Mingyi Xie

Northern blot is a technique that enables the detection and quantification of RNA. In Northern blotting, RNA samples are size separated by gel electrophoresis, immobilized on a nylon membrane, and identified with a sequence-specific probe. Traditional Northern blots use radioactive ³²P-labeled RNA or DNA oligonucleotide probes to detect the RNAs of interest. Here we describe a nonradioactive Northern blot method using near-infrared (IR) fluorescent probes. Use of IR-fluorescent probes enables this “irNorthern” method to achieve similar sensitivity as traditional radioactive Northern blot methods while limiting the complications associated with using radioactive material. This method also enables multiplexing such that different species of RNA can be detected at the same time.

MicroRNAs (miRNAs) are short RNAs that regulate gene expression, critical for development and disease. Residing in Argonaute (AGO) proteins, miRNAs target messenger RNAs via complementary base-pairing. Current miRNA-target databases rely on indirect data from AGO crosslinking immunoprecipitation (AGO-CLIP). In contrast, CLASH (Crosslinking, Ligation, and Sequencing of Hybrids) employs proximity ligation within AGO complexes, providing direct miRNA-target interaction evidence. Existing CLASH datasets remain limited to a few human and mouse samples. Here, we present CLASHub, which integrates CLASH-defined interactions with gene and miRNA expression data from human, mouse, Drosophila , and C. elegans , spanning 25 cell types and tissues, including 91 new CLASH datasets generated from 17 cell types/tissues. The datasets also include samples with knockout of ZSWIM8 , an essential component in target-directed miRNA degradation (TDMD), providing insights into miRNA turnover mechanisms. CLASHub features a user-friendly Analyzer interface for CLASH, RNA-seq, miRNA-seq, and cumulative fraction curve analyses. Leveraging these tools, we uncover a TDMD trigger in the ATP6V1G1 3′ UTR for miR-335-3p degradation, as well as multiple targets of miR-18a-5p. Thus, CLASHub is an online platform that enables cell/tissue-specific exploration of miRNA-target interactions, supporting miRNA and broader RNA biology research. The platform is publicly accessible at https://clashub.rc.ufl.edu/ .

Alternative polyadenylation (APA) is a widespread post-transcriptional regulatory mechanism that diversifies transcript isoforms and modulates mRNA stability, localization, and translation. Although single-cell RNA sequencing (scRNA-seq) provides an unprecedented opportunity to study cell-type-specific APA dynamics, existing computational tools are largely designed for bulk RNA-seq data or rely heavily on gene annotations, limiting their applicability to single-cell contexts. Here, we present scDeepAPA, a deep learning framework specifically optimized for scRNA-seq data to enable accurate polyadenylation site (PAS) detection, isoform quantification, and functional interpretation of APA events at single-cell resolution. Trained on high-confidence annotations from PolyASite v3.0, scDeepAPA integrates convolutional feature extraction with Mamba-based state-space modeling and bidirectional LSTM layers to capture both long-range and local sequence dependencies. Comprehensive benchmarking against five state-of-the-art PAS prediction models demonstrates that scDeepAPA consistently achieves superior performance across accuracy, F1 score, and area under the receiver operating characteristic metrics in both human and mouse datasets. Applying scDeepAPA to Alzheimer’s disease mouse brain data revealed widespread, cell-type-specific APA remodeling across immune and glial populations, including shifts toward proximal PAS usage and 3′ UTR shortening. In KRAS-mutant small cell lung cancer, scDeepAPA uncovered global proximal PAS activation and tumor-specific intronic polyadenylation events. Notably, several intronic APA events generated truncated transcripts encoding predicted neoantigenic peptides with strong major histocompatibility complex class I binding affinity, supported by structural modeling and tumor-specific expression patterns. By enabling accurate PAS identification and quantitative APA profiling, scDeepAPA facilitates in-depth downstream analyses of regulatory mechanisms and immunogenic consequences in single-cell transcriptomics, advancing the understanding of post-transcriptional regulation in neurodegeneration and cancer.

