Biology subjects

Jin, Y.

Publications and source records attributed to Jin, Y..

At least 109 records · Page 6Linked to original sources

bioRxiv2024

Metal fluorides - multi-functional tools for the study of phosphoryl transfer enzymes

Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein group across all kingdoms of life, making up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyse these reactions is essential in characterising the processes they regulate. Metal fluorides can be used as multifunctional tools for the study of these enzymes. These ionic species bear the same charge as phosphate and the transferring phosphoryl group and, in addition, allow the enzyme to be trapped in catalytically important states with spectroscopically sensitive atoms interacting directly with active site residues. The ionic nature of these phosphate surrogates also allows their removal and replacement with other analogues. Here, we describe the best practices to obtain these complexes, their use in NMR, X-ray crystallography, cryoEM and SAXS and describe a new metal fluoride, scandium tetrafluoride, which has significant anomalous signal with soft X-rays. Highlights Enzymes that catalyse phosphoryl transfer are the largest family of enzymes and are involved in the storage and transmission of genetic information, energy transfer, signalling and cellular differentiation Metal fluorides form a comprehensive tool kit to study the mechanisms of these enzymes by stabilizing the active conformation; mimicking both the transition state and ground state; and placing spectroscopically sensitive atoms into the active site A guide is presented to the optimal formation of these complexes and their use in a wide variety of techniques in structural biology

molecular biology↗

bioRxiv2024

Immunomagneto-activated hepatocyte-specific tissue vesicle profiling reveals message diversification from regenerating liver

Background & AimsThe liver possesses a remarkable regenerative capacity involving intricate intercellular communication, the mechanisms of which remain poorly understood. Extracellular vesicles (EVs) emerge as important messengers in diverse pathophysiological conditions. However, tissue-derived, cell-specific functional EV populations in regeneration have not been robustly investigated. MethodsBulk and single-cell RNA sequencing analyses of the regenerating liver after partial hepatectomy (PHx), ultrastructural examinations of in situ liver tissue EVs (LT-EVs), and nanoscale and proteomic profiling of hepatocyte-derived tissue EVs (Hep-EVs) were integrated. Targeted inhibition of Hep-EV release in vivo was performed via AAV-mediated shRNA knockdown of Rab27a, and Hep-EVs were intravenously infused for therapeutically use. Gross, histological, and immunofluorescent examinations of livers with evaluating in vivo and ex vivo hepatocyte proliferation were conducted. ResultsLT-EVs contribute to the liver regenerative process after PHx, and hepatocytes serve as the major origin of tissue EVs in the regenerating liver. Moreover, Hep-EVs play an indispensable role to orchestrate liver regeneration, which is strengthened to release with proliferative messages identified after PHx. Mechanistically, Hep-EVs from the regenerating liver reciprocally promote hepatocyte proliferation, which are hallmarked by and function based on the Cyclin dependent kinase 1 (Cdk1) activity. Importantly, replenishment of Hep-EVs from the regenerating liver holds translational promise and rescues insufficient liver regeneration. ConclusionsOur study establishes a functional and mechanistic framework that release of regenerative Hep-EVs govern rapid hepatocyte proliferation through cell cycle control, shedding light on investigation of physiological and endogenous tissue EV populations in organ regeneration and therapy.

cell biology↗

bioRxiv2024

Schlafen-11 and -9 are innate immune sensors for intracellular single-stranded DNA

Nucleic acids are major structures detected by the innate immune system. Although intracellular single-stranded DNA (ssDNA) is accumulated during pathogen infection or pathogenesis, it remains unclear whether and how intracellular ssDNA stimulates the immune system. We report that intracellular ssDNA triggers cytokine expression and cell death in a CGT motif-dependent manner. Through a genome-wide CRISPRLCas9 screen, we identified Schlafen-11 (SLFN11), which is essential for the response to endogenous and pathogen ssDNA. SLFN11 directly binds ssDNA containing CGT motifs and translocates to the cytoplasm upon ssDNA recognition. The mice deficient in SLFN9, the homologue of SLFN11, were resistant to CGT ssDNA-induced inflammation, acute hepatitis and septic shock. This study establishes CGT ssDNA and SLFN11/9 as a novel type of immunostimulatory nucleic acids and pattern recognition receptors, respectively. One-sentence summariessingle-stranded DNA inside cells can awaken Schlafens with a CGT motif to trigger cytokine expression and lytic cell death.

