RNA Transcripts Research Articles

M6A RNA methylation dynamics during in vitro maturation of cumulus-oocyte complexes derived from adult or prepubertal sheep.

N6-methyladenosine (m6A) is the most prevalent base epigenetic modification within eukaryotic mRNAs. It participates in post-transcriptional regulation, including maternal RNA maintenance and decay in mouse oocytes and during maternal-to-zygotic transition. The landscape in other mammalian species remains largely unexplored. The present work analyzed m6A dynamics in sheep cumulus oocyte complexes (COCs), during in vitro maturation. To explore potential relationships with oocyte developmental competence, a previously established model consisting of oocytes derived from adult and prepubertal sheep was adopted. m6a dynamics were analyzed in terms of m6A RNA methylation abundance in cumulus cells (CCs) by colorimetric assay and expression of key m6A methylation-related proteins (METTL3, METTL14, METTL16, VIRMA, YTHDC1, YTHDC2, YTHDF2, YTHDF3, ALKBH5, and FTO) in both cumulus cells and oocytes by real-time PCR. We report the dynamics of m6A in sheep COCs, and reveal alterations in both oocytes and cumulus cells derived from prepubertal donors. These changes were observed in terms of m6A RNA methylation levels and transcript dynamics of several m6A methylation-related proteins. Notably, our study shows that dysregulations occur after IVM. Overall, this work describes for the first time the dynamics of m6A in sheep COCs and uncovers the involvement of m6A RNA methylation in oocyte developmental potential.

Endothelial TDP-43 depletion disrupts core blood-brain barrier pathways in neurodegeneration.

Endothelial cells (ECs) help maintain the blood-brain barrier but deteriorate in many neurodegenerative disorders. Here we show, using a specialized method to isolate EC and microglial nuclei from postmortem human cortex (92 donors, 50 male and 42 female, aged 20-98 years), that intranuclear cellular indexing of transcriptomes and epitopes enables simultaneous profiling of nuclear proteins and RNA transcripts at a single-nucleus resolution. We identify a disease-associated subset of capillary ECs in Alzheimer's disease, amyotrophic lateral sclerosis and frontotemporal degeneration. These capillaries exhibit reduced nuclear β-catenin and β-catenin-downstream genes, along with elevated TNF/NF-κB markers. Notably, these transcriptional changes correlate with the loss of nuclear TDP-43, an RNA-binding protein also depleted in neuronal nuclei. TDP-43 disruption in human and mouse ECs replicates these alterations, suggesting that TDP-43 deficiency in ECs is an important factor contributing to blood-brain barrier breakdown in neurodegenerative diseases.

A systematic benchmark of Nanopore long-read RNA sequencing for transcript-level analysis in human cell lines.

The human genome contains instructions to transcribe more than 200,000 RNAs. However, many RNA transcripts are generated from the same gene, resulting in alternative isoforms that are highly similar and that remain difficult to quantify. To evaluate the ability to study RNA transcript expression, we profiled seven human cell lines with five different RNA-sequencing protocols, including short-read cDNA, Nanopore long-read direct RNA, amplification-free direct cDNA and PCR-amplified cDNA sequencing, and PacBio IsoSeq, with multiple spike-in controls, and additional transcriptome-wide N6-methyladenosine profiling data. We describe differences in read length, coverage, throughput and transcript expression, reporting that long-read RNA sequencing more robustly identifies major isoforms. We illustrate the value of the SG-NEx data to identify alternative isoforms, novel transcripts, fusion transcripts and N6-methyladenosine RNA modifications. Together, the SG-NEx data provide a comprehensive resource enabling the development and benchmarking of computational methods for profiling complex transcriptional events at isoform-level resolution.

Effect of N1-methyladenosine in the quantification of RNA.

The reverse transcription (RT) of RNA to cDNA is a key step for the quantification of nucleic acid molecules in numerous basic research and medical diagnosis. Although multiple sources of errors have been considered, little is known about the impact of RNA modifications on the validity of genes of interest for quantitative RT-PCR. Here, we evaluated the influence of RNA modifications of N1-methyladenosine (m1A) on the validity of the RT step by quantifying two RNAs with commercial reverse transcriptase and RNA sample from HEK-293T cells or in vitro transcription. Our findings prove that RNA modification of m1A is a source of RT variability as it acts as an arrest signal of RT at its position, in turn affecting the corresponding RNA quantification.

NBS1 facilitates preribosomal RNA biogenesis

Mutations in the NBS1 gene result in Nijmegen breakage syndrome (NBS), and the gene encodes NBS1 that forms a complex with MRE11 and RAD50 and participates in DNA damage repair. However, the molecular mechanism by which NBS1 mutations cause clinical phenotypes of NBS, such as craniofacial dysmorphism, is still unclear. Here, we show that NBS1 localizes at the ribosomal DNA (rDNA) loci in nucleoli and interacts with ribosomal RNA (rRNA) transcription machinery including RNA polymerase I (Pol I) and TCOF1. Loss of NBS1 impairs Pol I-dependent transcription of pre-rRNA and induces nucleolar stress. In particular, lacking Nbs1 in mouse neural crest cells not only leads to the reduction of ribosome biogenesis but also craniofacial abnormalities during prenatal development. Moreover, the C-terminus of NBS1 is associated with pre-rRNA and a number of pre-rRNA processing factors, which may also facilitate pre-rRNA maturation. Taken together, our study reveals the functions of NBS1 in rRNA biogenesis.

Abstract A035: Functional profiling of chimeric RNA transcripts identifies new biomarkers and therapeutic targets for precision medicine in cancer

Abstract Precision medicine in oncology is rapidly emerging as the next frontier for cancer therapy, and accordingly there exists a need to identify new therapeutically actionable targets. Gene fusions have long been established as biomarkers and therapeutic targets for cancer treatment, with targetable fusions such as BCR-ABL1, EML4-ALK, and TPM3-NTRK1 already having shown sustained success in the clinic. While historically studies have focused on DNA rearrangements as the source of fusion transcripts and proteins, recent advances in RNA sequencing have revealed a new landscape of fusions originating at the RNA level through intergenic cis and trans splicing. Combined, this class of molecule, termed “chimeric RNA”, presents new opportunities for molecular profiling of the fusion transcriptome. The purpose of this study is to systematically characterize chimeric RNAs across cancer to identify molecular drivers of cell growth. To build an atlas of chimeric transcripts, we applied multiple fusion-calling programs followed by stringent filtering to predict fusions in 9509 primary tumors from The Cancer Genome Atlas (TCGA) and 1019 cell lines from The Cancer Cell Line Encyclopedia (CCLE). From this we identified 7047 recurrent chimeric transcripts, 2085 of which were not found in normal tissue. To further characterize these fusions, we integrated matched structural variant predictions from whole-genome sequencing to establish the origin of formation at the DNA or RNA level and mapped transcript breakpoint coordinates to protein annotations to establish the potential for the formation of a fusion protein. To identify fusions driving growth and proliferation we developed a functional screen, integrating shRNA dependency screens from DepMap with our cell line chimeric RNA predictions. By mapping the shRNA probes to the sequences of the fusion parental genes, we are able to compare the difference in cell growth between the chimera-mapping and parent specific probes. Using this method, we are able to identify fusions functioning as cancer dependencies independently of the parental genes. Our method accurately detected well-established fusions such as EML4-ALK, FGFR3-TACC3, and TPM3-NTRK1 and showed a significant enrichment of oncogenic fusions from the COSMIC database, fusions containing established oncogenes, and fusions predicted to code for proteins. Out of the 2225 total fusion transcripts screened, we identified 47 cancer-specific transcripts predicted to function as promoters of growth in cancer. Based on degree of essentiality, expression across multiple cancer indications, and potential of forming a targetable fusion protein, we selected candidate fusions for further characterization through cell-based gain and loss of function studies to confirm oncogenicity. Our results reveal a new landscape of chimeric transcripts as drivers of cancer and hold potential for the development of new precision medicine-based treatments. Citation Format: Samir Lalani, Mason Ingram, Sehajroop Gadh, Hui Li. Functional profiling of chimeric RNA transcripts identifies new biomarkers and therapeutic targets for precision medicine in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr A035.