Most RNA molecules adopt multiple alternative structures, forming dynamic ensembles that cannot be captured by single-structure prediction. Recent advances in chemical probing methods (e.g. DMS-MaPseq and SHAPE-MaP sequencing) now provide single-molecule signals that reflect this structural heterogeneity, enabling computational reconstruction of RNA conformational states. However, existing ensemble-inference approaches based on expectation–maximization (EM) often suffer from instability, convergence to suboptimal local optima, and poor scalability on high-dimensional, sparse mutation matrices, particularly for complex or modification-dependent RNA ensembles. To address these limitations, we developed VIRSE, a variational Bayesian framework that uses coordinate ascent variational inference to achieve efficient, scalable, and noise-robust reconstruction of RNA conformational mixtures from chemical probing data. We evaluated VIRSE using extensive simulations, including mechanism-informed mutation simulations that mimic realistic DMS-MaP-seq behavior (A/C mutation bias, context-dependent dropouts, position-specific mutation rates) and idealized Bernoulli-mixture datasets without experimental artifacts. Across all conditions, especially in high-dimensional and long RNA regimes, VIRSE achieved superior ensemble separation and improved cluster identifiability compared with EM, while maintaining stable posteriors, resolving low-abundance states, and scaling to thousands of nucleotide positions. Applied to experimental datasets, including the human immunodeficiency virus-1 Rev response element, SARS-CoV-2 SHAPE-MaP measurements, and the Escherichia coli mgtL Mg2+-responsive riboswitch, VIRSE successfully recovered biologically meaningful and physically plausible RNA conformational ensembles. VIRSE is freely available at https://github.com/QSong-github/VIRSE.

RNA secondary structure plays a critical role in gene regulation, yet existing computational and experimental tools for structure analysis are often fragmented across prediction, ensemble modeling, and functional interpretation workflows. Here, we present ShapeRNA, a user-friendly web server for integrated RNA secondary structure prediction, ensemble inference, and structure-aware regulatory annotation. ShapeRNA supports three complementary analytical workflows, including sequence-based structure prediction, reactivity-guided modeling using SHAPE or DMS data, and sequencing–guided ensemble inference from high-throughput probing experiments. The platform integrates multiple established prediction algorithms and provides standardized data processing, ensemble clustering, and visualization. In addition, ShapeRNA enables mapping of RNA modification sites, microRNA target regions, and RNA-binding protein interaction motifs onto predicted RNA structures and representative ensemble conformations. We demonstrate the utility of ShapeRNA through applications including analysis of mutation-associated structural changes in MAPT exon 10, characterization of conformational heterogeneity in the HIV-1 Rev Response Element, and regulatory annotation of the oncogenic long non-coding RNA HULC. ShapeRNA provides an accessible and extensible platform for investigating RNA structural heterogeneity and regulatory mechanisms. This website is free and open to all users, and there is no login requirement. The server is accessible at https://shaperna.com.

The spatiotemporal expression patterns of microRNAs (miRNAs) are crucial to their function. Target-directed miRNA degradation (TDMD) is an emerging regulatory module that contributes to these expression patterns wherein a specialized RNA (a TDMD trigger) drives miRNA decay through base pairing and the resulting recruitment of E3 ubiquitin ligase ZSWIM8/EBAX-1. Extensive base pairing to the miRNA seed region and 3′ end has been proposed as a key feature that distinguishes TDMD triggers from conventional mRNA targets of miRNAs, which primarily pair with the seed. Here we identify the long noncoding RNA tts-2 as a TDMD trigger for mir-35-42 , the most abundant miRNA family in Caenorhabditis elegans early embryos. We demonstrate that a single site in tts-2 drives decay through base pairing with the seed sequence shared by all eight family members. A second site in tts-2 supports decay of mir-38 with incomplete seed complementarity. Retargeting tts-2 to other miRNAs suggests that the GC-rich seed of mir-35-42 allows for seed-sufficient TDMD. Our findings demonstrate that extended base pairing is not a universal requirement of TDMD and that TDMD drives developmentally timed clearance of abundant miRNAs at the exit of C. elegans embryogenesis.

Myogenesis is a stepwise process encompassing myogenic progenitor proliferation, lineage commitment, differentiation, myocyte fusion, and myotube maturation, and it is orchestrated by myogenic regulatory factors (MRFs) together with signaling pathways that coordinate these transitions. Long noncoding RNAs (lncRNAs) have emerged as important regulators of muscle development and regeneration, yet how lncRNAs integrate with canonical signaling networks to shape myogenic progression remains incompletely defined. Here, we identify a novel myocyte-enriched, Notch-repressed myogenic lncRNA ( NRMLncR, known as A930003A15Rik ), as a previously uncharacterized regulator of mouse myogenesis. The expression of NRMLncR is robustly induced during primary myoblast activation and differentiation. Loss-of-function analyses show that knockdown of NRMLncR impairs myogenic differentiation, accompanied by reduced expression of key myogenic genes. In contrast, adenovirus-mediated overexpression of NRMLncR enhances myogenic differentiation in vitro and is associated with increased muscle fiber size in vivo . Mechanistically, MyoD and MyoG occupy the NRMLncR promoter and promote its transcription during myogenic differentiation. NRMLncR knockdown alerts the transcription of nearby genes, suggesting its function through a cis-regulatory mechanism. RNA pull-down assays further identify an interaction between NRMLncR and the RNA-binding protein CELF1. Together, these findings establish NRMLncR as a novel Notch-associated lncRNA that promotes myogenic differentiation and provide insight into lncRNA-dependent regulation of the myogenic program.

