Showing papers on "Transcription (biology) published in 2021"

m 6 A RNA methylation: from mechanisms to therapeutic potential

Abstract: RNA carries a diverse array of chemical modifications that play important roles in the regulation of gene expression. N6 -methyladenosine (m6 A), installed onto mRNA by the METTL3/METTL14 methyltransferase complex, is the most prevalent mRNA modification. m6 A methylation regulates gene expression by influencing numerous aspects of mRNA metabolism, including pre-mRNA processing, nuclear export, decay, and translation. The importance of m6 A methylation as a mode of post-transcriptional gene expression regulation is evident in the crucial roles m6 A-mediated gene regulation plays in numerous physiological and pathophysiological processes. Here, we review current knowledge on the mechanisms by which m6 A exerts its functions and discuss recent advances that underscore the multifaceted role of m6 A in the regulation of gene expression. We highlight advances in our understanding of the regulation of m6 A deposition on mRNA and its context-dependent effects on mRNA decay and translation, the role of m6 A methylation of non-coding chromosomal-associated RNA species in regulating transcription, and the activities of the RNA demethylase FTO on diverse substrates. We also discuss emerging evidence for the therapeutic potential of targeting m6 A regulators in disease.

METTL3 regulates heterochromatin in mouse embryonic stem cells

Abstract: METTL3 (methyltransferase-like 3) mediates the N6-methyladenosine (m6A) methylation of mRNA, which affects the stability of mRNA and its translation into protein1. METTL3 also binds chromatin2-4, but the role of METTL3 and m6A methylation in chromatin is not fully understood. Here we show that METTL3 regulates mouse embryonic stem-cell heterochromatin, the integrity of which is critical for silencing retroviral elements and for mammalian development5. METTL3 predominantly localizes to the intracisternal A particle (IAP)-type family of endogenous retroviruses. Knockout of Mettl3 impairs the deposition of multiple heterochromatin marks onto METTL3-targeted IAPs, and upregulates IAP transcription, suggesting that METTL3 is important for the integrity of IAP heterochromatin. We provide further evidence that RNA transcripts derived from METTL3-bound IAPs are associated with chromatin and are m6A-methylated. These m6A-marked transcripts are bound by the m6A reader YTHDC1, which interacts with METTL3 and in turn promotes the association of METTL3 with chromatin. METTL3 also interacts physically with the histone 3 lysine 9 (H3K9) tri-methyltransferase SETDB1 and its cofactor TRIM28, and is important for their localization to IAPs. Our findings demonstrate that METTL3-catalysed m6A modification of RNA is important for the integrity of IAP heterochromatin in mouse embryonic stem cells, revealing a mechanism of heterochromatin regulation in mammals.

Structures and functions of coronavirus replication-transcription complexes and their relevance for SARS-CoV-2 drug design.

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed millions of people and continues to cause massive global upheaval. Coronaviruses are positive-strand RNA viruses with an unusually large genome of ~30 kb. They express an RNA-dependent RNA polymerase and a cohort of other replication enzymes and supporting factors to transcribe and replicate their genomes. The proteins performing these essential processes are prime antiviral drug targets, but drug discovery is hindered by our incomplete understanding of coronavirus RNA synthesis and processing. In infected cells, the RNA-dependent RNA polymerase must coordinate with other viral and host factors to produce both viral mRNAs and new genomes. Recent research aiming to decipher and contextualize the structures, functions and interplay of the subunits of the SARS-CoV-2 replication and transcription complex proteins has burgeoned. In this Review, we discuss recent advancements in our understanding of the molecular basis and complexity of the coronavirus RNA-synthesizing machinery. Specifically, we outline the mechanisms and regulation of RNA translation, replication and transcription. We also discuss the composition of the replication and transcription complexes and their suitability as targets for antiviral therapy. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses rely on a cohort of specialized viral proteins to transcribe and replicate their RNA genomes. Recent studies have improved our understanding of coronavirus RNA translation, replication and transcription, and offer new therapeutic targets.

Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues.

Abstract: Prolonged detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and recurrence of PCR-positive tests have been widely reported in patients after recovery from COVID-19, but some of these patients do not appear to shed infectious virus. We investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the DNA of human cells in culture and that transcription of the integrated sequences might account for some of the positive PCR tests seen in patients. In support of this hypothesis, we found that DNA copies of SARS-CoV-2 sequences can be integrated into the genome of infected human cells. We found target site duplications flanking the viral sequences and consensus LINE1 endonuclease recognition sequences at the integration sites, consistent with a LINE1 retrotransposon-mediated, target-primed reverse transcription and retroposition mechanism. We also found, in some patient-derived tissues, evidence suggesting that a large fraction of the viral sequences is transcribed from integrated DNA copies of viral sequences, generating viral-host chimeric transcripts. The integration and transcription of viral sequences may thus contribute to the detection of viral RNA by PCR in patients after infection and clinical recovery. Because we have detected only subgenomic sequences derived mainly from the 3' end of the viral genome integrated into the DNA of the host cell, infectious virus cannot be produced from the integrated subgenomic SARS-CoV-2 sequences.

