationally develop second generation inhibitors with a higher barrier to resistance. IMPORTANCELenacapavir (LEN) is a highly potent and long-acting antiretroviral that works by a unique mechanism of targeting the viral capsid protein. The inhibitor is used in combination with other antiretrovirals to treat multi-drug-resistant HIV-1 infection in heavily treatment-experienced adults. Furthermore, LEN is in clinical trials for preexposure prophylaxis (PrEP) with interim results indicating 100 % efficacy to prevent HIV-1 infections. However, one notable shortcoming is a relatively low barrier of viral resistance to LEN. Clinical trials and cell culture experiments identified emergent resistance mutations near the inhibitor binding site on capsid. The M66I variant was the most prevalent capsid substitution identified in patients receiving LEN to treat muti-drug resistant HIV-1 infections. The studies described here elucidate the underlying mechanism by which the M66I substitution confers a marked resistance to the inhibitor. Furthermore, our structural findings will aid future efforts to develop the next generation of capsid inhibitors with enhanced barriers to resistance.

microbiology↗

bioRxiv2024

A T6SS DNase effector induces nuclear DNA Damage to trigger apoptosis via activation of the cGAS-STING-TNF axis

In the relentless battle for survival, bacteria have evolved sophisticated weaponry like the Type VI secretion system (T6SS) to not only outcompete rivals but also manipulate host cell processes to their advantage. Apoptosis, a controlled form of programmed cell death without triggering inflammatory responses, is often exploited by pathogens. While many bacterial pathogens are known to induce apoptosis, the role of T6SS effectors in this process remains unexplored. In this study, we discovered that Yersinia pseudotuberculosis (Yptb) utilizes its T6SS to secrete a DNase effector, TkeA, which induces apoptosis in host cells. Mechanistically, the translocation of TkeA into host cells causes nuclear DNA damage, leading to the release of fragmented DNA into the cytoplasm. This, in turn, activates the DNA-sensing cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. The activation of the cGAS-STING pathway by TkeA subsequently triggers apoptosis in host cells via extrinsic pathways, with tumor necrosis factor (TNF) signaling playing a critical role. Additionally, TkeA also mediates interbacterial competition, promoting the colonization of Yptb in mices gut. Our findings reveal that the T6SS-secreted DNase effector TkeA acts as a trans-kingdom effector, enhancing interbacterial competition and inducing apoptosis in host cells by activating the cGAS-STING-TNF axis. This discovery adds a new dimension to the mechanisms employed by bacterial pathogens to manipulate host cell death pathways by using their secretion systems. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/613802v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@1bfd70org.highwire.dtl.DTLVardef@19bfc89org.highwire.dtl.DTLVardef@1e0bf9dorg.highwire.dtl.DTLVardef@4412b9_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTS* Yptb T6SS secretes a trans-kingdom DNase effector TkeA * TkeA degrades bacterial DNA to mediate interbacterial competition * TkeA causes nuclear DNA damage in host cells to induce apoptosis * TkeA induces apoptosis via the cGAS-STING-TNF axis

microbiology↗

bioRxiv2024

KMAP: Kmer Manifold Approximation and Projection for visualizing DNA sequences

Identifying and illustrating patterns in DNA sequences is a crucial task in various biological data analyses. In this task, patterns are often represented by sets of kmers, the fundamental building blocks of DNA sequences. To visually unveil these patterns, we could project each kmer onto a point in two-dimensional (2D) space. However, this projection poses challenges due to the high-dimensional nature of kmers and their unique mathematical properties. Here, we established a mathematical system to address the peculiarities of the kmer manifold. Leveraging this kmer manifold theory, we developed a statistical method named KMAP for detecting kmer patterns and visualizing them in 2D space. We applied KMAP to three distinct datasets to showcase its utility. KMAP achieved a comparable performance to the classical method MEME, with approximately 90% similarity in motif discovery from HT-SELEX data. In the analysis of H3K27ac ChIP-seq data from Ewing Sarcoma (EWS), we found that BACH1, OTX2 and ERG1 might affect EWS prognosis by binding to promoter and enhancer regions across the genome. We also found that FLI1 bound to the enhancer regions after ETV6 degradation, which showed the competitive binding between ETV6 and FLI1. Moreover, KMAP identified four prevalent patterns in gene editing data of the AAVS1 locus, aligning with findings reported in the literature. These applications underscore that KMAP could be a valuable tool across various biological contexts. KMAP is freely available at: https://github.com/chengl7-lab/kmap.

