Biology subjects

Wen, L.

Publications and source records attributed to Wen, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Affinity Maturation Enables Dual Targeting of Cancer-Associated MUC1-Tn/STn and Drives Potent Tumor Control

A major challenge in solid tumor immunotherapy is identifying tumor-restricted antigens while overcoming intratumoral antigen heterogeneity. The truncated O-glycans Tn (GalNAc-O-Ser/Thr) and STn (Neu5Ac2-6GalNAc-O-Ser/Thr) are aberrantly displayed on the transmembrane glycoprotein MUC1 across a broad spectrum of epithelial malignancies, making MUC1-Tn/STn an attractive shared tumor-associated antigen class. However, heterogeneous intratumoral Tn/STn expression renders single-glycoform-specific antibodies vulnerable to antigen-driven escape. To address this challenge, we used structure-guided directed evolution and yeast surface display to affinity-mature the anti-MUC1-Tn antibody 5E5, generating variants with high-affinity dual recognition of MUC1-Tn and MUC1-STn. Co-crystal structures revealed that heavy-chain CDR loops cooperatively engage the GalNAc moiety primarily through hydrogen-bonding networks, in which HCDR3 residue 98 fine-tunes glycoform preference: Tyr98 favors MUC1-Tn recognition, whereas His98 enhances MUC1-STn engagement, while both support high-affinity recognition of both glycoforms. When reformatted as bispecific T-cell engagers, the affinity-matured variants mediated potent and durable cytolytic activity against cancer cells with heterogeneous glycan profiles, suppressed mixed Tn/STn tumors in vivo where the parental antibody failed, and showed enhanced activity in patient-derived tumor-like clusters. Together, these findings establish functional glycoform coverage as a design principle for antibody engineering, whereby a single paratope is optimized to recognize a defined spectrum of tumor-associated glycoforms while preserving antigen specificity, providing a strategy to overcome glycan heterogeneity in solid tumor immunotherapy.

cancer biology↗

bioRxiv2026

CD59 Promotes SNARE Complex Assembly via Interaction with the Proline-Rich N-Terminal Domain of VAMP2

ObjectiveThe complement regulatory protein CD59 has been shown to promote SNARE complex assembly, yet its interaction with vesicle-associated membrane protein 2 (VAMP2) remains poorly characterized. This study aims to identify the key domain in VAMP2 that mediates the CD59 interaction and to evaluate whether CD59 point mutations affect SNARE complex assembly. MethodsThe interaction between CD59 and VAMP2 was examined by immunofluorescence confocal microscopy and co-immunoprecipitation (co-IP). The effect of CD59 on SNARE complex assembly was assessed by co-expressing CD59 with the three core SNARE proteins (syntaxin-1, SNAP-25, and VAMP2) and detecting complex formation by western blotting. Four CD59 single-point mutants were generated and evaluated in SNARE assembly assays. AlphaFold3 was employed to predict the interaction between CD59 and individual VAMP2 domains (confidence threshold: ipTM + pTM [≥] 0.75). Truncated VAMP2 constructs were further characterized by molecular dynamics simulations and co-IP. Results(1) CD59 directly bound VAMP2 and promoted SNARE complex assembly without altering individual SNARE protein levels. (2) All four CD59 single-point mutants retained the ability to promote SNARE assembly at a level comparable to wild-type CD59, despite showing differential effects on binding stability in molecular dynamics simulations. (3) The proline-rich (P-rich) N-terminal domain of VAMP2 was identified as the key binding interface; its deletion abolished the CD59 interaction, whereas deletion of the SNARE motif did not. ConclusionCD59 promotes SNARE complex assembly through interaction with the P-rich N-terminal domain of VAMP2. The examined point mutations do not impair this function, suggesting that these sites may tolerate substitutions or that redundant contact residues maintain the interaction.

