Biology subjects

Lin, C.-L.

Publications and source records attributed to Lin, C.-L..

7 recordsLinked to original sources

bioRxiv2026

Structural and Functional Principles of Hcp-Mediated Antibacterial Toxin Delivery by the Type VI Secretion System

The type VI secretion system (T6SS) is a bacterial contractile nanomachine that translocates toxic effectors into neighboring cells, helping bacteria to survive and compete in polymicrobial environments. The core T6SS structural protein Hcp forms the inner tube and mediates the loading and delivery of diverse effectors. Yet, how Hcp selectively recruits structurally and functionally distinct effectors in the absence of recognizable secretion signals remains poorly understood. Here, using Pseudomonas aeruginosa H1-T6SS as a model, we combined competition-coupled deep mutational scanning with cryo-EM based structural analysis to determine how Hcp accommodates distinct effectors. The resulting mutational landscape distinguishes structurally constrained residues required for Hcp tube assembly from lumen-facing residues specialized for cargo engagement. Near-atomic-resolution structures of Hcp bound to Tse2 and Tse4, together with a high-confidence Hcp-Tse1 model, revealed that while effector-specific contacts exist, Hcp engages different cargos through largely shared, dispersed interfaces characterized by polar-biased and variation-tolerant interactions. Comparative structural analyses further showed that effector-interacting residues are not governed by conserved primary-sequence motifs, but by conserved lumen-facing geometry and permissive physicochemical properties across distantly related bacteria. Together, our findings support a model in which Hcp acts as a selective yet adaptable scaffold that accommodates diverse antibacterial effectors through conserved structural features and flexible interaction surfaces, providing a mechanistic framework for understanding cargo selection by the T6SS and explaining how a conserved secretion nanomachine can evolve to deliver diverse toxic payloads.

microbiology↗

bioRxiv2025

Splicing regulation and intron evolution in the short-intron ciliate model of endosymbiosis Paramecium bursaria

The integration of symbionts into host cells during endosymbiosis significantly alters gene expression and cell physiology. Though alternative splicing facilitates cellular adaptation through rapid modulation of gene expression and protein isoform diversity, its regulatory role during endosymbiosis remains poorly understood. Paramecium bursaria, which harbors hundreds of Chlorella variabilis algae within its cytoplasm, offers a powerful model to study splicing during endosymbiosis, especially given its exceptionally short introns (median [~]24 nt). Using time-course RNA sequencing of symbiotic and aposymbiotic cells, we found that splicing, especially of 5 proximal introns, enhances gene expression. Moreover, we identified 883 genes with differentially spliced introns, particularly enriched in transmembrane transporters essential for establishing nutrient exchange between a host cell and algal symbionts. Splicing regulation correlated with expression changes in conserved spliceosome components, implicating that these factors act as splicing enhancers or repressors during symbiosis. By exploring intron orthology across ciliates, we found that conserved introns exhibited more efficient splicing, characterized by lower GC content and uniform length, suggesting that intron evolution favors features that optimize expression. Our study reveals how splicing contributes to host adaptation during endosymbiosis and highlights the evolutionary dynamics of short introns in eukaryotes. Key pointsParamecium retains conserved core spliceosome genes but employs unique splicing regulation. Alternative splicing regulates metabolism during endosymbiosis, especially for transporter genes. Older introns evolve toward [~]24 nt length and low GC for efficient splicing and expression.

evolutionary biology↗

bioRxiv2024

Elevated levels of intracellular RNA lariats suppress the antiviral response

Recent studies report the genetic loss of the lariat debranching enzyme (DBR1) activity increases susceptibility to viral infection. Here, we show that more than 25% of human introns contain large hairpin structures created by the folding of two Alu elements inserted in opposite orientation. In wildtype cells, this large reservoir of endogenous dsRNA is efficiently degraded. In DBR1-null cells, lariats accumulate in the cytosol and dsRNA becomes enriched. We demonstrate how the chronic exposure to these lariats attenuates the dsRNA sensors, reducing the response of the MDA5, RIG-I, RNase L and PKR sensing pathways. We observe evidence for both attenuation and endogenous dsRNA in anti-viral response and viral evasion. Lariats are transiently elevated during infection (e.g. HSV-1, influenza, KSHV). The HSV-1 genome expresses multiple, stable lariats that may attenuate dsRNA sensors during latency. HIGHLIGHTSO_LIIntronic inverted repeat Alu elements constitute largest source of endogenous dsRNA. C_LIO_LIIn the absence of DBR1, lariats accumulate in the cytoplasm and form dsRNA. C_LIO_LIChronic exposure to endogenous dsRNA in a DBR1-depleted environment desensitizes the dsRNA sensing pathway. C_LIO_LIICP0 intron 1 of HSV-1 has a highly-structured stable lariat. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/627371v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@3698bborg.highwire.dtl.DTLVardef@654727org.highwire.dtl.DTLVardef@1229245org.highwire.dtl.DTLVardef@b10622_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Massively Parallel Polyribosome Profiling Reveals Translation Defects of Human Disease-Relevant UTR Mutations

The untranslated regions (UTRs) of mRNAs harbor regulatory elements influencing translation efficiency. Although 3.7% of disease-relevant human mutations occur in UTRs, their exact role in pathogenesis remains unclear. Through metagene analysis, we mapped pathogenic UTR mutations to regions near coding sequences, with a focus on the upstream open reading frame (uORF) initiation site. Subsequently, we utilized massively parallel poly(ribo)some profiling to compare the ribosome associations of 6,555 pairs of wildtype and mutant UTR fragments. We identified 46 UTR variants that altered polysome profiles, with enrichment in pathogenic mutations. Both univariate analysis and the elastic net regression model highlighted the significance of motifs of short repeated sequences, including SRSF2 binding sites, as mutation hotspots that lead to aberrant translation. Furthermore, these polysome-shifting mutations exhibited considerable impact on RNA secondary structures, particularly for upstream AUG-containing 5 UTRs. Integrating these features, our model achieved high accuracy (AUROC > 0.8) in predicting polysome-shifting mutations in the test dataset. Additionally, several lines of evidence indicate that changes in uORF usage underlie the translation deficiency arising from these mutations. Illustrating this, we demonstrate that a pathogenic mutation in the IRF6 5 UTR suppresses translation of the primary open reading frame by creating a uORF. Remarkably, site- directed ADAR editing of the mutant mRNA rescued this translation deficiency. Overall, our study provides insights into the molecular mechanisms of UTR mutations and their links to clinical impacts through translation defects. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/589132v2_ufig1.gif" ALT="Figure 1000"> View larger version (50K): org.highwire.dtl.DTLVardef@57217corg.highwire.dtl.DTLVardef@575410org.highwire.dtl.DTLVardef@15013bforg.highwire.dtl.DTLVardef@408840_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

Multiplexed Assays of Human Disease-relevant Mutations Reveal UTR Dinucleotide Composition as a Major Determinant of RNA Stability

UTRs contain crucial regulatory elements for RNA stability, translation and localization, so their integrity is indispensable for gene expression. Approximately 3.7% of genetic variants associated with diseases occur in UTRs, yet a comprehensive understanding of UTR variant functions remains limited due to inefficient experimental and computational assessment methods. To systematically evaluate the effects of UTR variants on RNA stability, we established a massively parallel reporter assay on 6,555 UTR variants reported in human disease databases. We examined the RNA degradation patterns mediated by the UTR library in two cell lines, and then applied LASSO regression to model the influential regulators of RNA stability. We found that UA dinucleotides and UA-rich motifs are the most prominent destabilizing element. Gain of UA dinucleotide outlined mutant UTRs with reduced stability. Studies on endogenous transcripts indicate that high UA-dinucleotide ratios in UTRs promote RNA degradation. Conversely, elevated GC content and protein binding on UA dinucleotides protect high-UA RNA from degradation. Further analysis reveals polarized roles of UA- dinucleotide-binding proteins in RNA protection and degradation. Furthermore, the UA- dinucleotide ratio of both UTRs is a common characteristic of genes in innate immune response pathways, implying a coordinated stability regulation through UTRs at the transcriptomic level. We also demonstrate that stability-altering UTRs are associated with changes in biobank-based health indices, underscoring the importance of precise UTR regulation for wellness. Our study highlights the importance of RNA stability regulation through UTR primary sequences, paving the way for further exploration of their implications in gene networks and precision medicine.

genetics↗

bioRxiv2022

Chemoenzymatic Synthesis of Genetically-Encoded Multivalent Liquid N-glycan Arrays

A hallmark of cellular glycosylation is its chemical complexity and heterogeneity, which can be challenging to capture synthetically. Using chemoenzymatic synthesis on M13 phage, we produce a genetically-encoded liquid glycan array (LiGA) of biantennary complex type N-glycans. Ligation of azido-functionalized sialylglycosyl-asparagine derived from egg yolk to phage functionalized with 50-1000 copies of dibenzocyclooctyne produced divergent intermediate that can be trimmed by glycosidases and extended by glycosyltransferases to yield a library of phages with different N-glycans. Post-reaction analysis by MALDI-TOF MS provided a rigorous approach to confirm N-glycan structure and density, both of which were encoded in the bacteriophage DNA. The binding of this N-glycan LiGA by ten lectins, including CD22 or DC-SIGN expressed on live cells, uncovered an optimal structure/density combination for recognition. Injection of the LiGA into mice identified glycoconjugates with structures and avidity necessary for enrichment in specific organs. This work provides an unprecedented quantitative evaluation of the interaction of complex N-glycans with GBPs in vitro and in vivo.

biochemistry↗

bioRxiv2020

Single-cell chromatin profiling reveals demethylation-dependent metabolic vulnerabilities of breast cancer epigenome

Metabolic reprogramming in cancer cells not only sustains bioenergetic and biosynthetic needs but also influences transcriptional programs, yet how chromatin regulatory networks are rewired by altered metabolism remains elusive. Here we investigate genome-scale chromatin remodeling in response to 2-hydroxyglutarate (2HG) oncometabolite using single-cell assay for transposase accessible chromatin with sequencing (scATAC-seq). We find that 2HG enantiomers differentially disrupt exquisite control of epigenome integrity by limiting -ketoglutarate (KG)-dependent DNA and histone demethylation, while enhanced cell-to-cell variability in the chromatin regulatory landscape is most evident upon exposure to L2HG enantiomer. Despite the highly heterogeneous responses, 2HG largely recapitulates two prominent hallmarks of the breast cancer epigenome, i.e., global loss of 5-hydroxymethylcytosine (5hmC) and promoter hypermethylation, particularly at tumor suppressor genes involved in DNA damage repair and checkpoint control. Single-cell mass cytometry further demonstrates downregulation of BRCA1, MSH2 and MLH1 in 2HG-responsive subpopulations, along with acute reversal of chromatin remodeling upon withdrawal. Collectively, this study provides a molecular basis for metabolism-epigenome coupling and identifies metabolic vulnerabilities imposed on the breast cancer epigenome.

cancer biology↗