The Warburg effect has long suggested that oxidative phosphorylation (OXPHOS) is dispensable for tumor growth. However, recent studies have shown that the mitochondrial RNA polymerase inhibitors IMT1 and IMT1b, which impair OXPHOS, are potent anticancer agents. Here, we demonstrate that ionomycin, a selective ionophore known to modulate mitochondrial homeostasis, similarly inhibits mitochondrial gene expression across cancer cell lines. Specifically, gene expression and nascent RNA profiling revealed a global downregulation of mitochondrial gene transcription in Jurkat T, THP-1, HeLa, and NCI-H441 cells. Thus, we conclude that ionomycin suppressed mitochondrial gene transcription, impaired OXPHOS, and thereby inhibited cancer cell proliferation and growth, providing a novel insight into the function of ionomycin.

Apoptosis is indispensable for a variety of physio-pathological processes. RNA is one of essential macromolecules for life. Extensive RNA decay is a characteristic feature of apoptosis. However, it is unknown whether there is de novo RNA synthesis in apoptotic cells and metabolites. In this study, we show that apoptotic mesenchymal stem cells (MSCs) and their apoptotic vesicles (apoVs) synthesize de novo RNAs. Nascent RNA-seq showed apoptotic MSCs and apoVs produced numerous nascent RNAs that were different from those in living MSCs, including protein-coding and non-coding RNAs. Mechanistically, apoptotic de novo RNA synthesis was related to the caspase-3/Sp1/RNA polymerase axis. Additionally, we found the LINE-2a (L2a) and LINE-2b (L2b) RNAs were specifically transcribed in apoptotic MSCs and transferred into apoVs to prevent virus infection. Altogether, this study reveals a previously unknown phenomenon that apoptotic cells synthesize various de novo RNAs and identifies that apoptotic LINE-2 RNAs can regulate innate immunity to prevent virus infection.

Oct4 is a key transcription factor essential for maintaining pluripotency and self-renewal in embryonic stem cells (ESCs), where it activates pluripotency-related genes and represses differentiation-associated genes. While previous studies have identified OCT4 target genes using methods such as chromatin immunoprecipitation sequencing (ChIP-seq) and RNA interference (RNAi), these approaches may not fully capture direct transcriptional regulation. The auxin-inducible degron (AID) system, which enables rapid and reversible protein degradation, combined with nascent RNA sequencing, provides a refined method for identifying direct transcriptional targets by detecting immediate transcriptional changes in both protein-coding genes and non-coding RNAs. In this study, we utilized mouse Oct4-mAID ESCs and nascent RNA sequencing with 5-ethynyl uridine (5-EU) labeling to systematically identify direct OCT4 targets. Our results uncovered novel potential OCT4 targets, providing a dataset for further research into the functions and regulatory networks of Oct4 and related transcription factors.

Nascent RNA-seq is pivotal for elucidating the dynamics and transcriptional regulation of enhancer RNA and protein-coding genes, and for revealing the regulatory linkages between enhancer-gene pairs in diverse physiological and pathological contexts. However, despite the rapid growth of nascent RNA-seq datasets, a comprehensive database for the collection and analysis of these datasets remains absent. Here, we developed the Nascent RNA database (NaRaDa, http://www.narada.bio), which compiles and categorizes 3,664 global or precision run-on sequencing (GRO/PRO-seq) datasets from 415 studies across 22 species. NaRaDa provides a user-friendly interface for searching, visualizing, and assessing the quality of integrated GRO/PRO-seq datasets. Furthermore, NaRaDa identifies transcriptional regulatory elements (TREs) along with other annotation information from ENCODE, FANTOM5, and disease-associated risk SNPs, which are accessible by either “Gene-Centric” or “Project ID-Centric” modes. The database also furnishes researchers with web-based tools to identify differentially expressed genes, TREs, transcription pausing index changes, and more importantly, potential functional transcription factors, in response to specific treatments. These results can be readily downloaded for further independent analysis. NaRaDa serves as an invaluable resource for deepening our understanding of the regulatory mechanisms governing transcription and for identifying previously unrecognized correlations between genomic loci and diseases.