neuroscience↗

bioRxiv2026

Development of a BM7G((TKO/hCD46/hCD55/hTHBD/hEPCR) Donor Pig with Endogenous Promoter-Driven Transgenes for Xenotransplantation

Xenotransplantation offers a potential solution to the organ shortage crisis. Multi-gene modification of pigs, such as knockout of three carbohydrate antigen-related genes and expression of immunoprotective proteins, can significantly improve xenograft survival. However, existing strategies face challenges: transposon-based transgenesis may lead to unstable expression, while exogenous promoters used in site-specific integration are susceptible to epigenetic silencing, hindering long-term stable expression. Therefore, developing a donor pig model capable of sustained multi-gene expression is critical. To address this, CRISPR-Cas9 was used to knockout three major glycan antigen genes to eliminate hyperacute rejection. Subsequently, four human protective genes were site-specifically integrated into the porcine Rosa26 safe-harbor locus, with expression driven by the endogenous Rosa26 promoter and the THBD core promoter for long-term stable and tissue-specific expression, and the selection marker was removed using Cre/loxP. Results showed complete absence of the three glycan antigens in BM7G pigs, while the four protective proteins were stably expressed in vascular endothelial cells and major organs. Among them, hCD55 and hCD46 were widely expressed, while hTHBD and hEPCR showed vascular-specific expression. In-vitro assays confirmed that BM7G porcine endothelial cells significantly reduced human antibody binding, effectively inhibited complement-dependent cytotoxicity, and decreased thrombin-antithrombin complex formation. In conclusion, by combining xenoantigen knockout with endogenous promoter-driven expression of multiple human protective genes, a seven-gene modified pig model with low immunogenicity and synergistic protective function was successfully constructed, providing an important donor resource for xenotransplantation preclinical research.

immunology↗

bioRxiv2026

Co-evolution of Oncogenic KRAS Signaling and LILRBhigh Macrophages Drives Pancreatic Cancer Recurrence

Pancreatic ductal adenocarcinoma (PDAC) frequently recurs after surgical resection, indicating that residual disease is sustained by coordinated tumor-microenvironment interactions. To define the biological basis of recurrence, we leverage large-scale clinical data from 2,710 patients, deeply characterized multi-omics profiling (whole-exome, bulk RNA, and single-nucleus sequencing) of 36 matched primary and locally recurrent PDACs, an in-house multiplex spatial imaging cohort of 190 patients, and extensive public datasets. Recurrent tumors were characterized by increased KRAS mutant allele dosage and reinforced KRAS signaling, accompanied by expansion of basal-like malignant cell states. In parallel, we identified an immunosuppressive macrophage population marked by high LILRB expression that spatially co-localized with KRAS-activated tumor cells. Functional studies showed that LILRB4+ macrophages enhanced tumor cell plasticity and progression, whereas inhibition of macrophage LILRB4 suppressed these phenotypes. Notably, a first-in-class human anti-LILRB4 antibody reduced macrophage-driven tumor traits, and dual targeting of KRAS signaling and LILRB4 achieved superior tumor control in macrophage-containing mouse models. These findings reveal a co-evolved tumor-immune niche underlying PDAC recurrence and nominate the KRAS-LILRB4 axis as a therapeutic vulnerability.

cancer biology↗

bioRxiv2026

Porcine extended pluripotent stem cell-derived somite-like mesoderm cells with Dox-driven PAX7 are robust seed cell resource for facilitating production of cultured meat

Cultured meat technology, with its significant advantages of shortening meat production cycles, reducing natural resource consumption, minimizing the risk of zoonotic disease transmission, and enabling precise control over nutritional composition and texture, offers a novel alternative source for human meat consumption. One of the major challenges to produce cultured meat in large scale is how to establish high.quality seed cells, which should have long term proliferative capacities and are able to differentiate into muscles efficienuy with simple procedures. Here, we first established an engineered porcine expanded potential stem cells (Tet-On-PAX7 EPSCs) containing Tet-On regulated PAX7 gene. Then the Tet-On-PAX7 EPSCs were induced to somite-liKe mesodermal cells. These somite-liKe mesodermal cells can be expanded over 1025-fold even after 40 passages in-vitro culture while retaining strong myogenic potential. The somite-like mesodermal cells treated with DOX for one day would differentiate into muscle stem cells (Muses), and the later were able to differentiate into muscles with an efficiency of up to 90% within just 7 days in 11-FSDeDa without Dox. Moreover, when somite-liKe mesodermal cells were seeded on patterned scaffolds, microcarrier scaffolds, or cultured in anchorage-independent suspension, they maintained high efficiency in muscle differentiation, confirming their potential to be used as seed cells for scaled cultured meat production.

developmental biology↗

bioRxiv2025

Exudate-Guided Janus Trilayer Bioelectronic Dressing for Multimodal Sensing and Therapy of Chronic Wounds

Chronic wound treatment remains challenging because conventional dressings and systemic therapies typically lack real-time adaptability to the dynamic changes in the wound microenvironment. Here, we report a breathable self-adhesive multifunctional wound dressing based on a gradient-wettable Janus trilayer fibrous membrane, which integrates real-time biochemical sensing, on-demand drug release, and low-voltage electrical stimulation therapy simultaneously. The dressing features a vertically integrated trilayer architecture with hierarchical partitioning, where the three vertically stacked layers integrate a multiplex electrochemical sensor array (glucose, lactate, pH), a silver-sulfadiazine drug reservoir, and stimulation electrodes. Specifically, a hydrophilic sensing layer, intermediate drug-storage layer, and hydrophobic adhesivelayer are assembled to achieve spatially resolved functionalities. The membranes gradient wettability and asymmetric porous structure enable directional exudate transport, thereby triggering stepwise activation of sensing, drug release, and electrical stimulation in a spatiotemporally controlled manner. The dressing is thin, breathable, stretchable, self-adhesive and biocompatible, enabling conformal contact and stable signal acquisition on deformable skin without tapes. This design ensures continuous bio-signal monitoring while maintaining a favorable wound healing microenvironment. Preclinical tests demonstrate its effectiveness in promoting wound closure, reducing infection, and providing continuous wound status monitoring. This integrated system offers a promising approach for next-generation wound care and regenerative medicine.

bioengineering↗

bioRxiv2025

Integrative spatial multi-omics reveals prognostic tumor niches in female genital tumors

Female genital tumors (FGTs), including ovarian, endometrial, and cervical cancers, pose a major global health challenge, yet their spatial and molecular determinants of progression and therapy resistance remain poorly understood. Here we present a large-scale integrative spatial and multi-omics atlas of FGTs, combining exome, transcriptome, methylome, and miRNA sequencing with high-resolution Visium HD spatial transcriptomics, profiling over 62 million cells from 100 tumors. We show that HPV-driven suppression of TGF{beta} signaling in cervical cancer disrupts myofibroblast (myCAF) formation and reshapes immune infiltration, while serous tumors exhibit extensive oncogene amplifications that activate MYC and PI3K pathways, linking chromosomal instability (CIN) to proliferative, immune-cold phenotypes. In high-grade serous ovarian cancer, we identify a previously unrecognized malignant domain, the Proliferating Core Desert (PCD) niche, defined by CIN-high epithelial enrichment, immune exclusion, stromal protection, and chemotherapy resistance, maintained through crosstalk with macrophages and fibroblasts. Finally, we develop POWER, a pathology foundation model that predicts PCD niche abundance from routine H&E slides, enabling robust stratification of prognosis and anti-angiogenic therapy response.

genomics↗

bioRxiv2025

Efficient Identification of Short Tandem Repeats via Context-Aware Motif Discovery and Ultra-Fast Sequence Alignment