immunology↗

bioRxiv2024

Increased capacity to maintain glucose homeostasis in a transgenic mouse expressing human but not mouse growth hormone with developing high fat diet-related insulin resistance, steatosis and adipose dysfunction

ObjectiveDifferences in primate and non-primate growth hormone (GH) genes can affect their regulation and function. This includes the ability of human (h) but not mouse (m) GH to bind the prolactin (PRL) receptor (PRLR) as well as the GH receptor (GHR). Potential differential effects were assessed in male hGH- or mGH-expressing mice and fed high fat diet (HFD) versus regular chow diet (RCD). Pancreas and epididymal white adipose tissue (eWAT) gene expression and/or related function were targeted as the pancreas responds to both PRLR and GHR signaling and catabolic effects like lipolytic activity are more directly attributable to GH and GHR signaling. DesignA transgenic CD-1 mouse expressing hGH but not mGH under hypothalamic control was generated to compare with wild type CD-1 mice and size and bone density determined. Glucose clearance, islet area, insulin and insulin-like growth factor (IGF) -2 gene expression were assessed as well as serum glucose and insulin levels in mice fed a HFD versus RCD for 8 and 24 weeks. Adiposity, liver and serum triglycerides as well as eWAT cell area, cytokine (leptin and adiponectin) and senescence-related marker (p21CIP1 and p16INK4a) RNA levels were also assessed. ResultsMale hGH-expressing transgenic CD-1[{Delta}mGH.hGH] mice have significantly lower liver IGF-1 RNA levels and are smaller based on length and weight than wild type CD-1[mGH] mice. They also have [~]1.5-fold higher total body fat and serum triglyceride levels. However, CD-1[{Delta}mGH.hGH] and CD-1[mGH] mice grow at the same rate with similar cortical and trabecular bone densities. Unlike CD-1[mGH] mice, there was no significant delay in glucose clearance in CD-1[{Delta}mGH.hGH] mice after 8 weeks on a HFD versus RCD; while basal (RCD) serum insulin levels were similar, fasting glucose levels were lower and pancreas IGF-2 RNA levels were increased in CD-1[{Delta}mGH.hGH] mice. However, both CD-1[{Delta}mGH.hGH] and CD-1[mGH] showed evidence of increased insulin resistance after 24 weeks on HFD, including delayed glucose clearance in spite of increased pancreatic islet area and insulin production as well as signs of liver steatosis and increased hepatic triglyceride levels. These increases correlated with elevated PRLR but not GHR RNA levels. Assessment of eWAT revealed >2-fold larger adipocytes in CD-1[{Delta}mGH.hGH] compared to CD-1 [mGH] mice fed RCD at both 12 and 28 weeks. This was associated with an [~]2.6-fold increase in leptin RNA levels at 12 weeks and [~]58% lower adiponectin RNA levels at 28 weeks. A >2-fold increase in p21CIP1 transcript levels was also detected in eWAT from both CD-1[{Delta}mGH.hGH] and CD-1 [mGH] mice fed RCD with age (28 versus 12 weeks) but were unaffected by diet. However, a >2-fold increase in p16INK4a RNA levels was observed after 24 weeks on HFD. ConclusionsWhile limited to observations in the male, transgenic CD-1[{Delta}mGH.hGH] mice exhibit signs of GH insufficiency and eWAT adipocyte dysfunction. These mice also show an initial resistance to the negative effects of HFD on glucose clearance when compared to CD-1[mGH] mice, which is potentially related to a differential effect of hGH versus mGH on pancreas development and/or function.

physiology↗

bioRxiv2024

C. elegans LIN-66 mediates EIF-3.G-dependent protein translation via a cold-shock domain

Protein translation initiation is a complex and conserved process involving many proteins acting in concert. The eukaryotic initiation factor 3 (eIF3) complex is essential for the assembly of the pre-initiation complex that scans and positions mRNA at the initiation codon. eIF3 complex consists of 13 subunits. In addition to their essential roles in general translation initiation, emerging studies suggest that individual subunits of eIF3 can provide specialized functions in response to specific stimuli. We have previously reported that a gain-of-function (gf) mutation in the G subunit of C. elegans eIF3 complex, eif-3.g(gf), selectively modulates protein translation in the ventral cord cholinergic motor neurons. Here, through unbiased genetic suppressor screening, we have identified the lin-66 gene that mediates the eif-3.g(gf)-dependent protein translation in the motor neurons. LIN-66 is previously reported to be a nematode-specific protein composed of largely low complexity amino acid sequences with unknown functional domains. We combined bioinformatic analysis with in vivo functional dissection and identified a cold-shock domain in LIN-66 to be critical for its function. In the cholinergic motor neurons, LIN-66 shows somatic cytoplasmic localization and close association with EIF-3.G. The low complexity amino acid sequences of LIN-66 modulate its subcellular pattern. Cold-shock-domains are known to interact with RNA and have broad functions in RNA metabolism and protein translation. We propose that LIN-66 mediates stimuli-dependent protein translation by facilitating the interaction of mRNAs with EIF-3.G.

genetics↗

bioRxiv2024

Coordinated Immune Cell Networks in the Bone Marrow Microenvironment Define the Graft versus Leukemia Response with Adoptive Cellular Therapy

Understanding how intra-tumoral immune populations coordinate to generate anti-tumor responses following therapy can guide precise treatment prioritization. We performed systematic dissection of an established adoptive cellular therapy, donor lymphocyte infusion (DLI), by analyzing 348,905 single-cell transcriptomes from 74 longitudinal bone-marrow samples of 25 patients with relapsed myeloid leukemia; a subset was evaluated by protein-based spatial analysis. In acute myelogenous leukemia (AML) responders, diverse immune cell types within the bone-marrow microenvironment (BME) were predicted to interact with a clonally expanded population of ZNF683+GZMB+ CD8+ cytotoxic T lymphocytes (CTLs) which demonstrated in vitro specificity for autologous leukemia. This population, originating predominantly from the DLI product, expanded concurrently with NK and B cells. AML nonresponder BME revealed a paucity of crosstalk and elevated TIGIT expression in CD8+ CTLs. Our study highlights recipient BME differences as a key determinant of effective anti-leukemia response and opens new opportunities to modulate cell-based leukemia-directed therapy.

cancer biology↗

bioRxiv2024

Phenotypic screening of covalent compound libraries identifies chloromethyl ketone antibiotics and MiaA as a new target

The emerging antibiotic resistance requires the development of new antibiotics working on novel bacterial targets. Here, we reported an antibiotic discovery workflow by combining the cysteine-reactive compound library phenotypic screening with activity-based protein profiling, which enables the rapid identification of lead compounds as well as new druggable targets in pathogens. Compounds featuring chloromethyl ketone scaffolds exhibited a notably high hit rate against both gram-negative and gram-positive bacterial strains, but not the more commonly used warheads such as acrylamide or chloroacetamide. Target identification of the lead compound, 10-F05, revealed that its primary targets in S. flexneri are FabH Cys112 and MiaA Cys273. We validated the target relevance through biochemical and genetic interactions. Mechanistic studies revealed modification of MiaA by 10-F05 impair substrate tRNA binding, leading to decreased bacterial stress resistance and virulence. Our findings underscore chloromethyl ketone as a novel antibacterial warhead in covalent antibiotic design. The study showcases that combining covalent compound library phenotypic screening with chemoproteomics is an efficient way to identify new drug targets as well as lead compounds, with the potential to open new research directions in drug discovery and chemical biology. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/576576v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@8d6420org.highwire.dtl.DTLVardef@a89d44org.highwire.dtl.DTLVardef@1ed0ae2org.highwire.dtl.DTLVardef@1659d3e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Beige adipose tissue-derived extracellular vesicles: a potent metabolic regulator and a novel remedy for nonalcoholic fatty liver disease

Adipose tissue (AT) is an essential metabolic and endocrine organ, which are categorized into the white adipose tissue (WAT) and the brown adipose tissue (BAT), as well as the beige adipose tissue (BeAT) that derives from WAT browning. Notably, extracellular vesicles (EVs), especially tissue-produced EVs, have been recognized to be critical players in multiple physiological and pathophysiological settings and provide efficient strategy for disease treatment. However, little is known about the BeAT-derived EVs (BeEVs). In this study, we induced BeEV formation by cold stimulation and extracted EVs from three kinds of AT via enzyme digestion combined with gradient centrifugation. The isolated BeEVs possess typical morphological, structural and marker characteristics of EVs. Proteomics analysis further demonstrated that the BeEVs hold a distinct protein profile while are enriched with multiple metabolic regulatory proteins. Importantly, systemic injection of BeEVs into mice improved metabolic dysfunction in a nonalcoholic fatty liver disease (NAFLD) model induced by high-fat diet (HFD). Moreover, we demonstrated that the BeEVs alleviated liver steatosis via decreasing the lipogenesis. These findings promote the understanding of AT-EVs and shed light on the isolation, identification and applications of BeEVs as a potent metabolic regulator and novel remedy for metabolic diseases.

cell biology↗

bioRxiv2024

DMSO derives Trophectoderm and Clonal Blastoid from Single Human Pluripotent Stem Cell

Human naive pluripotent stem cells (nPSCs) can differentiate into extra-embryonic trophectoderm (TE), a critical step in the generation of the integrated embryo model termed blastoid. The current paradigm of blastoid generation necessitates the aggregation of dozens of nPSCs treated with multiple small molecule inhibitors, growth factors, or genetic modifications to initiate TE differentiation. The presence of complex crosstalk among pathways and cellular heterogeneity in these models complicates mechanistic study and genetic screens. Here, we show that a single small molecule, dimethyl sulfoxide (DMSO), potently induces TE differentiation in basal medium without pharmacological and genetic perturbations. DMSO enhances blastoid generation and, more importantly, is sufficient for blastoid generation by itself. DMSO blastoids resemble blastocysts in morphology and lineage composition. DMSO induces blastoid formation through PKC signaling and cell cycle regulation. Lastly, DMSO enables single nPSC-derived clonal blastoids, which could facilitate genetic screens for mechanistic understanding of human embryogenesis.

developmental biology↗

bioRxiv2023

CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

Pooled genetic screens are powerful tools to study gene function in a high-throughput manner. Typically, sequencing-based screens require cell lysis, which limits the examination of critical phenotypes such as cell morphology, protein subcellular localization, and cell-cell/tissue interactions. In contrast, emerging optical pooled screening methods enable the investigation of these spatial phenotypes in response to targeted CRISPR perturbations. In this study, we report a multi-omic optical pooled CRISPR screening method, which we have named CRISPRmap. Our method combines a novel in situ CRISPR guide identifying barcode readout approach with concurrent multiplexed immunofluorescence and in situ RNA detection. CRISPRmap barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency, while reducing both dependency on third party proprietary sequencing reagents and assay cost. Notably, we conducted a multi-omic base-editing screen in a breast cancer cell line on core DNA damage repair genes involved in the homologous recombination and Fanconi anemia pathways investigating how nucleotide variants in those genes influence DNA damage signaling and cell cycle regulation following treatment with ionizing radiation or DNA damaging agents commonly used for cancer therapy. Approximately a million cells were profiled with our multi-omic approach, providing a comprehensive phenotypic assessment of the functional consequences of the studied variants. CRISPRmap enabled us to pinpoint likely-pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance. Furthermore, our approach effectively distinguished barcodes of a pooled library in tumor tissue, and we coupled it with cell-type and molecular phenotyping by cyclic immunofluorescence. Multi-omic spatial analysis of how CRISPR-perturbed cells respond to various environmental cues in the tissue context offers the potential to significantly expand our understanding of tissue biology in both health and disease.