Master transcription factors (MTFs) are key regulators in cell fate determination. However, an approach to profile MTF's direct transcriptional targets together with their associated RNA-binding proteins (RBPs), is still lacking. Here, we applied 5-ethynyluridine RNA metabolic labeling and click chemistry to map the dynamics of the transcriptional targets and the RBPs interacting with the newly transcribed RNAs upon acute NANOG degradation in mouse embryonic stem cells (mESCs). We identified UTP15, a classic rRNA-biogenesis regulator, acts as a key activator of pluripotency-associated gene transcription independently of rRNA biogenesis. Importantly, NANOG-regulated transcription enhances UTP15 binding to transcription start sites (TSSs), associated with increased Pol II binding and more active transcription. Moreover, UTP15 promotes the assembly of Pol II biomolecular condensates, thereby potentially driving pluripotency gene transcription. Collectively, our study uncovers a NANOG-nascent transcript-UTP15 regulatory axis to activate pluripotency gene transcription, providing a distinct approach for studying MTF's function during cell fate determination.

METTL3 is a crucial mRNA methyltransferase in mammals, essential for the regulation of gene expression and various biological processes. Here, we demonstrate that Mettl3 knockout (KO) in mouse embryonic stem cells (mESCs) leads to widespread upregulation of transposable elements (TEs) and 2-cell (2C)-like genes in a m6A enzyme activity-dependent manner, independent of culture conditions. Furthermore, embryo chimera experiments using a transient METTL3 degradation system (dTAG) revealed that METTL3-deficient mESCs can contribute to trophectoderm lineages at the blastocyst stage, indicative of expanded developmental potential. These findings highlight the role of METTL3-mediated m6A modifications in regulating the transcriptional and developmental plasticity of mESCs and suggest a link between m6A loss and the acquisition of a 2C-like state with features of extended potency.

N6-methyladenosine 6-methyladenosine (m6A) 6 A) modification is deemed to be co-transcriptionally installed on pre-mRNAs, thereby influencing various downstream RNA metabolism events. However, the causal relationship between m6A 6 A modification and RNA processing is often unclear, resulting in premature or even misleading generalizations on the function of m6A 6 A modification. Here, we develop 4sU-coupled m6A-level 6 A-level and isoform-characterization sequencing (4sU-m6A-LAIC-seq) 6 A-LAIC-seq) and 4sU-GLORI to quantify the m6A 6 A levels for both newly synthesized and steady-state RNAs at transcript and single-base-resolution levels, respectively, which enable dissecting the relationship between m6A 6 A modification and alternative RNA polyadenylation. Unexpectedly, our results show that many m6A 6 A addition events occur post-transcriptionally, especially on transcripts with high m6A 6 A levels. Importantly, we find higher m6A 6 A levels on shorter 30 0 UTR isoforms, which likely result from sequential polyadenylation of longer 30 0 UTR isoforms with prolonged nuclear dwelling time. Therefore, m6A 6 A modification can also take place post-transcriptionally to intimately couple with other key RNA metabolism processes to establish and dynamically regulate epi-transcriptomics in mammalian cells.

AbstractInfluenza viruses and thogotoviruses account for most recognized orthomyxoviruses. Thogotoviruses, exemplified by Thogoto virus (THOV), are capable of infecting humans using ticks as vectors. THOV transcribes mRNA without the extraneous 5′ end sequences derived from cap-snatching in influenza virus mRNA. Here, we report cryo-EM structures to characterize THOV polymerase RNA synthesis initiation and elongation. The structures demonstrate that THOV RNA transcription and replication are able to start with short dinucleotide primers and that the polymerase cap-snatching machinery is likely non-functional. Triggered by RNA synthesis, asymmetric THOV polymerase dimers can form without the involvement of host factors. We confirm that, distinctive from influenza viruses, THOV-polymerase RNA synthesis is weakly dependent of the host factors ANP32A/B/E in human cells. This study demonstrates varied mechanisms in RNA synthesis and host factor utilization among orthomyxoviruses, providing insights into the mechanisms behind thogotoviruses’ broad-infectivity range.

Pluripotent stem cells possess a unique nuclear architecture characterized by a larger nucleus and more open chromatin, which underpins their ability to self-renew and differentiate. Here, we show that the nucleolus-specific RNA helicase DDX18 is essential for maintaining the pluripotency of human embryonic stem cells. Using techniques such as Hi-C, DNA/RNA-FISH, and biomolecular condensate analysis, we demonstrate that DDX18 regulates nucleolus phase separation and nuclear organization by interacting with NPM1 in the granular nucleolar component, driven by specific nucleolar RNAs. Loss of DDX18 disrupts nucleolar substructures, impairing centromere clustering and perinucleolar heterochromatin (PNH) formation. To probe this further, we develop NoCasDrop, a tool enabling precise nucleolar targeting and controlled liquid condensation, which restores centromere clustering and PNH integrity while modulating developmental gene expression. This study reveals how nucleolar phase separation dynamics govern chromatin organization and cell fate, offering fresh insights into the molecular regulation of stem cell pluripotency.

