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Single-cell quantification of a broad rna spectrum reveals unique noncoding patterns associated with cell types and states

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2021年第51期118卷 e2113568118-e2113568118页

作者： Isakova, Alina Neff, Norma Quake, Stephen R. Stanford Univ Dept Bioengn Stanford CA 94305 USA Chan Zuckerberg Biohub San Francisco CA 94158 USA Stanford Univ Dept Appl Phys Stanford CA 94305 USA

The ability to interrogate total rna content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states. However, current single-cell rna-sequencing (rna-seq) methods are unable to simultaneously monitor all forms of rna transcripts at the single-cell level, and thus deliver only a partial snapshot of the cellular rnaome. Here we describe Smart-seq-total, a method capable of assaying a broad spectrum of coding and noncoding rna from a single cell. Smart-seq-total does not require splitting the rna content of a cell and allows the incorporation of unique molecular identifiers into short and long rna molecules for absolute quantification. It outperforms current poly(A)-independent total rna-seq protocols by capturing transcripts of a broad size range, thus enabling simultaneous analysis of protein-coding, long-noncoding, microrna, and other noncoding rna transcripts from single cells. We used Smart-seq-total to analyze the total rnaome of human primary fibroblasts, HEK293T, and MCF7 cells, as well as that of induced murine embryonic stem cells differentiated into embryoid bodies. By analyzing the coexpression patterns of both noncoding rna and mrna from the same cell, we were able to discover new roles of noncoding rna throughout essential processes, such as cell cycle and lineage commitment during embryonic development. Moreover, we show that independent classes of short-noncoding rna can be used to determine cell-type identity.

关键词： single-cell rna-seq noncoding rna cell cycle differentiation

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High-Throughput Single-Nucleus rna Profiling of Minimal Puncture FFPE Samples Reveals Spatiotemporal Heterogeneity of Cancer

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ADVANCED SCIENCE 2025年第4期12卷 e2410713页

作者： Jiang, Weiqin Zhang, Xiang Xu, Ziye Cheng, Qing Li, Xiaohan Zhu, Yuyi Lu, Fangru Dong, Ling Zeng, Linghui Zhong, Weixiang Wang, Yongcheng Fan, Longjiang Chen, Hongyu Zhejiang Univ Affiliated Hosp 1 Sch Med Dept Colorectal Surg Hangzhou 310003 Peoples R China Lanzhou Univ Clin Med Coll 1 Lanzhou 730000 Peoples R China Zhejiang Univ Med Ctr Liangzhu Lab Hangzhou 311121 Peoples R China Zhejiang Univ Affiliated Hosp 1 Sch Med Dept Lab Med Hangzhou 311121 Peoples R China Zhejiang Univ Inst Bioinformat Hangzhou Chin Myanmar Zhejiang Univ James D Watson Inst Genome Sci Hangzhou 310058 Peoples R China M20 Genom Hangzhou 310030 Peoples R China Hangzhou City Univ Sch Med Hangzhou 316021 Peoples R China Zhejiang Univ Affiliated Hosp 1 Coll Med Dept Pathol Hangzhou 310003 Peoples R China Zhejiang Univ Coll Biomed Engn & Instrument Sci Hangzhou 310027 Peoples R China

Puncture biopsy, especially those preserved by formalin fixed paraffin embedding (FFPE) samples, play an important role in various research purposes. Diverse single-nucleus rna sequencing (snrna-seq) techniques have been developed for FFPE samples, however, how to perform high-throughput snrna-seq on small FFPE puncture samples is still a challenge. Here, the previously developed snrna-seq technique (snRandom-seq) is optimized by implementing a pre-indexing procedure for the minimal puncture FFPE samples. In analyzing 20 samples from various solid tumors, optimized snRandom-seq still detected approximate to 17 000 genes and 12 000 long non-coding rnas (lncrnas), achieving precise clustering based on tissue origin. A head-to-head comparison with 10x Genomics on fresh biopsy samples showed a similar gene detection rate, with significantly enhanced lncrna detection, indicating that the optimized snRandom-seq technique maintains its established gene detection advantages even when applied to small samples. Utilizing 7 puncture FFPE samples of liver metastases from 3 colorectal cancer patients pre- and post-immunotherapy, the cellular developmental trajectories are reconstructed and revealed dynamic spatiotemporal heterogeneity during treatment, including insights into pseudoprogression of immunotherapy. Therefore, the optimized snRandom-seq offers a solution for high-throughput single-cell rna and non-coding rna analysis in minimal puncture FFPE sample.

关键词： FFPE sample noncoding rna puncture biopsy pseudoprogression spatiotemporal heterogeneity

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LINC00892 as a Prognostic Biomarker in Lung Adenocarcinoma: Role in Immune Infiltration and EMT Suppression

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JOUrnaL OF IMMUNOLOGY RESEARCH 2025年第1期2025卷 4341348页

作者： Luan, Xinyu Peng, Xuxing Hou, Qinghua Liu, Jixian Peking Univ Shenzhen Hosp Dept Thorac Surg Shenzhen Guangdong Peoples R China

Lung adenocarcinoma (LUAD) is a prevalent and aggressive form of lung cancer with poor prognosis, largely due to late-stage diagnosis and limited therapeutic options. Recent studies suggest that long noncoding rnas (lncrnas) play critical roles in cancer progression and immune modulation, emerging as potential therapeutic targets. In this study, we investigated the expression and functional role of LINC00892 in LUAD using rna sequencing data from The Cancer Genome Atlas (TCGA) and functional assays in vitro and in vivo. We found that LINC00892 is significantly downregulated in LUAD tissues compared to normal tissues, and lower LINC00892 expression correlates with poorer overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI), particularly in younger patients and those with early-stage disease. Bioinformatic analyses revealed that LINC00892 expression is positively correlated with immune cell infiltration, including CD4+ and CD8+ T cells, and negatively correlated with tumor-promoting Th2 cells, suggesting its role in shaping the tumor immune microenvironment. In vitro functional assays showed that LINC00892 overexpression inhibits LUAD cell proliferation, migration, and invasion while promoting apoptosis. Mechanistically, LINC00892 upregulation was found to suppress epithelial-mesenchymal transition (EMT) by increasing E-cadherin expression and decreasing levels of N-cadherin, vimentin, and slug. Additionally, in an in vivo mouse xenograft model, LINC00892 overexpression suppressed tumor growth and metastasis, accompanied by enhanced immune cell infiltration such as CD4+ and CD8+ T cells. Collectively, these findings suggest that LINC00892 acts as a tumor suppressor in LUAD by modulating immune infiltration and EMT, highlighting its potential as a prognostic biomarker and therapeutic target.

关键词： biomarker immune cells lung adenocarcinoma noncoding rna prognosis

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Broad noncoding transcription suggests genome surveillance by rna polymerase V

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2020年第48期117卷 30799-30804页

作者： Tsuzuki, Masayuki Sethuraman, Shriya Coke, Adriana N. Rothi, M. Hafiz Boyle, Alan P. Wierzbicki, Andrzej T. Univ Michigan Dept Mol Cellular & Dev Biol Ann Arbor MI 48109 USA Univ Tokyo Grad Sch Arts & Sci Dept Life Sci Tokyo 1538902 Japan Univ Michigan Bioinformat Grad Program Ann Arbor MI 48109 USA Univ Michigan Dept Computat Med & Bioinformat Ann Arbor MI 48109 USA Univ Michigan Dept Human Genet Ann Arbor MI 48109 USA

Eukaryotic genomes are pervasively transcribed, yet most transcribed sequences lack conservation or known biological functions. In Arabidopsis thaliana, rna polymerase V (Pol V) produces noncoding transcripts, which base pair with small interfering rna (sirna) and allow specific establishment of rna-directed DNA methylation (RdDM) on transposable elements. Here, we show that Pol V transcribes much more broadly than previously expected, including subsets of both heterochromatic and euchromatic regions. At already established RdDM targets, Pol V and sirna work together to maintain silencing. In contrast, some euchromatic sequences do not give rise to sirna but are covered by low levels of Pol V transcription, which is needed to establish RdDM de novo if a transposon is reactivated. We propose a model where Pol V surveils the genome to make it competent to silence newly activated or integrated transposons. This indicates that pervasive transcription of nonconserved sequences may serve an essential role in maintenance of genome integrity.

关键词： Arabidopsis thaliana rna polymerase V noncoding rna RdDM

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Differential Expression of Long noncoding rnas in Murine Myoblasts After Short Hairpin rna-Mediated Dysferlin Silencing In Vitro: Microarray Profiling

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JMIR Bioinformatics and Biotechnology 2022年第1期3卷 e33186页

作者： Singhal, Richa Lukose, Rachel Carr, Gwenyth Moktar, Afsoon Gonzales-Urday, Ana Lucia Rouchka, Eric C. Vajravelu, Bathri N. Department of Biochemistry and Molecular Genetics KY IDeA Networks of Biomedical Research Excellence Bioinformatics Core University of Louisville Louisville KY United States Department of Physician Assistant Studies Massachusetts College of Pharmacy and Health Sciences Boston MA United States Department of Medical and Molecular Biology School of Arts and Sciences Massachusetts College of Pharmacy and Health Sciences Boston MA United States Department of Pharmaceutical Sciences Massachusetts College of Pharmacy and Health Sciences Boston MA United States

Background: Long noncoding rnas (lncrnas) are noncoding rna transcripts greater than 200 nucleotides in length and are known to play a role in regulating the transcription of genes involved in vital cellular functions. We hypothesized the disease process in dysferlinopathy is linked to an aberrant expression of lncrnas and messenger rnas (mrnas). Objective: In this study, we compared the lncrna and mrna expression profiles between wild-type and dysferlin-deficient murine myoblasts (C2C12 cells). Methods: Lncrna and mrna expression profiling were performed using a microarray. Several lncrnas with differential expression were validated using quantitative real-time polymerase chain reaction. Gene Ontology (GO) analysis was performed to understand the functional role of the differentially expressed mrnas. Further bioinformatics analysis was used to explore the potential function, lncrna-mrna correlation, and potential targets of the differentially expressed lncrnas. Results: We found 3195 lncrnas and 1966 mrnas that were differentially expressed. The chromosomal distribution of the differentially expressed lncrnas and mrnas was unequal, with chromosome 2 having the highest number of lncrnas and chromosome 7 having the highest number of mrnas that were differentially expressed. Pathway analysis of the differentially expressed genes indicated the involvement of several signaling pathways including PI3K-Akt, Hippo, and pathways regulating the pluripotency of stem cells. The differentially expressed genes were also enriched for the GO terms, developmental process and muscle system process. Network analysis identified 8 statistically significant (P<.05) network objects from the upregulated lncrnas and 3 statistically significant network objects from the downregulated lncrnas. Conclusions: Our results thus far imply that dysferlinopathy is associated with an aberrant expression of multiple lncrnas, many of which may have a specific function in the disease process. GO terms an

关键词： abnormal expression bioinformatics dysferlinopathy gene ontology LGMD-2B limb-girdle muscular dystrophy 2B lncrna long noncoding rnas messenger rna mrna muscular dystrophy noncoding rna protein expression qRT-PCR quantitative real-time polymerase chain reaction transcription

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Intravenous and oral administration of the synthetic rna drug, TY1, reverses heart failure with preserved ejection fraction in mice

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BASIC RESEARCH IN CARDIOLOGY 2025年第2期120卷 363-371页

作者： Miyamoto, Kazutaka Jones, Xaviar M. Yamaguchi, Shukuro Ciullo, Alessandra Li, Chang Coto, Joshua Godoy Tsi, Kara Anderson, Jessica Morris, Ashley Marban, Eduardo Ibrahim, Ahmed Gamal-Eldin Cedars Sinai Med Ctr Smidt Heart Inst 8700 Beverly Blvd Los Angeles CA 90048 USA

TY1, a synthetic non-coding rna (ncrna) bioinspired by small Y rnas abundant in extracellular vesicles (EVs), decreases cGAS/STING activation in myocardial infarction and thereby attenuates inflammation. Motivated by the concept that heart failure with preserved ejection fraction (HFpEF) is a systemic inflammatory disease, we tested TY1 in a murine model of HFpEF. Intravenous TY1, packaged in a transfection reagent, reversed the cardiac and systemic manifestations of HFpEF in two-hit obese-hypertensive mice, without inducing weight loss. The effects of TY1 were specific, insofar as they were not reproduced by a control rna of the same nucleotide content but in scrambled order. TY1 consistently suppressed myocardial stress-induced MAP kinase signaling, as well as downstream inflammatory, fibrotic, and hypertrophic gene pathways in heart tissue. TY1 not only prevented but actually reversed key pathological processes underlying HFpEF, with no evidence of toxicity. Most noteworthy from a practical perspective, the effects of intravenous TY1 were reproduced by feeding HFpEF mice an oral micellar formulation of TY1. As the prototype for a novel class of ncrna drugs which target cell stress, TY1 exhibits exceptional disease-modifying bioactivity in HFpEF.