bioinformatics↗

bioRxiv2024

Modeling Hereditary Diffuse Leukoencephalopathy with Axonal Spheroids using microglia-sufficient brain organoids

Hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) is a rare, fatal, adult-onset neurodegenerative disease that is most often caused by mutations affecting the Colony Stimulating factor-1 Receptor (CSF-1R). To understand how CSF-1R-mutation affects human microglia - the specialized brain-resident macrophages of the central nervous system - and the downstream consequences for neuronal cells, we used a macrophage and forebrain organoid co-culture system based on induced pluripotent stem cells generated from two patients with HDLS, with CSF-1R gene-corrected isogenic organoids as controls. Macrophages derived from iPSC (iMacs) of patients exhibited a metabolic shift towards the glycolytic pathway and reduced CSF-1 sensitivity, which was associated with higher levels of IL-1{beta} production and an activated inflammatory phenotype. Single-cell RNA sequencing revealed that iMacs adopt a reactive state that leads to impaired regulation of neuronal cell populations in organoid cultures, thereby identifying microglial dysregulation and specifically IL-1{beta} production as key contributors to the degenerative neuro-environment in HDLS.

neuroscience↗

bioRxiv2024

Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

Pseudouridine ({Psi}) is one of the most abundant modifications in cellular RNA. However, its function remains elusive, mainly due to the lack of highly sensitive and accurate detection methods. To address this challenge, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS) for quantitative profiling of {Psi} at single-base resolution. Based on novel bromoacrylamide cyclization chemistry, BACS enables a {Psi}-to-C transition. Compared to previous methods, BACS allowed the precise identification of {Psi} positions, especially in densely modified {Psi} regions and consecutive uridine sequences. BACS successfully detected all known {Psi} sites in human rRNA and spliceosomal snRNAs and generated the first quantitative {Psi} map of human snoRNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine (A-to-I) editing sites and N1-methyladenosine (m1A). Depletion of three key pseudouridine synthases (PUS) enabled us to elucidate the targets and sequence motifs of TRUB1, PUS7, and PUS1 in HeLa cells. We further applied BACS to Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) and identified a highly abundant {Psi}114 site in EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of {Psi} in their viral transcripts or genomes, shedding light on differences in pseudouridylation between virus families. We anticipate BACS to serve as a powerful tool to uncover the biological importance of {Psi} in future studies.

molecular biology↗

bioRxiv2023

Calibrating Bayesian decoders of neural spiking activity

Accurately decoding external variables from observations of neural activity is a major challenge in systems neuroscience. Bayesian decoders, that provide probabilistic estimates, are some of the most widely used. Here we show how, in many common settings, the probabilistic predictions made by traditional Bayesian decoders are overconfident. That is, the estimates for the decoded stimulus or movement variables are more certain than they should be. We then show how Bayesian decoding with latent variables, taking account of low-dimensional shared variability in the observations, can improve calibration, although additional correction for overconfidence is still needed. We examine: 1) decoding the direction of grating stimuli from spike recordings in primary visual cortex in monkeys, 2) decoding movement direction from recordings in primary motor cortex in monkeys, 3) decoding natural images from multi-region recordings in mice, and 4) decoding position from hippocampal recordings in rats. For each setting we characterize the overconfidence, and we describe a possible method to correct miscalibration post-hoc. Properly calibrated Bayesian decoders may alter theoretical results on probabilistic population coding and lead to brain machine interfaces that more accurately reflect confidence levels when identifying external variables. Significance StatementBayesian decoding is a statistical technique for making probabilistic predictions about external stimuli or movements based on recordings of neural activity. These predictions may be useful for robust brain machine interfaces or for understanding perceptual or behavioral confidence. However, the probabilities produced by these models do not always match the observed outcomes. Just as a weather forecast predicting a 50% chance of rain may not accurately correspond to an outcome of rain 50% of the time, Bayesian decoders of neural activity can be miscalibrated as well. Here we identify and measure miscalibration of Bayesian decoders for neural spiking activity in a range of experimental settings. We compare multiple statistical models and demonstrate how overconfidence can be corrected.