Tandem repeats (TRs) are highly polymorphic genomic elements, associated with diverse molecular traits and implicated in numerous human diseases. However, large-scale analysis of TRs has been limited by computational challenges, including motif recognition, detection in complex regions, and excessive computational cost. Here we present FastSTR, a computationally efficient tool for precise detection and characterization of TRs. FastSTR integrates a context-aware N-gram motif model with a segmented global alignment algorithm to enable accurate motif identification and boundary definition, even for repeat units up to 8 bp. Across 13 species, FastSTR achieved >90% recall and 99% precision, running several times faster than existing methods white outperforming them in both sensitivity and accuracy. Applied to the human genome, FastSTR uncovered previously unannotated HSATII elements, resolved population-specific TR demonstrate, and identified recurrent STR alterations in lung cancer. These results demonstrate FastSTR as a versatile framework for TR annotation and discovery, advancing studies of genome evolution, genetic diversity, and disease.

bioinformatics↗

bioRxiv2025

RESM: Capturing sequence and structure encoding of RNAs by mapped transfer learning from ESM (evolutionary scale modeling) protein language model

RNA sequences exhibit lower evolutionary conservation than proteins due to their informationally constrained four-letter alphabet, compared to the 20-letter code of proteins. More limited information makes unsupervised learning of structural and functional evolutionary patterns more challenging from single RNA sequences. We overcame this limitation by mapping RNA sequences to pseudo-protein sequences to allow effective transfer training from a protein language model (protein Evolution-Scale Model 2, protESM-2). The resulting RNA ESM (RESM) outperforms 12 existing RNA language models in zero-shot prediction, not only in sequence classification but also in RNA secondary structure and RNA-RNA interaction prediction. Further supervised fine-tuning demonstrates RESMs generalizability and superior performance over the existing models compared across multiple downstream tasks, including mRNA ribosome loading efficiency and gene expression prediction, despite RESM being trained exclusively on noncoding RNAs. Moreover, RESM can generalize to unseen sequences beyond its 1,024-nucleotide training limit, achieving 81.3% improvement over state-of-the-art methods in supervised secondary structure prediction for RNAs up to 4,000 nucleotides, limited only by the available GPU memory, while providing >1000-fold speedup compared to MSA-based approaches. RESM provides a robust foundation for deciphering RNA sequence-structure-function relationships, with broad implications for RNA biology.

bioinformatics↗

bioRxiv2025

Nanopore-Based Glycan Sequencing via Fragmentation-Reassembly Strategy

Glycan sequencing remains a major analytical challenge due to the high structural complexity, extensive branching, and isomeric diversity of glycans. Nanopore sensing has emerged as a promising single-molecule platform for glycan analysis, but existing strategies--such as hydrolysis and strand sequencing, face significant limitations when applied to highly branched or non-linear glycans. Here we report the first experimental realization of glycan assembly sequencing, a nanopore-based strategy that reconstructs full glycan structures from individually analyzed fragments. Using a biantennary complex-type N-glycan as a model, we performed controlled enzymatic digestion to generate structurally defined oligosaccharide fragments. An engineered -HL nanopore was developed for enhanced glycan recognition, enabling the construction of a multidimensional electrical fingerprinting library. Fragment signals from unknown samples were matched to this reference dataset, and full-sequence reconstruction was achieved via fragment reassembly based on set-theoretic integration of structural candidates. This study establishes glycan assembly sequencing as a viable and modular approach to resolve complex glycoforms using nanopore technologies. The method expands the accessible sequence space beyond what is achievable with conventional strategies and provides a foundation for scalable, high-resolution glycomic analysis.

bioengineering↗

bioRxiv2025

RNA localization to nuclear speckles follows splicing logic

Nuclear speckles are membraneless organelles implicated in multiple RNA processing steps. In this work, we systematically characterize the sequence logic determining RNA localization to nuclear speckles. We find extensive similarities between the speckle localization code and the RNA splicing code, even for transcripts that do not undergo splicing. Specifically, speckle localization is enhanced by the presence of unspliced exon-like or intron-like sequence features. We demonstrate that interactions required for early splicesomal complex assembly contribute to speckle localization. We also show that speckle localization of isolated endogenous exons is reduced by disease-associated single nucleotide variants. Finally, we find that speckle localization strongly correlates with splicing kinetics of splicing-competent constructs and is tightly linked to the decision between exon inclusion and skipping. Together, these results suggest a model in which RNA speckle localization is associated with the formation of the early spliceosomal complex and enhances the efficiency of splicing reactions. HighlightsO_LISequences containing hallmarks of pre-mRNA dictate speckle localization C_LIO_LIRNA speckle localization is coupled to early spliceosome assembly C_LIO_LIDisease-associated single nucleotide variants reduce localization of isolated exons C_LIO_LIRNA speckle localization strongly correlates with splicing kinetics C_LI O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/656493v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1efbb02org.highwire.dtl.DTLVardef@1deb7aaorg.highwire.dtl.DTLVardef@ec1ec2org.highwire.dtl.DTLVardef@1d78b72_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

biochemistry↗

bioRxiv2024

Motor protein-assisted glycan translocation and sequencing using nanopores

The glycan sequencing remains a significant bottleneck in glycoscience. While nanopore platforms have achieved substantial progress in single-molecule nucleic acid sequencing, their application to glycan sequencing has faced considerable challenges, with limited advancements to date. In this study, we propose a novel strategy for controlling glycan translocation through the MspA nanopore as an initial step toward glycan sequencing. By conjugating the target glycan with a helicase-controlled single-stranded DNA, we successfully achieved sequencing reads of up to eleven glycans. For the first time, we isolated glycan-associated electrical signals, enabling the translocation, stretching, and controlled speed of neutral glycans through the nanopore. This method provides a platform for obtaining glycan read lengths and identifying different glycan modifications, demonstrating the capability to resolve monosaccharide composition and glycosidic linkages. To further improve resolution, we propose engineered M2-MspA to reduce the pore constriction size and enhance precision by minimizing the random thermal motion of the translocating glycan. These modifications are expected to increase the sequencing accuracy and reliability. This work represents the first proof-of-concept demonstration of glycan chain nanopore sequencing and lays a promising foundation for the development of single-molecule glycan fingerprinting and sequencing technologies. We anticipate that this approach will significantly advance the development and commercialization of nanopore-based glycan sequencing techniques.

bioengineering↗

bioRxiv2024

Combining Glycosidases and Nanopore Technology for Glycan Sequencing

The efficient characterization of glycan structures remains a critical challenge. Direct glycan sequencing technologies with improved sensitivity and throughput are still needed. Here, we propose a glycan sequencing strategy based on glycosidase -assisted nanopore sensing. We used engineered nanopore -hemolysin (M113R/T115A), which provided high-resolution discrimination of monosaccharide volume differences and sensitivity to variations in glycan chain length. By utilizing the specificity of glycosidases, we sequentially hydrolyzed the terminal residues of the glycan chains and detected the characteristic shifts in electrical signals generated by the translocation of hydrolysis products. The accuracy of recognition of hydrolysis fragments achieved over 90 % using machine learning. This allowed us to efficiently and conveniently determine the sequence of consecutive monosaccharide units in the glycan chains. Based on this principle, we conducted a proof-of-concept demonstration on actual samples and verified the accuracy via HPLC-MS. We achieved the sequencing of consecutive units in glycan chains, confirming the theoretical feasibility of glycosidase -assisted nanopore glycan sequencing. With future optimization, the development of arrayed nanopore glycan sequencing technology based on hydrolysis strategy will provide an innovative approach for rapid decoding of glycans, which could promote the progress of glycomics research and glycobiology.