Effect of knockdown LncRNA SNHG1 on autophagic function in SH-SY5Y cells: a model of Alzheimer’s disease (AD)

ABSTRACT Alzheimer ‘s disease, a neurodegenerative disease, is considered a serious global type of dementia affecting predominantly elderly associated with progressive memory loss. Alzheimer ‘s disease exhibits typical pathological manifestations including neuronal loss, β-amyloid deposition, and tau protein neurofibrillary tangles. Significantly increased expression of long-non -coding transcript RNA, LncRNA SNHG1, was detected in the brain of AD patients. However, it is not clear whether knockdown of LncRNA SNHG1 might improve autophagy function in SH-SY5Y cells and reduce the number of apoptotic cells. The aim of this study was to (1) examine the role of LncRNA SNHG1 on autophagic function of SH-SY5Y cells following induction by Aβ1–42 and (2) elucidate the underlying mechanisms. SH-SY5Y cells were transfected with lentiviral vectors to construct a cell line with stable genetic ability to knock down LncRNA SNHG1 and compared to control empty vector cell line. Following induction with Aβ1–42 for 24 hr, an AD cell model was constructed. Downregulation with LncRNA SNHG1 significantly increased cell viability and lowered the number of apoptotic cells. Concomitantly downregulation of the expression of LncRNA SNHG1 in SH-SY5Y cells induced significant decrease in expression of p-tau and caspase3 associated with elevated expression of Beclin1 and AMBRA1. Our results showed that knockdown of LncRNA SNHG1 in SH-SY5Y cells reduced the number of apoptotic cells by enhancing expression of Beclin1 and AMBRA1. Data suggest that by knocking down the expression of LncRNA SNHG1 may be considered a potential target for compounds to treat AD.

FEN1-Aided RPA (FARPA) Coupled with Autosampling Microfluidic Chip Enables Highly Multiplexed On-Site Pathogen Screening.

A simple, rapid, low-cost, and multiplex detection platform is crucial for the diagnosis of infectious diseases, especially for on-site pathogen screening. However, current methods are difficult to satisfy the requirements for minimal instrument and multiplexed point-of-care testing (POCT). Herein, we propose a versatile and easy-to-use platform (FARPA-chip) by combining multiplex FARPA with an autosampling microfluidic chip. A pair of universal recombinase polymerase amplification (RPA) primers introduced during double-stranded cDNA (ds-cDNA) preparation are employed to amplify multiple targets, followed by amplicon-decoding with the chip, indicating no bias in amplifying different targets due to the universal RPA primers. FARPA-chip exhibits that as low as 10 copies of each target RNA in the starting sample can be sensitively detected by 12-plex detection of vector-borne viruses within 45 min and no cross-talk is observed between different targets. The feasibility of this platform is confirmed by designing a 9-plex FARPA-chip to detect 6 kinds of clinically common respiratory viruses from 16 clinical samples of nasopharyngeal swabs, and the results are completely consistent with RT-qPCR. Furthermore, by integrating quick extraction reagent, the turnaround time can be significantly decreased to <50 min, highlighting that our FARPA-chip enables a cost-effective on-site pathogen screening with a relatively high level of multiplexing. Depending on the number of chambers in the chip, the current design is theoretically capable of detecting up to 24 different pathogens, which should fulfill most clinical purposes. We believe that the proposed platform could provide an effective way for a series of healthcare-related applications in resource-limited settings.

Antisense-mediated regulation of exon usage in the elastic spring region of Titin modulates sarcomere function.

Alternative splicing of Titin (TTN) I-band exons produce protein isoforms with variable size and elasticity, but the mechanisms whereby TTN splice factors regulate exon usage and thereby determining cardiomyocyte passive stiffness and diastolic function, is not well understood. Non-coding RNA transcripts from the antisense strand of protein-coding genes have been shown to regulate alternative splicing of the sense gene. The TTN gene locus harbours >80 natural antisense transcripts (NATs) with unknown function in the human heart. The aim of this study was to determine if TTN antisense transcripts play a role in alternative splicing of TTN. RNA-sequencing and RNA in situ hybridization (ISH) of cardiac tissue from heart failure patients (HF), unused donor hearts and human iPS-derived cardiomyocytes (iPS-CMs) were used to determine the expression and localization of TTN NATs. Live cell imaging was used to analyze the effect of NATs on sarcomere properties. RNA ISH, immunofluorescence was performed in iPS-CMs to study the interaction between NATs, TTN mRNA and splice factor protein RBM20.We found that TTN-AS1-276 was the predominant TTN NAT in the human heart and that it was upregulated in HF. Knock down of TTN-AS1-276 in human iPS-CMs resulted in decreased interaction between the splicing factor RBM20 and TTN pre-mRNA, decreased TTN I-band exon skipping, and markedly lower expression of the less compliant TTN isoform N2B. The effect on TTN exon usage was independent of sense-antisense exon overlap and polymerase II elongation rate. Furthermore, knockdown resulted in longer sarcomeres with preserved alignment, improved fractional shortening and relaxation times. We demonstrate a role for TTN-AS1-276 in facilitating alternative splicing of TTN and regulating sarcomere properties. This transcript could constitute a target for improving cardiac passive stiffness and diastolic function in conditions such as heart failure with preserved ejection fraction.