N6-methyladenosine (m6A) modification is deemed to be co-transcriptionally installed on pre-mRNAs, thereby influencing various downstream RNA metabolism events. However, the causal relationship between m6A modification and RNA processing is often unclear, resulting in premature or even misleading generalizations on the function of m6A modification. Here, we develop 4sU-coupled m6A-level and isoform-characterization sequencing (4sU-m6A-LAIC-seq) and 4sU-GLORI to quantify the m6A levels for both newly synthesized and steady-state RNAs at transcript and single-base-resolution levels, respectively, which enable dissecting the relationship between m6A modification and alternative RNA polyadenylation. Unexpectedly, our results show that many m6A addition events occur post-transcriptionally, especially on transcripts with high m6A levels. Importantly, we find higher m6A levels on shorter 3' UTR isoforms, which likely result from sequential polyadenylation of longer 3' UTR isoforms with prolonged nuclear dwelling time. Therefore, m6A modification can also take place post-transcriptionally to intimately couple with other key RNA metabolism processes to establish and dynamically regulate epi-transcriptomics in mammalian cells.

EDITORIAL article Front. Cell Dev. Biol., 16 October 2023Sec. Signaling Volume 11 - 2023 | https://doi.org/10.3389/fcell.2023.1304817

RNA-protein interactions are precisely regulated by RNA secondary structures in various biological processes. Large-scale identification of proteins that interact with particular RNA structure is important to the RBPome. Herein, a kethoxal assisted single-stranded RNA interactome capture (KASRIC) strategy was developed to globally identify single-stranded RNA binding proteins (ssRBPs). This approach combines RNA secondary structure probing technology with the conventional method of RNA-binding proteins profiling, realizing the transcriptome-wide identification of ssRBPs. Applying KASRIC, we identified 3180 candidate RBPs and 244 candidate ssRBPs in HeLa cells. Importantly, the 244 candidate ssRBPs contained 55 previously reported ssRBPs and 189 novel ssRBPs. Function analysis of the candidate ssRBPs exhibited enrichment in cellular processes related to RNA splicing and RNA degradation. The KASRIC strategy will facilitate the investigation of RNA-protein interactions.

Cellular RNAs undergo dynamic changes during RNA biological processes, which are tightly orchestrated by RNA-binding proteins (RBPs). Yet, the investigation of RNA dynamics is hurdled by highly abundant steady-state RNAs, which make the signals of dynamic RNAs less detectable. Notably, the exert of nucleoside or nucleotide analogue-based RNA technologies has provided a remarkable platform for RNA dynamics research, revealing diverse unnoticed features in RNA metabolism. In this review, we focus on the application of two types of analogue-based RNA sequencing, antigen-/antibody- and click chemistry-based methodologies, and summarize the RNA dynamics features revealed. Moreover, we discuss emerging single-cell newly transcribed RNA sequencing methodologies based on nucleoside analogue labeling, which provides novel insights into RNA dynamics regulation at single-cell resolution. On the other hand, we also emphasize the identification of RBPs that interact with polyA, non-polyA RNAs, or newly transcribed RNAs and also their associated RNA-binding domains at genomewide level through ultraviolet crosslinking and mass spectrometry in different contexts. We anticipated that further modification and development of these analogue-based RNA and RBP capture technologies will aid in obtaining an unprecedented understanding of RNA biology. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Methods > RNA Analyses in Cells.

The RNA modification N-6-methyladenosine (m(6)A) has critical roles in many biological processes(1,2). However, the function of m(6)A in the early phase of mammalian development remains poorly understood. Here we show that the m(6)A reader YT521-B homology-domain-containing protein 1 (YTHDC1) is required for the maintenance of mouse embryonic stem (ES) cells in an m(6)A-dependent manner, and that its deletion initiates cellular reprogramming to a 2C-like state. Mechanistically, YTHDC1 binds to the transcripts of retrotransposons (such as intracisternal A particles, ERVK and LINE1) in mouse ES cells and its depletion results in the reactivation of these silenced retrotransposons, accompanied by a global decrease in SETDB1-mediated trimethylation at lysine 9 of histone H3 (H3K9me3). We further demonstrate that YTHDC1 and its target m(6)A RNAs act upstream of SETDB1 to repress retrotransposons and Dux, the master inducer of the two-cell stage (2C)-like program. This study reveals an essential role for m(6)A RNA and YTHDC1 in chromatin modification and retrotransposon repression.