neuroscience↗

bioRxiv2022

The first host-associated anaerobic isolate of Psychrilyobacter provides insights into its potential roles in the abalone gut

Gut bacteria are essential to their hosts, but few studies have focused on marine animals. Psychrilyobacter is frequently related to various marine animals, but the relationship with host remains unknown due to lack of host-associated isolate or genomic information. Here, we combined of high-throughput sequencing, isolating and genomic analyses to uncover its potential role in host abalone. The high-throughput sequencing and literature compiling results indicated that Psychrilyobacter is widely distributed in marine and terrestrial ecosystems with both host-associated and free-living lifestyles. It showed a strong preference for the guts of marine invertebrates, especially abalone, which generally persisted with high relative abundances. By mimicking the gut inner environment for enrichment, the first host-related pure culture of Psychrilyobacter was isolated from the abalone intestine. Phylogenetic, physiological and biochemical characterizations suggested that it represents a novel species named Psychrilyobacter haliotis B1. Carbohydrate utilization experiments and genomic evidence indicated that B1 is good at fermenting diverse host-food-related monosaccharides and disaccharides but not polysaccharides, implying its critical roles in downstream fermentation instead of upstream food degradation in the gut. Furthermore, this strain showed potential to colonize the gut and benefit the host via different strategies, such as short-chain fatty acids (SCFAs) generation by fermenting carbohydrates and amino acids, and the production of diverse vitamins and antibiotics to support the host growth and antipathogenicity. To our knowledge, strain B1 represents the first host-related pure culture of Psychrilyobacter, acting as a potential probiotic gut anaerobe dominating in diverse marine invertebrates. ImportancePsychrilyobacter is a globally distributing bacterial genus and with an inhabiting preference for guts of marine invertebrates. Due to the difficulty of cultivation and the limited genomic information, its role in host remains largely unknown. We isolated the first host-associated Psychrilyobacter species from abalone gut and uncovered its functional potentials to the host through different mechanisms. Our findings provide some insights into the understanding of host-microbe interactions on a core taxon with the marine invertebrates, and the isolate has an application prospect as a probiotic in protection of marine animals.

microbiology↗

bioRxiv2022

High-resolution small RNAs landscape provides insights into alkane adaptation in the marine alkane-degrader Alcanivorax dieselolei B-5

Alkanes are widespread in the ocean, and Alcanivorax is one of the most ubiquitous alkane-degrading bacteria in the marine ecosystem. Small RNAs (sRNAs) are usually at the heart of regulatory pathways, but sRNA-mediated alkane metabolic adaptability still remains largely unknown due to the difficulties of identification. Here, differential RNA sequencing (dRNA-seq) modified with a size selection ([~]50-nt to 500-nt) strategy was used to generate high-resolution sRNAs profiling in the model species Alcanivorax dieselolei B-5 under alkane (n-hexadecane) and non-alkane (acetate) conditions. As a result, we identified 549 sRNA candidates at single-nucleotide resolution of 5-ends, with 63.4% of transcription start sites (TSSs) and 36.6% of processing sites (PSSs). These sRNAs originated from almost any locations in the genome, regardless of intragenic (65.8%), antisense (20.6%) and intergenic (6.2%) regions, and RNase E may function in the maturation of sRNAs. Most sRNAs locally distribute across the 15 reference genomes of Alcanivorax, and only 7.5% of sRNAs are broadly conserved in this genus. Expression responses to alkane of several core conserved sRNAs, including 6S RNA, M1 RNA and tmRNA, indicate that they may participate in alkane metabolisms and result in more actively global transcription, RNA processing and stresses mitigation. Two novel CsrA-related sRNAs are identified, which may be involved in the translational activation of alkane metabolism-related genes by sequestering the global repressor CsrA. The relationships of sRNAs with characterized genes of the alkane sensing (ompS), chemotaxis (mcp, cheR, cheW2), transporting (ompT1, ompT2, ompT3) and hydroxylation (alkB1, alkB2, almA) were created based on the genome-wide targets prediction. Overall, the sRNAs landscape lays the ground for uncovering cryptic regulations in the critical marine bacterium, among which both core and species-specific sRNAs are implicated in the alkane adaptive metabolisms.

microbiology↗

bioRxiv2022

Vascular tortuosity quantification as an outcome metric of the oxygen-induced retinopathy model of ischemic retinopathy