PARL regulates porcine oocyte meiotic maturation by mediating mitochondrial activity.

PARL regulates porcine oocyte meiotic maturation by mediating mitochondrial activity.

Protocol for recombinant rabies virus titration by quantitative PCR.

Protocol for recombinant rabies virus titration by quantitative PCR.

Structural insights and milestones in TDP-43 research: A comprehensive review of its pathological and therapeutic advances.

Transactive response (TAR) DNA-binding protein 43 (TDP-43) is a critical RNA/DNA-binding protein involved in various cellular processes, including RNA splicing, transcription regulation, and RNA stability. Mislocalization and aggregation of TDP-43 in the cytoplasm are key features of the pathogenesis of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD). This review provides a comprehensive retrospective and prospective analysis of TDP-43 research, highlighting structural insights, significant milestones, and the evolving understanding of its physiological and pathological functions. We delineate five major stages in TDP-43 research, from its initial discovery as a pathological hallmark in neurodegeneration to the recent advances in understanding its liquid-liquid phase separation (LLPS) behavior and interactions with cellular processes. Furthermore, we assess therapeutic strategies targeting TDP-43 pathology, categorizing approaches into direct and indirect interventions, alongside modulating aberrant TDP-43 LLPS. We propose that future research will focus on three critical areas: targeting TDP-43 structural polymorphisms for disease-specific therapeutics, exploring dual temporal-spatial modulation of TDP-43, and advancing nano-therapy. More importantly, we emphasize the importance of understanding TDP-43's functional repertoire at the mesoscale, which bridges its molecular functions with broader cellular processes. This review offers a foundational framework for advancing TDP-43 research and therapeutic development.

Totipotent-like reprogramming: Molecular machineries and chemical manipulations.

Totipotent-like reprogramming: Molecular machineries and chemical manipulations.

Cell death-associated lncRNAs in cancer immunopathogenesis: An exploration of molecular mechanisms and signaling pathways.

Cell death-associated lncRNAs in cancer immunopathogenesis: An exploration of molecular mechanisms and signaling pathways.

Protocol for direct cDNA cap analysis of gene expression for paired-end patterned flow cell sequencing.

Protocol for direct cDNA cap analysis of gene expression for paired-end patterned flow cell sequencing.

Establishment of a Novel Caco-2-Based Cell Culture System for Human Sapovirus Propagation.

Human sapovirus (HuSaV), first identified in the 1970s, is a significant cause of acute gastroenteritis, particularly in young children. Despite its clinical significance, research on HuSaV has been limited due to the absence of a reliable cell culture system. In 2020, a breakthrough study reported that HuSaV GI.1 and GII.3 strains could be cultured and serially propagated using HuTu80 cells in the presence of bile acids. However, in 2024, a subsequent study reported that effective replication in HuTu80 cells requires specialized cells that have undergone over 100 passages. In this study, we sought to identify an alternative cell culture system for HuSaV. HuSaV GI.1 can replicate and be serially propagated using Caco-2 cells under bile acid supplementation. Importantly, the Caco-2 cells were freshly sourced from the American Type Culture Collection, ensuring reproducibility for laboratories worldwide. Furthermore, Caco-2MC cells were established via single-cell cloning from in-house Caco-2/Cas9 cells with 91.5% HuSaV-susceptible. HuSaV strains GI.1, GI.2, GI.3, GII.1, GII.3, and GV.1 were successfully propagated using Caco-2MC cells, with RNA copy numbers increasing up to 4.4 log10-fold within 5 days post-infection. This efficient HuSaV cell culture system represents a significant advancement in HuSaV research.

Genomic and transcriptomic sequencing in prostate cancer.