关键词： noncoding rna rna therapeutics Heart failure with preserved ejection fraction Cell stress Inflammation Fibrosis Hypertrophy

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A degenerate telomerase rna directs telomeric DNA synthesis in lepidopteran insects

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2025年第9期122卷 e2424443122页

作者： Chou, Yu - Shu Logeswaran, Dhenugen Chow, Chi - Nga Dunn, Phoebe L. Podlevsky, Joshua D. Liu, Tianxiang Akhter, Khadiza Chen, Julian J. - L. Arizona State Univ Sch Mol Sci Tempe AZ 85281 USA Arizona State Univ Sch Life Sci Tempe AZ 85281 USA Sandia Natl Labs Mol & Microbiol Dept Albuquerque NM 87185 USA

Telomerase elongates telomeres to maintain chromosome stability in most eukaryotes. Despite extensive studies across eukaryotic kingdoms, the telomerase holoenzyme in arthropods remains poorly understood. In this study, we purify the telomerase ribonucleoprotein complex from the lepidopteran insect Spodoptera frugiperda (fall army- worm) and identify a copurified 135- nucleotide telomerase rna (TR) component. This miniature S. frugiperda TR (sfTR), the smallest TR known to date, retains a universal pseudoknot structure and a structurally defined template. Despite its small size, sfTR assembles with the recombinant S. frugiperda telomerase reverse transcriptase (sfTERT) protein in vivo to reconstitute telomerase activity for the synthesis of insect telomeric DNA repeats (TTAGG)n. The sfTR gene, like other animal TR genes, features an snrna-type rna polymerase II promoter. Uniquely, the sfTR transcript harbors a 5 '- 7- methylguanosine (M7G) cap, as opposed to the more typical snrna-type 2,2,7- trimethylguanosine (TMG) cap. The difference in 5 '-cap is likely because sfTR lacks the H/ACA snorna biogenesis domain necessary for cap hypermethylation. Moreover, sfTR also lacks the CR4/5 regulatory domain that is indispensable in vertebrate TRs for telomerase activity. This degenerate sfTR complements an enigmatic sfTERT that is missing certain telomerase- specific elements yet catalytically active in the absence of sfTR. Thus, insects have evolved a simplified telomerase, consisting of a small noncoding rna that retains only minimal attributes essential for telomerase function. The simplified insect telomerase demonstrates a plausible evolutionary pathway for the emergence of telomerase ribonucleoprotein complex, arising from an ancient reverse transcriptase associated with a simple templating rna component in early eukaryotes.

关键词： noncoding rna arthropod evolution telomere rna biogenesis

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The Emerging Roles of Alternative Splicing in Human Oncovirus Infection

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JOUrnaL OF MEDICAL VIROLOGY 2025年第4期97卷 e70346页

作者： Liao, Xuefei Yang, Li Jiang, Mingjuan Xin, Yujie Yan, Huirong Qin, Qingshuang Chen, Mengdi Lu, Jianhong Cent South Univ Xiangya Sch Basic Med Sci Dept Microbiol Changsha Hunan Peoples R China

Alternative splicing (AS) is one of the most potent mechanisms for expanding the diversity of proteomes. During infection, human oncogenic viruses may exploit the AS to facilitate their replication cycle. Moreover, persistently infecting viruses can target key genes involved in classical signaling pathways to promote viral persistence and tumor progression. Here, we highlight how oncogenic viruses hijack AS system to manipulate host biological processes, and the host's AS system in turn modulates viral infection and replication. In addition, we have summarized the relatively underexplored involvement of noncoding rnas in AS following tumor virus infection. This bidirectional interaction provides novel insights into interaction of virus-host and opens new avenues for therapeutic strategies targeting oncogenic viral infections.

关键词： alternative splicing alternative splicing factor noncoding rna oncogenic virus

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Vitexin mitigates oxidative stress, mitochondrial damage, pyroptosis and regulates small nucleolar rna host gene 1/DNA methyltransferase 1/microrna-495 axis in sepsis-associated acute lung injury

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INFLAMMOPHARMACOLOGY 2025年第3期33卷 1435-1454页

作者： Zaki, Almaz Mohsin, Mohd Khan, Salman Khan, Aman Ahmad, Shaniya Verma, Amit Ali, Shakir Fatma, Tasneem Syed, Mansoor Ali Jamia Millia Islamia Fac Life Sci Dept Biotechnol Translat Res Lab Srinivas Ramanujan Block New Delhi 110025 India Jamia Millia Islamia Fac Life Sci Dept Biosci New Delhi India Jamia Hamdard Sch Chem & Life Sci Dept Biochem New Delhi India Univ Penn Dept Pathol & Lab Med Div Diagnost Innovat Philadelphia PA USA

Aim of the studyThis study examined vitexin's effect on sepsis-induced acute lung injury. We used network pharmacology and in vivo and in vitro experiments were performed to elucidate vitexin's role in preventing pyroptosis and regulating small nucleolar rna host gene 1 (SNHG1)/DNA methyltransferase 1 (DNMT1)/microrna-495 (miR-495 *** and methodsWe developed an acute lung injury model using C57BL/6 mice and MLE-12 cells. Through a combination of network pharmacology and in vitro screening, vitexin was identified as the most promising anti-inflammatory compound. Multiple techniques such as western blotting, real-time PCR, Hematoxylin and eosin staining, immunohistochemistry, and TUNEL assay were used. Additionally, immunofluorescence, DCFDA and TMRE staining, flow cytometry, methylation-specific PCR, and gene transfection techniques were performed to elucidate vitexin's potential targets and underlying *** treatment significantly reduced lung damage, neutrophil infiltration, and inflammation while improving tight junction integrity. In LPS-treated RAW264.7 macrophages and a septic mouse BALF-induced MLE-12 cell injury model, vitexin demonstrated anti-inflammatory effects, promoted M2 macrophage polarization, and enhanced regenerative markers. It also decreased oxidative stress, mitigated apoptosis and pyroptosis, and improved mitochondrial function. Our research uncovered a novel epigenetic regulatory mechanism involving lncrna SNHG1, DNMT1, and ***'s ability to reduce inflammation, counteract oxidative stress, and modulate epigenetic processes. These findings underscore the promising role of vitexin as a treatment for ALI generated by sepsis. The SNHG1/miR-495 axis, which has been identified, represents a new target for future therapies in acute lung injury.

关键词： Sepsis Acute Lung Injury Inflammation Mitochondria noncoding rna

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Microrna regulation of enteric nervous system development and disease

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TRENDS IN NEUROSCIENCES 2025年第4期48卷 268-282页

作者： Holland, Amy Marie Jehoul, Reindert Vranken, Jorunn Wohl, Stefanie Gabriele Boesmans, Werend Hasselt Univ Biomed Res Inst BIOMED Diepenbeek Belgium Maastricht Univ Res Inst Oncol & Reprod Med Ctr Dept Pathol Maastricht Netherlands SUNY Coll Optometry Dept Biol & Vis Sci New York NY USA

The enteric nervous system (ENS), an elaborate network of neurons and glia woven through the gastrointestinal tract, is integral for digestive physiology and broader human health. Commensurate with its importance, ENS dysfunction is linked to a range of debilitating gastrointestinal disorders. Micrornas (mirnas), with their pleiotropic roles in post-transcriptional gene regulation, serve as key developmental effectors within the ENS. Herein, we review the regulatory dynamics of mirnas in ENS ontogeny, showcasing specific mirnas implicated in both congenital and acquired enteric neuropathies, such as Hirschsprung's disease (HSCR), achalasia, and slow transit constipation (STC). By delineating mirna-mediated mechanisms in these diseases, we underscore their importance for ENS homeostasis and highlight their potential as therapeutic targets.

关键词： gastrointestinal Hirschsprung’s disease gut neural crest cells noncoding rna epigenetics

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