Biology subjects

TAN, L.

Publications and source records attributed to TAN, L..

3 recordsLinked to original sources

bioRxiv2025

Nanopore direct-RNA sequencing reveals TGEV epitranscriptomic and transcriptomic landscapes modulated by gene 7

Viral non-structural proteins have gained increasing attention for their roles in regulating host-virus interplay and reported to act as a key mediator of host and virus RNA modification dynamics. Transmissible gastroenteritis virus (TGEV) gene 7 has been implicated in virulence, but its other molecular functions remain unclear. Here, we compared wild-type TGEV (TGEV-wt) with a recombinant strain lacking gene 7 (TGEV-{Delta}7) in swine testis cells using Oxford Nanopore direct RNA sequencing. High-coverage datasets enabled simultaneous profiling of the full-length transcriptome, N6-methyladenosine (m6A) modifications, and polyA tail length. Deletion of TGEV gene 7 halved viral RNA replication yet increased m6A modification by [~]32 % across the viral genome, and elevated host m6A levels by [~]17 %, accompanied by reciprocal shifts in the m6A regulators FTO (eraser) and RBM15 (writer). Despite bulk transcriptome changes were comparable between strains, gene 7 deletion introduced additional DEGs beyond WT infection, showing stronger enrichment of antiviral and chemokine pathways, indicating heightened innate immunity. PolyA analysis uncovered the polyA features of TGEV gRNA and sgRNAs, and revealed a gene 7 dependent extension of viral by 7 nt, but not host polyA tails. These findings highlight RNA-modification machinery as a potential target for coronavirus control and provide a framework for vaccine strategies exploiting gene 7 attenuation.

microbiology↗

bioRxiv2025

Enhanced Detection of RNA Modifications in Escherichia coli Utilizing Nanopore RNA004 Technology

RNA modifications are critical regulators of diverse cellular processes, yet their roles in prokaryotic mRNAs remain poorly understood. Recent advances in Oxford Nanopore sequencing--especially the RNA004 kit--have enabled higher yields, reduced signal-to-noise ratio, and improved read accuracy, making them promising tools for investigating bacterial epitranscriptomes. Here, we presented a comprehensive walkthrough for Escherichia coli RNA modification analysis based on RNA004. Using both native (WT) and in vitro-transcribed (IVT) RNA samples, we first evaluated the Dorado modification detection models ({Psi}, mA, mC, and A-to-I). While each model successfully identified known rRNA modification sites, it also generated many false positives, emphasizing the need for careful data interpretation. To address these limitations, we introduced nanoSundial (https://github.com/lrslab/nanoSundial), a new comparative method that leveraged raw current features from WT and IVT samples to detect multiple types of RNA modifications in prokaryotes. We optimized nanoSundial on well-studied rRNA sites and validated its effectiveness with tRNA modifications. Through technical and biological replicate analyses, nanoSundial demonstrated reproducibility exceeding 95% in tRNA, rRNA, and ncRNA regions, albeit with lower reproducibility ([~]61%) in mRNA. We further found enrichment of mRNA modifications at the start or end of coding sequences. In total, 190 stably modified CDS regions were identified in E. coli, many of which cluster near the end of highly expressed transcriptional units (TUs) in each operon. Overall, this study highlighted the strengths and limitations of current nanopore-based modification detection methods on bacterial RNA, introduced a robust new comparative tool, and elucidated previously uncharacterized mRNA modification landscapes. Our findings open new avenues for understanding the functional impacts of bacterial RNA modifications and advancing epitranscriptomic research in prokaryotes.

bioinformatics↗

bioRxiv2021

Heparan Sulfate-dependent RAGE oligomerization is indispensable for pathophysiological functions of RAGE

RAGE, a druggable inflammatory receptor, is known to function as an oligomer but the exact oligomerization mechanism remains poorly understood. Previously we have shown that heparan sulfate (HS) plays an active role in RAGE oligomerization. To understand the physiological significance of HS-induced RAGE oligomerization in vivo, we generated RAGE knock-in mice (RageAHA/AHA) by introducing point mutations to specifically disrupt HS-RAGE interaction. The RAGE mutant demonstrated normal ligand-binding but impaired capacity of HS-binding and oligomerization. Remarkably, RageAHA/AHA mice phenocopied Rage-/- mice in two different pathophysiological processes, namely bone remodeling and neutrophil-mediated liver injury, which demonstrates that HS-induced RAGE oligomerization is essential for RAGE signaling. Our findings suggest that it should be possible to block RAGE signaling by inhibiting HS-RAGE interaction. To test this, we generated a monoclonal antibody that targets the HS-binding site of RAGE. This antibody blocks RAGE signaling in vitro and in vivo, recapitulating the phenotype of RageAHA/AHA mice. By inhibiting HS-RAGE interaction genetically and pharmacologically, our work validated an alternative strategy to antagonize RAGE. Finally, we have performed RNA-seq analysis of neutrophils and lungs and found that while Rage-/- mice had a broad alteration of transcriptome in both tissues compared to wild-type mice, the changes of transcriptome in RageAHA/AHA mice were much more restricted. This unexpected finding suggests that by preserving the expression of RAGE protein (in a dominant-negative form), RageAHA/AHA mouse might represent a cleaner genetic model to study physiological roles of RAGE in vivo compared to Rage-/- mice.

cell biology↗