genomics↗

bioRxiv2023

Ca2+ Oscillation in Vascular Smooth Muscle Cells Control Myogenic Spontaneous Vasomotion and Counteract Post-ischemic No-reflow

Ischemic stroke produces the highest adult disability. Despite successful recanalization, no-reflow, or the futile restoration of the cerebral perfusion after ischemia, is a major cause of brain lesion expansion. However, the vascular mechanism underlying this hypoperfusion is largely unknown, and no approach is available to actively promote optimal reperfusion to treat no-reflow. Here, by combining two-photon laser scanning microscopy (2PLSM) and a mouse middle cerebral arteriolar occlusion (MCAO) model, we found myogenic vasomotion deficits correlated with post-ischemic cerebral circulation interruptions and no-reflow. Transient occlusion-induced transient loss of mitochondrial membrane potential ({Delta}{Psi}m) permanently impaired mitochondria-endoplasmic reticulum (ER) contacts and abolished Ca2+ oscillation in smooth muscle cells (SMCs), the driving force of myogenic spontaneous vasomotion. Furthermore, tethering mitochondria and ER by specific overexpression of ME-Linker in SMCs restored cytosolic Ca2+ homeostasis, remotivated myogenic spontaneous vasomotion, achieved optimal reperfusion, and ameliorated neurological injury. Collectively, the maintaining of arteriolar myogenic vasomotion and mitochondria-ER contacts in SMCs, are of critical importance in preventing post-ischemic no-reflow.

neuroscience↗

bioRxiv2023

UBE2N is essential for maintenance of skin homeostasis and suppression of inflammation

UBE2N, a Lys63-ubiquitin conjugating enzyme, plays critical roles in embryogenesis and immune system development and function. However, its roles in adult epithelial tissue homeostasis and pathogenesis are unclear. We generated conditional mouse models that deleted Ube2n in skin cells in a temporally and spatially controlled manner. We found that Ube2n- knockout (KO) in the adult skin keratinocytes induced a range of inflammatory skin defects characteristic of psoriatic and actinic keratosis. These included eczematous inflammation, epidermal and dermal thickening, parakeratosis, and increased immune cell infiltration, as well as signs of edema and blistering. Single cell transcriptomic analyses and RT-qPCR showed that Ube2n KO keratinocytes expressed elevated myeloid cell chemo-attractants such as Cxcl1 and Cxcl2 and decreased the homeostatic T lymphocyte chemo-attractant, Ccl27a. Consistently, the infiltrating immune cells of Ube2n-KO skin were predominantly myeloid-derived cells including neutrophils and M1-like macrophages that were highly inflammatory, as indicated by expression of Il1{beta} and Il24. Pharmacological blockade of the IL-1 receptor associated kinases (IRAK1/4) alleviated eczema, epidermal and dermal thickening, and immune infiltration of the Ube2n mutant skin. Together, these findings highlight a key role of keratinocyte-UBE2N in maintenance of epidermal homeostasis and skin immunity and identify IRAK1/4 as potential therapeutic target for inflammatory skin disorders.

pathology↗

bioRxiv2023

Tau P301L mutation promotes core 4R tauopathy fibril fold through near-surface water structuring and conformational rearrangement

Tau forms toxic fibrillar aggregates in a family of neurodegenerative diseases known as tauopathies. The faithful replication of tauopathy-specific fibril structures is a critical gap for developing diagnostic and therapeutic tools. This study debuts a strategy of identifying a critical segment of tau that forms a folding motif that is characteristic of a family of tauopathies and isolating it as a standalone peptide that form seeding-competent fibrils. The 19-residue jR2R3 peptide (295-313) spanning the R2/R3 splice junction of tau, in the presence of P301L, forms seeding-competent amyloid fibrils. This tau fragment contains the hydrophobic VQIVYK hexapeptide that is part of the core of every pathological tau fibril structure solved to-date and an intramolecular counter-strand that stabilizes the strand-loop-strand (SLS) motif observed in 4R tauopathy fibrils. This study shows that P301L exhibits a duality of effects: it lowers the barrier for the peptide to adopt aggregation-prone conformations and enhances the local structuring of water around the mutation site that facilitates site-specific dewetting and in-register stacking of tau to form cross {beta}-sheets. We solve a 3 [A] cryo-EM structure of jR2R3-P301L fibrils with a pseudo 21 screw symmetry in which each half of the fibrils cross-section contains two jR2R3-P301L peptides. One chain adopts a SLS fold found in 4R tauopathies that is stabilized by a second chain wrapping around the SLS fold, reminiscent of the 3-fold and 4-fold structures observed in 4R tauopathies. These jR2R3-P301L fibrils are able to template full length tau in a prion-like fashion. Significance StatementThis study presents a first step towards designing a tauopathy specific aggregation pathway by engineering a minimal tau prion building block, jR2R3, that can template and propagate distinct disease folds. We present the discovery that P301L--among the widest used mutations in cell and animal models of Alzheimers Disease--destabilizes an aggregation-prohibiting internal hairpin and enhances the local surface water structure that serves as an entropic hotspot to exert a hyper-localized effect in jR2R3. Our study suggests that P301L may be a more suitable mutation to include in modeling 4R tauopathies than for modelling Alzheimers Disease, and that mutations are powerful tools for the purpose of designing of tau prion models as therapeutic tools.

biophysics↗

bioRxiv2023

High-resolution vasomotion analysis reveals novel venous physiological features and progressive modulation of cerebral vascular networks by stroke

Spontaneous cerebral vasomotion, characterized by [~]0.1 Hz rhythmic contractility, is crucial for brain homeostasis. However, our understanding of vasomotion is limited due to a lack of high-precision analytical methods to determine single vasomotion events at basal levels. Here, we developed a novel strategy that integrates a baseline smoothing algorithm, allowing precise measurements of vasodynamics and concomitant Ca2+ dynamics in mouse cerebrovasculature imaged by two-photon microscopy. We identified several previously unrecognized vasomotion properties under different physiological and pathological conditions, especially in ischemic stroke, which is a highly harmful brain disease that results from vessel occlusion. First, the dynamic characteristics between SMCs Ca2+ and corresponding arteriolar vasomotion are interplayed. Second, compared to previous diameter-based estimations, our radius-based measurements reveal nonisotropic vascular movements, enabling a more precise determination of the latency between smooth muscle cell (SMC) Ca2+ activity and vasocontraction. Third, we characterized single vasomotion event kinetics at scales of less than 4 seconds. Finally, following pathological vasoconstrictions induced by ischemic stroke, vasoactive arterioles transitioned to an inert state and persisted despite recanalization. In summary, we developed a highly accurate technique for analyzing spontaneous vasomotion, and we suggest a potential strategy to reduce stroke damage by promoting vasomotion recovery.

neuroscience↗

bioRxiv2023

A high-cholesterol zebrafish diet promotes hypercholesterolemia and fasting-associated liver triglycerides accumulation