Genomic and transcriptomic sequencing technologies have revolutionized our ability to characterize prostate cancer at the molecular level. The underlying premise of next-generation sequencing technologies and their current and evolving applications in prostate cancer management are provided in the review. Improved methodologies are allowing timely sequencing of the coding regions or both the coding and noncoding regions of the genome to help identify potential mutations and structural variations in the prostate cancer genome, some of which are currently also targetable therapeutically. DNA microarray- based differential gene expression has been supplanted by RNA sequencing (RNA-seq), which not only allows for more accurate quantitation but also nucleotide-level resolution to investigate the entire transcriptome, including alternative gene spliced transcripts and noncoding RNA transcripts, whose full clinical implications have yet to be fully understood and realized. Gene classifier platforms that predict risk of recurrence or metastasis are being incorporated into prostate cancer management algorithms. In the appropriate clinical context, not only somatic but also germline mutation testing is being recommended. Continued clinical integration of sequencing technologies and ongoing research will lead to improved understanding of prostate cancer biology and prostate cancer treatment.

Evidence of a novel gene locus ARHGAP44 for longitudinal change in hemoglobin A1c levels among non-diabetic subjects from the Long Life Family Study.

Glycated hemoglobin A1c (HbA1c) indicates average glucose levels over three months and is associated with insulin resistance and type 2 diabetes (T2D). Longitudinal change in circulating HbA1c (ΔHbA1c) are also associated with aging processes, cognitive performance, and mortality. We analyzed ΔHbA1c in 1,886 non-diabetic Europeans from the Long Life Family Study (LLFS) to uncover gene loci influencing ΔHbA1c. Using growth curve modeling adjusted for multiple covariates, we derived ΔHbA1c and conducted linkage-guided sequence analysis. Our genome-wide linkage scan identified a significant locus on 17p12. In-depth analysis revealed a gene locus ARHGAP44 (rs56340929, explaining 27% of the linkage peak) that was significantly associated with ΔHbA1c. Interestingly, RNA transcription of ARHGAP44 was also significantly associated with ΔHbA1c in the LLFS, and this discovery was replicable on the gene level in the Framingham Offspring Study (FOS). Taking together, we successfully identified a novel gene locus ARHGAP44 for ΔHbA1c in family members without T2D. Further follow-up studies using longitudinal omics data in large independent cohorts are warranted.

Stress-Related LncRNAs and Their Roles in Diabetes and Diabetic Complications

Diabetes mellitus (DM) is a chronic metabolic disorder and one of the most significant global health burdens worldwide. Key pathophysiological mechanisms underlying its onset and associated complications include hyperglycemia-related stresses, such as oxidative stress and endoplasmic reticulum stress (ER stress). Long non-coding RNAs (lncRNAs), defined as RNA transcripts longer than 200 nucleotides and lacking protein-coding capacity, play crucial roles in various biological processes and have emerged as crucial regulators in the pathogenesis of diabetes. This review provides a comprehensive overview of lncRNA biogenesis and its functional roles, emphasizing recent findings that link stress-related lncRNAs to diabetic pathology and complications. Also, we discuss how lncRNAs influence diabetes and its complications by modulating pathways involved in cell death, proliferation, inflammation, and fibrosis, which contribute to pancreatic β cell dysfunction, insulin resistance, diabetic nephropathy, and retinopathy. By analyzing current research, we aim to enhance understanding of lncRNA involvement in diabetes while identifying potential therapeutic targets and guiding future research directions to elucidate the complex mechanisms underlying this pervasive condition.

Emergent 3D genome reorganization from the stepwise assembly of transcriptional condensates.

Transcriptional condensates are clusters of transcription factors, coactivators, and RNA Pol II associated with high-level gene expression, yet how they assemble and function within the cell remains unclear. Here we show that transcriptional condensates form in a stepwise manner to enable both graded and three-dimensional (3D) gene control in the yeast heat shock response. Molecular dissection revealed a condensate cascade. First, the transcription factor Hsf1 clusters upon partial dissociation from the chaperone Hsp70. Next, the coactivator Mediator partitions following further Hsp70 dissociation and Hsf1 phosphorylation. Finally, Pol II condenses, driving the emergent coalescence of HSR genes. Molecular analysis of a series of Hsf1 mutants revealed graded, rather than switch-like, transcriptional activity. Separation-of-function mutants showed that condensate formation can be decoupled from gene activation. Instead, fully assembled HSR condensates promote adaptive 3D genome reconfiguration, suggesting a role of transcriptional condensates beyond gene activation.