biochemistry↗

bioRxiv2024

Strong segregation promotes self-destructive cooperation

Self-destructive cooperator (SDC), where individuals sacrifice themselves for others, poses significant personal costs yet is widespread in nature. Traditional group selection theory struggles to explain its persistence, as the extreme cost of self-destruction typically exceeds the benefit to these individuals. Here, we predict that this behavior can endure in structured environments with strong segregation, where populations are divided into homogenous groups originating from one or two founding members. In such contexts, the benefits derived from SDC are primarily confined within the homogenous SDC groups, thus preserving the value of this altruistic sacrifice and ensuring its maintenance. To validate our hypothesis, we employ a synthetic system with engineered bacteria to mimic both SDC and non-sacrificing cheaters. We then conduct automated experiments facilitated by biofoundry technology to monitor and operate the subgroups with diverse growth behaviors stemming from a restricted number of initial cells because of strong segregation. Eventually, we demonstrate that SDC indeed maintains under strong segregation, and high stress benefits SDC because it reduces the benefits received by cheaters in heterogenous subgroups. This study extends the group selection theory to encompass even the most extreme manifestations of altruism and highlights the potential of automation in evolutionary research.

microbiology↗

bioRxiv2024

Hepatic aldose reductase drives a Warburg effect-like metabolic reprogramming to promote insulin resistance, fatty liver and obesity

Background & AimsEmerging evidence suggest that abnormal activation of aldose reductase/the polyol pathway (Ar/PP) is associated with the pathogenesis or development of fatty liver, obesity and metabolic syndrome. However, the underlying mechanisms were unclear. In this study, we investigated the metabolic reprogramming following activation or inhibition of Ar, the first and the rate-limiting enzyme of PP. We also investigated the long-term effects of Ar/PP-mediated metabolic shift in vivo. MethodsMetabolomic analyses were performed with the AB-SCIE QTRAP-5500 LC-MS/MS System for control mouse hepatocytes and hepatocytes stably overexpressing Ar and exposed to 25 mM glucose. Glycolysis stress tests and mitochondrial stress tests were performed using the Seahorse Bioscience Extracellular Flux Analyzer. The in vivo long-term effects of Ar overexpression and inhibition were evaluated in either transgenic mice overexpressing AR or a line of double transgenic mice carrying an Ar-null mutation and an Agouti-yellow Ay mutation. ResultsAbnormal activation of Ar in hepatocytes was found to trigger and drive a drastic Warburg effect-like metabolic reprogramming, induce de novo lipogenesis, and alter insulin and AMP-activated protein kinase signaling. In glucose-fed AR-overexpressing transgenic mice, AR activation causes systemic alterations in physiological parameters and the development of overt phenotypes of insulin resistance, fatty liver, obesity. In the yellow obese syndrome mice, Ar deficiency greatly improves Agouti Ay mutation-induced abnormalities. ConclusionsCollectively, the results highlight the important contribution of Ar/PP or the putative pseudo-glycolysis in hepatic metabolic homeostasis and the development of metabolic diseases. These findings have profound implications for the development of therapeutic strategies or drugs against metabolic diseases and cancer. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/614395v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@de91ddorg.highwire.dtl.DTLVardef@3a801corg.highwire.dtl.DTLVardef@b0cf35org.highwire.dtl.DTLVardef@1f3c1c1_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIActivation of aldose reductase triggers and drives a Warburg effect-like metabolic eprogramming in hepatocytes. C_LIO_LILiver-specific activation of the polyol pathway leads to insulin resistance, fatty liver and obesity. C_LIO_LIInhibition of aldose reductase greatly ameliorates Agouti Ay-induced metabolic abnormalities. C_LI Impact and implicationsThis study reveals that abnormal activation of Ar/PP will trigger and drive a Warburg effect-like metabolic reprogramming in hepatocytes. In normal subjects, Ar/PP mediated metabolic reprogramming tends to promote lipogenesis, insulin resistance, fatty liver and obesity. In cancer cells, Ar/PP mediated metabolic reprogramming will be part of the Warburg effect to support the growth and proliferation of cancer cells. These findings imply that Ar and its down-stream metabolic enzymes are important therapeutic targets for cancers and metabolic diseases.

biochemistry↗

bioRxiv2024

Decoding protein glycosylation by an integrative mass spectrometry-based de novo sequencing strategy

Glycoproteins, representing over 50% of human proteins and most biopharmaceuticals, are crucial for regulating various biological processes. The complexity of multiple glycosylation sites, often leading to incomplete sequence coverage and ambiguous glycan modification profiles. Here, we developed an integrative mass spectrometry-based approach for decoding unknown glycoproteins, which is featured with the combination of deglycosylation-mediated de novo sequencing with glycosylation site characterization. We utilized enzymatic deglycosylation of N-/ O-glycan to achieve comprehensive sequence coverage. Additionally, EThcD fragmentation enables the identification of high-quality long peptides, facilitating precise protein assembly. We subsequently applied this method to de novo sequencing of the highly glycosylated therapeutic fusion protein Etanercept (Enbrel(R)). We also sequenced three new tumor necrosis factor receptor (TNFR): Fc-fusion biologics with largely unknown sequences, unveiling subtle distinctions in the primary sequences. Furthermore, we characterized N- and O-glycosylation modifications of these proteins at subunit, glycopeptide, and glycan levels. This strategy bridges the gap between the de novo sequencing and glycosylation modification, providing complete information of the primary structure and glycosylation modifications for glycoproteins. Notably, our method could be a robust solution for accurate sequencing of the glycoproteins and has practical value not only in basic research but also in the biopharmaceutical industry.

biochemistry↗

bioRxiv2024

Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

Nuclear speckles, a type of membraneless nuclear organelle in higher eukaryotic cells, play a vital role in gene expression regulation. Using the reverse transcription-based RNA-binding protein binding sites sequencing (ARTR-seq) method, we study human transcripts associated with nuclear speckles. We identify three gene groups whose transcripts demonstrate different speckle localization properties and dynamics: stably enriched in nuclear speckles, transiently enriched in speckles at the pre-mRNA stage, and not enriched in speckles. Specifically, we find that stably-enriched transcripts contain inefficiently spliced introns. We show that nuclear speckles specifically facilitate splicing of speckle-enriched transcripts. We further reveal RNA sequence features contributing to transcript speckle localization, underscoring a tight interplay between genome organization, RNA cis-elements, and transcript speckle enrichment, and connecting transcript speckle localization with splicing efficiency. Finally, we show that speckles can act as hubs for the regulated retention of introns during cellular stress. Collectively, our data highlight a role of nuclear speckles in both co- and post-transcriptional splicing regulation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/581881v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1a55b13org.highwire.dtl.DTLVardef@fe50baorg.highwire.dtl.DTLVardef@6610fborg.highwire.dtl.DTLVardef@e4df5b_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

cell biology↗

bioRxiv2023

Discrimination of Isomeric Glycans via High-sensitive Engineered Nanopore

Glycan isomers are the basis for forming the spatial structure of glycans, which poses great challenges to glycan structural analysis and hinders the study of the relationship between structure and function. An economical and convenient analytical method with high resolution is still in demand. Here, we designed an engineered -hemolysin nanopore with double-mutants (-HL M113R/T115A) to achieve sensing of glycans with different glycosidic linkages. By extracting three parameters to depict a 3D scatter plot, four glycans with different degrees of polymerization and glycosidic bonds could be distinguished. Through molecular dynamics (MD), we further elucidated the motion trajectories of two different glycosidic bonds in the pore region. In addition, the sensing ability was also applied in the direct identification of glycans in mixture systems. The obtained nanopore data permitted us to achieve a separation accuracy of over 90% independent of the assistance of machine learning algorithms. Designing biological nanopores with high discrimination and sensitivity for glycans with different lineages paves the way for nanopore glycan profiling and potentially sequencing.

bioengineering↗