Zebrafish are an ideal model organism to study lipid metabolism and to elucidate the molecular underpinnings of human lipid-associated disorders. In this study, we provide an improved protocol to assay the impact of a high-cholesterol diet (HCD) on zebrafish lipid deposition and lipoprotein regulation. Fish fed HCD developed hypercholesterolemia as indicated by significantly elevated ApoB-containing lipoproteins (ApoB-LP) and increased plasma levels of cholesterol and cholesterol esters. Feeding of the HCD to larvae (8 days followed by a 1 day fast) and adult female fish (2 weeks, followed by 3 days of fasting) was also associated with a fatty liver phenotype that presented as severe hepatic steatosis. The HCD feeding paradigm doubled the levels of liver triacylglycerol (TG), which was striking because our HCD was only supplemented with cholesterol. The accumulated liver TG was unlikely due to increased de novo lipogenesis or inhibited {beta}-oxidation since no differentially expressed genes in these pathways were found between the livers of fish fed the HCD versus control diets. However, fasted HCD fish had significantly increased lipogenesis gene fasn in adipose tissue and higher free fatty acids (FFA) in plasma. This suggested that elevated dietary cholesterol resulted in lipid accumulation in adipocytes, which supplied more FFA during fasting, promoting hepatic steatosis. In conclusion, our HCD zebrafish protocol represents an effective and reliable approach for studying the temporal characteristics of the physiological and biochemical responses to high levels of dietary cholesterol and provides insights into the mechanisms that may underlie fatty liver disease.

physiology↗

bioRxiv2023

The More Unclear the Hearing, the Faster the Feeling: Speech Comprehension Changes Subjective Speech Rate

Imagine being in a foreign country, surrounded by locals speaking a language entirely alien to you. Does their conversation seem to race like a machine gun? The perception of speech rate, as a facet of time perception, greatly impacts our daily lives. However, the factors influencing this perception remain poorly understood. Through a series of three experiments (with sample sizes of 31, 27, and 25 respectively), we discovered that speech comprehension significantly alters listeners perception of speech rate. As comprehensibility diminishes, listeners tend to overestimate the speech rate. Intriguingly, this effect is further influenced by the listeners language proficiency. Specifically, the better the listeners language proficiency, the more likely their perception of speech rate is influenced by the speech comprehension. Our study corroborates the notion that biases in time perception may be prevalent in our everyday communication. These subjective experiences could prompt listeners to modify their behavior or processing strategies.

molecular biology↗

bioRxiv2023

A conserved N-terminal motif of CUL3 mediates assembly and licenses E3 ligase activation of CRL3KLHL22

The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through E3-E3 dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3KLHL22 complex and reveal a conserved N-terminal motif in CUL3 that mediates the dimerization assembly and licenses the activation of CRL3KLHL22. Deletion of the CUL3 N-terminal motif leads to non-stochiometric assembly and impairs E3 activity of both CRL3KLHL22 and several other CRL3s. In addition, we found that the dynamics of dimeric E3-E3 superassembly generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate a CUL3 N-terminal motif-dependent E3-E3 superassembly mechanism and provide insights into the assembly and activation of CRL3s.

molecular biology↗

bioRxiv2023

BATTER: Accurate Prediction of Rho-dependent and Rho-independent Transcription Terminators in Metagenomes

Bacterial transcription termination is a critical yet underexplored mechanism of gene regulation in microbial ecosystems. Existing computational tools, however, primarily focus on predicting transcript 3 ends generated by Rho-independent terminators (RITs) in model species, leaving significant gaps in understanding those generated by Rho-dependent terminators (RDTs), especially in non-model species. To address these limitations, we developed BATTER (BActeria Transcript Three Prime End Recognizer), a comprehensive computational tool for bacterial transcript 3 termini prediction. BATTER builds on the observation that conserved stem-loop structures are frequently associated with 3 ends of primary transcripts generated by both RIT and RDT mechanisms across distantly related bacterial species. BATTER demonstrated its advantage compared to existing tools. It enabled a comprehensive analysis of 42,905 representative bacterial genomes, uncovering that stem-loop structures exhibit clade-specific properties with greater variations between species than between gene families. Notably, BATTER uncovered that certain Cyanobacteria lineages, despite lacking Rho homologs, harbor Rho utilization (RUT) site-like sequences near 3 ends, with preliminary experimental validation in E. coli suggesting their partial functionality in transcription termination. Additionally, BATTER systematically identified pervasive premature termination events in antimicrobial resistance (AMR) genes, highlighting their regulatory roles in translation protection and drug efflux. This study advances our understanding of transcription termination across diverse bacterial lineages and provides a robust computational approach for exploring transcription regulation in complex microbial ecosystems.

bioinformatics↗