Pluripotent stem cells (PSCs) can be expanded in vitro in different culture conditions, resulting in a spectrum of cell states with distinct properties. Understanding how PSCs transition from one state to another, ultimately leading to lineage-specific differentiation, is important for developmental biology and regenerative medicine. Although there is significant information regarding gene expression changes controlling these transitions, less is known about post-translational modifications of proteins. Protein crotonylation is a newly discovered post-translational modification where lysine residues are modified with a crotonyl group. Here, we employed affinity purification of crotonylated peptides and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to systematically profile protein crotonylation in mouse PSCs in different states including ground, metastable, and primed states, as well as metastable PSCs undergoing early pluripotency exit. We successfully identified 3628 high-confidence crotonylated sites in 1426 proteins. These crotonylated proteins are enriched for factors involved in functions/processes related to pluripotency such as RNA biogenesis, central carbon metabolism, and proteasome function. Moreover, we found that increasing the cellular levels of crotonyl-coenzyme A (crotonyl-CoA) through crotonic acid treatment promotes proteasome activity in metastable PSCs and delays their differentiation, consistent with previous observations showing that enhanced proteasome activity helps to sustain pluripotency. Our atlas of protein crotonylation will be valuable for further studies of pluripotency regulation and may also provide insights into the role of metabolism in other cell fate transitions.

Some transcription factors that specifically bind double-stranded DNA appear to also function as RNA-binding proteins. Here, we demonstrate that the transcription factor Sox2 is able to directly bind RNA in vitro as well as in mouse and human cells. Sox2 targets RNA via a 60-amino-acid RNA binding motif (RBM) positioned C-terminally of the DNA binding high mobility group (HMG) box. Sox2 can associate with RNA and DNA simultaneously to form ternary RNA/Sox2/DNA complexes. Deletion of the RBM does not affect selection of target genes but mitigates binding to pluripotency related transcripts, switches exon usage and impairs the reprogramming of somatic cells to a pluripotent state. Our findings designate Sox2 as a multi-functional factor that associates with RNA whilst binding to cognate DNA sequences, suggesting that it may co-transcriptionally regulate RNA metabolism during somatic cell reprogramming.

The interplay between the Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) and transcriptional/epigenetic co-regulators in somatic cell reprogramming is incompletely understood. Here, we demonstrate that the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3 plays conflicting roles in mouse reprogramming. On one side, JMJD3 induces the pro-senescence factor Ink4a and degrades the pluripotency regulator PHF20 in a reprogramming factor-independent manner. On the other side, JMJD3 is specifically recruited by KLF4 to reduce H3K27me3 at both enhancers and promoters of epithelial and pluripotency genes. JMJD3 also promotes enhancer-promoter looping through the cohesin loading factor NIPBL and ultimately transcriptional elongation. This competition of forces can be shifted towards improved reprogramming by using early passage fibroblasts or boosting JMJD3's catalytic activity with vitamin C. Our work, thus, establishes a multifaceted role for JMJD3, placing it as a key partner of KLF4 and a scaffold that assists chromatin interactions and activates gene transcription.

N6-methyladenosine (m6A), the most abundant reversible modification on eukaryote messenger RNA, is recognized by a series of readers, including the YT521-B homology domain family (YTHDF) proteins, which are coupled to perform physiological functions. Here, we report that YTHDF2 and YTHDF3, but not YTHDF1, are required for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). Mechanistically, we found that YTHDF3 recruits the PAN2-PAN3 deadenylase complex and conduces to reprogramming by promoting mRNA clearance of somatic genes, including Tead2 and Tgfb1, which parallels the activity of the YTHDF2-CCR4-NOT deadenylase complex. Ythdf2/3 deficiency represses mesenchymal-to-epithelial transition (MET) and chromatin silencing at loci containing the TEAD motif, contributing to decreased reprogramming efficiency. Moreover, RNA interference of Tgfb1 or the Hippo signaling effectors Yap1, Taz, and Tead2 rescues Ythdf2/3-defective reprogramming. Overall, YTHDF2/3 couples RNA deadenylation and regulation with the clearance of somatic genes and provides insights into iPSC reprogramming at the posttranscriptional level.

Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that β-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by β-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts.