Dynamic information of intracellular transcripts is essential to understand their functional roles. Routine RNA-sequencing (RNA-seq) methods only measure RNA species at a steady state and do not provide RNA dynamic information. Here, we develop addition-elimination mechanism-activated nucleotide transition sequencing (AENT-seq) for transcriptome-wide profiling of RNA dynamics. In AENT-seq, nascent transcripts are metabolically labeled with 4-thiouridine (4sU). The total RNA is treated with NH·HO under aqueous conditions. NH·HO is demonstrated to convert 4sU to 4-hydrazino cytosine (C*) based on an addition-elimination chemistry. C* is regarded as cytosine (C) during the DNA extension process. This 4sU-to-C transition marks nascent transcripts, so it enables sequencing analysis of RNA dynamics. We apply our AENT-seq to investigate transcript dynamic information of several genes involved in cancer progression and metastasis. This method uses a simple chemical reaction in aqueous solutions and will be rapidly disseminated with extensive applications.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1021/acs.analchem.1c03361 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
High-resolution fleezers reveal duplex opening and stepwise assembly by an oligomer of the DEAD-box helicase Ded1p.
Nat Commun
January 2025
Department of Physics and Astronomy, Michigan State University, East Lansing, MI, USA.
DEAD-box RNA-dependent ATPases are ubiquitous in all domains of life where they bind and remodel RNA and RNA-protein complexes. DEAD-box ATPases with helicase activity unwind RNA duplexes by local opening of helical regions without directional movement through the duplexes and some of these enzymes, including Ded1p from Saccharomyces cerevisiae, oligomerize to effectively unwind RNA duplexes. Whether and how DEAD-box helicases coordinate oligomerization and unwinding is not known and it is unclear how many base pairs are actively opened.
View Article and Find Full Text PDFThe 40S ribosomal subunit recycling complex modulates mitochondrial dynamics and endoplasmic reticulum - mitochondria tethering at mitochondrial fission/fusion hotspots.
Nat Commun
January 2025
Department of Biological Sciences, Dedman College of Humanities and Sciences, Southern Methodist University, Dallas, TX, 75275, USA.
The 40S ribosomal subunit recycling pathway is an integral link in the cellular quality control network, occurring after translational errors have been corrected by the ribosome-associated quality control (RQC) machinery. Despite our understanding of its role, the impact of translation quality control on cellular metabolism remains poorly understood. Here, we reveal a conserved role of the 40S ribosomal subunit recycling (USP10-G3BP1) complex in regulating mitochondrial dynamics and function.
View Article and Find Full Text PDFHemaScope: A Tool for Analyzing Single-cell and Spatial Transcriptomics Data of Hematopoietic Cells.
Genomics Proteomics Bioinformatics
January 2025
Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Research Unit of Hematologic Malignancies Genomics and Translational Research of Chinese Academy of Medical Sciences, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) techniques hold great value in evaluating the heterogeneity and spatial characteristics of hematopoietic cells within tissues. These two techniques are highly complementary, with scRNA-seq offering single-cell resolution and ST retaining spatial information. However, there is an urgent demand for well-organized and user-friendly toolkits capable of handling single-cell and spatial information.
View Article and Find Full Text PDFTelomerase in cancer- ongoing quest and future discoveries.
Mol Biol Rep
January 2025
Department of Integrative Biology, School of Bio-Sciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, 632014, India.
Telomerase, constituted by the dynamic duo of telomerase reverse transcriptase (TERT), the catalytic entity, and an integral RNA component (TERC), is predominantly suppressed in differentiated human cells due to postnatal transcriptional repression of the TERT gene. Dysregulation of telomerase significantly contributes to cancer development via telomere-dependent and independent mechanisms. Telomerase activity is often elevated in advanced cancers, with TERT reactivation and upregulation of TERC observed in early tumorigenesis.
View Article and Find Full Text PDFDynamic Roles of RNA and RNA Epigenetics in HTLV-1 Biology.
Viruses
January 2025
Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210, USA.
Since the discovery of RNA in the early 1900s, scientific understanding of RNA form and function has evolved beyond protein coding. Viruses, particularly retroviruses like human T-cell leukemia virus type 1 (HTLV-1), rely heavily on RNA and RNA post-transcriptional modifications to regulate the viral lifecycle, pathogenesis, and evasion of host immune responses. With the emergence of new sequencing technologies in the last decade, our ability to dissect the intricacies of RNA has flourished.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!