The integrator complex (INT) is an essential regulator of RNA biogenesis across evolution. Most current findings describe INT’s function in states of equilibrium, presenting a research gap in INT’s role in dynamic states, such as in infections and cancers. Viruses hijack cellular RNA machinery to transcribe their genes and produce viral progeny, presenting a unique condition to investigate INT-dependent RNA regulation under perturbation. Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic DNA virus that causes two deadly cancers, Kaposi’s sarcoma and primary effusion lymphoma. KSHV undergoes a highly regulated and robust transcription of viral genes upon lytic reactivation, providing a complex and dynamic system to investigate integrator-mediated viral/host RNA regulation. We find that integrator subunit 11 (INTS11), the enzymatic core of INT, is essential for KSHV lytic replication triggered by reactivation or primary infection. Further RNA-seq analyses revealed a dynamic and unique signature of human transcriptomes during each lytic stage, respectively. Although the knockdown of INTS11 resulted in selective upregulation and downregulation of certain human gene transcription, INTS11’s loss globally repressed the KSHV transcriptome throughout KSHV lytic replication. This inhibited viral lytic gene expression, viral genome replication, and virion production. Integrator subunits 9 and 6 are also important for KSHV lytic replication. Mechanistically, ChIP-seq analysis showed that INTS11 is increasingly recruited to the KSHV genome with some unique binding patterns as the lytic cycle progresses, suggesting that KSHV hijacks INTS11 during lytic gene transcriptions. In all, our findings reveal the essential roles of the Integrator complex in KSHV lytic replication. IMPORTANCE: The integrator complex (INT) is essential for RNA metabolism and is fundamental to all organisms, but its function and regulation during viral infection are not well described. Kaposi’s sarcoma-associated herpesvirus (KSHV) infection establishes lifelong infection and causes two deadly cancers; however, no vaccine is available. Using KSHV as a model, we found that integrator subunit 11 (INTS11), the enzymatic core of INT, is recruited to the KSHV genome under lytic phases and plays an essential role in facilitating global KSHV lytic mRNA transcription and viral production. This reveals the critical role of INT in viral infection, a common and inevitable event in human life.

Background: Epithelial-mesenchymal transition (EMT) is prevalent in human cancer and facilitates tumor metastasis and therapy resistance by enhancing cancer cell motility, invasiveness, survival, and immune evasion. However, the molecular mechanisms underlying the cellular changes during EMT remain largely elusive, making it challenging to simultaneously target these diverse malignant phenotypes. Results: Here, we show that the EMT-inducing ZEB transcription factors directly repressed WWC1 (also known as KIBRA), a key upstream activating component of the Hippo signaling pathway. The EMT program thus inherently downregulated WWC1, leading to impaired Hippo signaling and constitutive activation of the downstream effector and transcriptional coactivator YAP. The YAP-dependent transcriptional program promotes manifold cellular phenotypes that resemble those induced during EMT. Indeed, pharmacological inhibition of YAP suppressed EMT-stimulated cell migration and invasion, apoptosis resistance, and cell size growth, identifying active YAP as a common essential mediator of multiple EMT-associated phenotypes. Moreover, YAP activation directly induced transcription of B7 family immune checkpoint proteins VSIR (VISTA) and PD-L2, and rendered cancer cells resistant to effector CD8 T cells. Conclusions: Collectively, the results suggest that EMT intrinsically activates YAP by repressing WWC1, providing a non-genetic mechanism for pervasive YAP activation in cancer. Activated YAP, in turn, critically contributes to diverse EMT-enhanced malignant phenotypes and immune evasion. Therefore, pharmacological targeting of YAP may suppress various EMT-associated malignant properties and improve the efficacy of anti-PD-1 immunotherapy, offering a promising therapeutic strategy against cancer cells exhibiting EMT characteristics.