The murine oxygen-induced retinopathy (OIR) model is one of the most widely used animal models of ischemic retinopathy, mimicking hallmark pathophysiology of initial vaso-obliteration (VO) resulting in ischemia that drives neovascularization (NV). In addition to NV and VO, human ischemic retinopathies including Retinopathy of Prematurity (ROP) are characterized by increased vascular tortuosity. Vascular tortuosity is an indicator of disease severity, need to treat, and treatment response in ROP. Current literature investigating novel therapeutics in the OIR model report their effects on NV and VO, but no standardized quantification of vascular tortuosity exists to date despite this metrics relevance to human disease in clinics. The current proof-of-concept study applied a computer-based image analysis algorithm capable of calculating standardized measurements of vascular tortuosity. Quantification of vascular tortuosity correlated with disease activity in OIR analogously to that observed in infants with ROP. Treatment of OIR mice with anti-Vascular Endothelial Growth Factor (aflibercept) rescued vascular tortuosity in the model. Altogether, these data demonstrated that vascular tortuosity is a quantifiable feature of the OIR model and may be used as an outcome measurement in future studies investigating new treatment modalities for retinal ischemia.

neuroscience↗

bioRxiv2022

Structural insights into the elevator-type transport mechanism of a bacterial ZIP metal transporter

The Zrt-/Irt-like protein (ZIP) family consists of ubiquitously expressed divalent metal transporters critically involved in maintaining systemic and cellular homeostasis of zinc, iron, and manganese. Here, we present a study on a prokaryotic ZIP from Bordetella bronchiseptica (BbZIP) by combining structural biology, evolutionary covariance, computational modeling, and a variety of biochemical assays to tackle the issue of the transport mechanism which has not been established for the ZIP family. The apo state structure in an inward-facing conformation revealed a disassembled transport site, altered inter-helical interactions, and importantly, a rigid body movement of a 4-transmembrane helix (TM) bundle relative to the other TMs. The computationally generated and biochemically validated outward-facing conformation model revealed a slide of the 4-TM bundle, which carries the transport site(s), by approximately 8 [A] toward the extracellular side against the static TMs which mediate dimerization. These findings allowed us to conclude that BbZIP is an elevator-type transporter.

biochemistry↗

bioRxiv2022

MATR3-antisense LINE1 RNA meshwork scaffolds higher-order chromatin organization

Long interspersed nuclear elements (LINEs) play essential role in shaping chromatin state, while the factors that cooperate with LINEs and their roles in higher-order chromatin organization remain poorly understood. Here we show that MATR3, a nuclear matrix protein, interplays with antisense LINE1 (AS L1) RNAs to form into a gel-like meshwork via phase-separation, providing a partially dynamic platform for chromatin spatial organization. Either depletion of MATR3 or AS L1 RNAs changes nuclear distribution of each other and leads to chromatin reorganization in the nucleus. After MATR3 depletion, topologically associating domains (TADs) that highly transcribed MATR3-associated AS L1 RNAs showed a decrease on local chromatin interactions. Furthermore, amyotrophic lateral sclerosis (ALS)-associated MATR3 mutants alter biophysical features of the MATR3-AS L1 RNA meshwork and cause chromatin reorganization. Collectively, we revealed an essential role of meshwork formed by nuclear matrix and retrotransposon-derived RNAs in gathering chromatin in the nucleus.

cell biology↗

bioRxiv2022

Void volume fraction of granular scaffolds

Void volume fraction (VVF) of granular biomaterials is a global measurement frequently used to characterize void space. There is currently no gold standard for measuring the VVF of granular scaffolds made in lab. To help the biomaterials field, we provide a library of different simulated scaffolds with known VVF for easy look-up. We use our simulated data to explore the relationship between microscope magnification and VVF, and we study the accuracy of approximating VVF using 2-D z-slice images, which is the most common approach for computing VVF of real scaffolds. Lastly, we test four different approaches for computing VVF using microscope images of real granular scaffolds and reveal that VVF may be an unreliable descriptor.

bioengineering↗

bioRxiv2022

Predictable fluctuations in excitatory synaptic strength due to natural variation in presynaptic firing rate

Many controlled, in vitro studies have demonstrated how postsynaptic responses to presynaptic spikes are not constant but depend on short-term synaptic plasticity (STP) and the detailed timing of presynaptic spikes. However, the effects of short-term plasticity (depression and facilitation) are not limited to short, sub-second timescales. The effects of STP appear on long timescales as changes in presynaptic firing rates lead to changes in steady-state synaptic transmission. Here we examine the relationship between natural variations in the presynaptic firing rates and spike transmission in vivo. Using large-scale spike recordings in awake mice from the Allen Institute Neuropixels dataset, we first detect putative excitatory synaptic connections based on cross-correlations between the spike trains of millions of pairs of neurons. For the subset of pairs where a transient, excitatory effect was detected, we use a model-based approach to track fluctuations in synaptic efficacy and find that efficacy varies substantially on slow ([~]1 minute) timescales over the course of these recordings. For many connections, the efficacy fluctuations are correlated with fluctuations in the presynaptic firing rate. To understand the potential mechanisms underlying this relationship, we then model the detailed probability of postsynaptic spiking on a millisecond timescale, including both slow changes in postsynaptic excitability and monosynaptic inputs with short-term plasticity. The detailed model reproduces the slow efficacy fluctuations observed with many putative excitatory connections, suggesting that these fluctuations can be both directly predicted based on the time-varying presynaptic firing rate and, at least partly, explained by the cumulative effects of STP.

neuroscience↗

bioRxiv2021

Xist-mediated silencing requires additive functions of SPEN and Polycomb together with differentiation-dependent recruitment of SmcHD1

X chromosome inactivation (XCI) is mediated by the non-coding RNA Xist which directs chromatin modification and gene silencing in cis. The RNA binding protein SPEN and associated corepressors have a central role in Xist-mediated gene silencing. Other silencing factors, notably the Polycomb system, have been reported to function downstream of SPEN. In recent work we found that SPEN has an additional role in correct localisation of Xist RNA in cis, indicating that its contribution to chromatin-mediated gene silencing needs to be reappraised. Making use of a SPEN separation-of-function mutation we show that SPEN and Polycomb pathways in fact function in parallel to establish gene silencing. Additionally, we find that differentiation-dependent recruitment of the chromosomal protein SmcHD1 is required for silencing many X-linked genes. Our results provide important insights into the mechanism of X inactivation and the coordination of chromatin-based gene regulation with cellular differentiation and development.

molecular biology↗

bioRxiv2021

Small but complete: liquid-liquid phase separation in minimalistic peptides

Liquid-liquid phase separation (LLPS) of proteins mediates the assembly of biomolecular condensates involved in physiological and pathological processes. Identifying the minimalistic building blocks and the sequence determinant of protein phase separation is of urgent importance but remains challenging due to the enormous sequence space and difficulties of existing methodologies in characterizing the phase behavior of ultrashort peptides. Here we demonstrate computational tools to efficiently quantify the microscopic fluidity and density of liquid-condensates/solid-aggregates and the temperature-dependent phase diagram of peptides. Utilizing our approaches, we comprehensively predict the LLPS abilities of all 400 dipeptide combinations of coded amino acids based on 492 micro-second molecular dynamics simulations, and observe the occurrences of spontaneous LLPS. We identify 54 dipeptides that form solid-like aggregates and three categories of dipeptides with high LLPS propensity. Our predictions are validated by turbidity assays and differential interference contrast (DIC) microscopy on four representative dipeptides (WW, QW, GF, and VI). Phase coexistence diagrams are constructed to explore the temperature dependence of LLPS. Our results reveal that aromatic moieties are crucial for a dipeptide to undergo LLPS, and hydrophobic and polar components are indispensable. We demonstrate for the first time that dipeptides, minimal but complete, possess multivalent interactions sufficient for LLPS, suggesting that LLPS is a general property of peptides/proteins, independent of their sequence length. This study provides a computational and experimental approach to the prediction and characterization of the phase behavior of minimalistic peptides, and will be helpful for understanding the sequence-dependence and molecular mechanism of protein phase separation. SignificanceProtein liquid-liquid phase separation (LLPS) is associated with human health and diseases. Identifying the minimalistic building blocks and sequence determinants of LLPS is of urgent importance but remains computationally challenging partially due to the lack of methodologies characterizing the liquid condensates. Herein we provide approaches to evaluate LLPS ability of dipeptides, and screen all 400 dipeptides by MD simulations combined with multi-bead-per-residue models which capture key interactions driving LLPS that are missing in one-bead-per-residue models. Three categories of LLPS dipeptides are identified and the experimentally-verified QW dipeptide is by far the smallest LLPS system. Our results suggest that dipeptides, minimal but complete, possess multivalent interactions sufficient for LLPS, and LLPS is a general property of peptides/proteins, independent of their length.

biophysics↗

bioRxiv2021

Unaffected functional recovery after spinal cord contusions at different circadian times

The circadian rhythms of gene expression drive diurnal oscillations of physiological processes that determine the acute injury response including immunity, inflammation and hemostasis. While outcomes of various acute injuries are affected by the time of day at which the original insult occurred, such diurnal influences on recovery after spinal cord injury (SCI) are unknown. We report that several key regulators of circadian gene expression are differentially expressed in uninjured spinal cord tissue of naive mice at Zeitgeber time 1 (ZT1) or ZT12, where ZT0 or ZT12 are times when lights are turned on or off, respectively. However, mice that received moderate, T9 contusive SCI at ZT0 or ZT12 showed similar recovery of locomotion as determined using the ladder walking test and the Basso mouse scale (BMS) over a 6 week post-injury period. Consistent with those findings, terminal histological analysis revealed no significant differences in white matter sparing at the injury epicenter. Therefore, locomotor recovery after thoracic contusive SCI is not affected by the time of day at which the neurotrauma occurred at least when comparing the beginning to the end of the mouse active period.

neuroscience↗

bioRxiv2020

Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading

Xist RNA directs the process of X-chromosome inactivation in mammals by spreading in cis along the chromosome from which it is transcribed and recruiting chromatin modifiers to silence gene transcription. To elucidate mechanisms of Xist RNA cis-confinement, we established a sequential dual color labeling, super-resolution imaging approach to trace individual Xist RNA molecules over time, enabling us to define fundamental parameters of spreading. We demonstrate a feedback mechanism linking Xist RNA synthesis and degradation, and an unexpected physical coupling between preceding and newly synthesized Xist RNA molecules. Additionally, we show that the protein SPEN, a key factor for Xist-mediated gene-silencing, has a distinct function in Xist RNA localization, stability and in coupling behavior. Our results provide important insights towards understanding the unique dynamic properties of Xist RNA. One Sentence SummaryVisualizing Xist RNA dynamics in single cells during X chromosome inactivation

developmental biology↗

bioRxiv2020

LncRNA Snhg1-driven self-reinforcing regulatory network promotes cardiomyocyte cytokinesis

Most of current cardiac regenerative approaches result in very limited cell division. Positive feedback loops are vital for cell division, but their role in CM regeneration remains unclear. We aimed to demonstrate that lncRNA Snhg1 formed a positive feedback loop with c-Myc to induce stable CM cytokinesis. We found that Snhg1 expression was increased in human and mouse fetal and myocardial infarction (MI) hearts, particularly in CMs. Snhg1 overexpression elicited stable CM proliferation and improved post-MI cardiac function. Antagonism of Snhg1 in neonatal mice inhibited CM proliferation and impaired cardiac repair after MI. Proliferative effect was confirmed using cardiac-specific transgenic mice. RNA pull-down assays showed that Snhg1 directly bound to PTEN and activated PI3K-Akt pathway, resulting in c-Myc activation. Chromatin immunoprecipitation experiments showed that Snhg1 expression was upregulated by c-Myc binding to the Snhg1 promoter region, indicating a positive feedback loop between c-Myc and Snhg1. In conclusion, c-Myc/Snhg1/PI3k-Akt positive feedback loop drove sustained activation of cell cycle re-entry and induced stable CM cytokinesis, and thus may be an attractive strategy for promoting heart regenerative response. Clinical PerspectivesMost of the current cardiac regenerative approaches result in very limited cell division and little new cardiomyocyte (CM) mass. Positive feedback loops are vital for cell division, but their role in CM regeneration remain unclear. Here, we identified the long noncoding RNA Snhg1 as a driver to induce stable CM division and improve cardiac function after myocardial infarction (MI) by forming a positive feedback loop to sustain PI3K-Akt signaling activation. This finding might provide a novel therapeutic of Snhg1 as a promising regenerative approach to improve the prognosis of patients with heart failure.

developmental biology↗