Nuclear sensing of breaks in mitochondrial DNA enhances immune surveillance

Abstract: Mitochondrial DNA double-strand breaks (mtDSBs) are toxic lesions that compromise the integrity of mitochondrial DNA (mtDNA) and alter mitochondrial function1. Communication between mitochondria and the nucleus is essential to maintain cellular homeostasis; however, the nuclear response to mtDSBs remains unknown2. Here, using mitochondrial-targeted transcription activator-like effector nucleases (TALENs)1,3,4, we show that mtDSBs activate a type-I interferon response that involves the phosphorylation of STAT1 and activation of interferon-stimulated genes. After the formation of breaks in the mtDNA, herniation5 mediated by BAX and BAK releases mitochondrial RNA into the cytoplasm and triggers a RIG-I–MAVS-dependent immune response. We further investigated the effect of mtDSBs on interferon signalling after treatment with ionizing radiation and found a reduction in the activation of interferon-stimulated genes when cells that lack mtDNA are exposed to gamma irradiation. We also show that mtDNA breaks synergize with nuclear DNA damage to mount a robust cellular immune response. Taken together, we conclude that cytoplasmic accumulation of mitochondrial RNA is an intrinsic immune surveillance mechanism for cells to cope with mtDSBs, including breaks produced by genotoxic agents. Breaks in mitochondrial DNA cause leakage of mitochondrial RNA into the cytoplasm, enhancing immune surveillance and synergizing with nuclear DNA damage to mount a robust type-I interferon immune response.

How transcription factors drive choice of the T cell fate

Abstract: Recent evidence has elucidated how multipotent blood progenitors transform their identities in the thymus and undergo commitment to become T cells. Together with environmental signals, a core group of transcription factors have essential roles in this process by directly activating and repressing specific genes. Many of these transcription factors also function in later T cell development, but control different genes. Here, we review how these transcription factors work to change the activities of specific genomic loci during early intrathymic development to establish T cell lineage identity. We introduce the key regulators and highlight newly emergent insights into the rules that govern their actions. Whole-genome deep sequencing-based analysis has revealed unexpectedly rich relationships between inherited epigenetic states, transcription factor-DNA binding affinity thresholds and influences of given transcription factors on the activities of other factors in the same cells. Together, these mechanisms determine T cell identity and make the lineage choice irreversible.

Cross-talk of four types of RNA modification writers defines tumor microenvironment and pharmacogenomic landscape in colorectal cancer

Abstract: The four major RNA adenosine modifications, ie, m6A, m1A, alternative polyadenylation, and adenosine-to-inosine RNA editing, are mediated mostly by the “writer” enzymes and constitute critical mechanisms of epigenetic regulation in immune response and tumorigenesis However, the cross-talk and potential roles of these “writers” in the tumor microenvironment (TME), drug sensitivity, and immunotherapy remain unknown We systematically characterized mRNA expression and genetic alterations of 26 RNA modification “writers” in colorectal cancer (CRC), and evaluated their expression pattern in 1697 CRC samples from 8 datasets We used an unsupervised clustering method to assign the samples into two patterns of expression of RNA modification “writers” Subsequently, we constructed the RNA modification “writer” Score (WM_Score) model based on differentially expressed genes (DEGs) responsible for the RNA modification patterns to quantify the RNA modification-related subtypes of individual tumors Furthermore, we performed association analysis for WM_Score and characteristics of TME, consensus molecular subtypes (CMSs), clinical features, transcriptional and post-transcriptional regulation, drug response, and the efficacy of immunotherapy We demonstrated that multi-layer alterations of RNA modification “writer” are associated with patient survival and TME cell-infiltrating characteristics We identified two distinct RNA modification patterns, characterized by a high and a low WM_Score The WM_Score-high group was associated with worse patient overall survival and with the infiltration of inhibitory immune cells, such as M2 macrophages, EMT activation, and metastasis, while the WM_Score-low group was associated with a survival advantage, apoptosis, and cell cycle signaling pathways WM_Score correlated highly with the regulation of transcription and post-transcriptional events contributing to the development of CRC In response to anti-cancer drugs, WM_Score highly negatively correlated (drug sensitive) with drugs which targeted oncogenic related pathways, such as MAPK, EGFR, and mTOR signaling pathways, positively correlated (drug resistance) with drugs which targeted in apoptosis and cell cycle Importantly, the WM_Score was associated with the therapeutic efficacy of PD-L1 blockade, suggesting that the development of potential drugs targeting these “writers” to aid the clinical benefits of immunotherapy Our study is the first to provide a comprehensive analysis of four RNA modifications in CRC We revealed the potential function of these writers in TME, transcriptional and post-transcriptional events, and identified their therapeutic liability in targeted therapy and immunotherapy This work highlights the cross-talk and potential clinical utility of RNA modification “writers” in cancer therapy

Detection of SARS-CoV-2 and Its Mutated Variants via CRISPR-Cas13-Based Transcription Amplification.

Abstract: The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global health emergency, and its gene mutation and evolution further posed uncertainty of epidemic risk. Herein, we reported a light-up CRISPR-Cas13 transcription amplification method, which enables the detection of SARS-CoV-2 and its mutated variants. Sequence specificity was ensured by both the ligation process and Cas13a/crRNA recognition, allowing us to identify viral RNA mutation. Light-up RNA aptamer allows sensitive output of amplification signals via target-activated ribonuclease activity of CRISPR-Cas13a. The RNA virus assay has been designed to detect coronavirus, SARS-CoV-2, Middle East respiratory syndrome (MERS), and SARS, as well as the influenza viruses such as, H1N1, H7N9, and H9N2. It was accommodated to sense as low as 82 copies of SARS-CoV-2. Particularly, it allowed us to strictly discriminate key mutation of the SARS-CoV-2 variant, D614G, which may induce higher epidemic and pathogenetic risk. The proposed RNA virus assays are promising for point-of-care monitoring of SARS-CoV-2 and its risking variants.

The BET family in immunity and disease

Abstract: Innate immunity serves as the rapid and first-line defense against invading pathogens, and this process can be regulated at various levels, including epigenetic mechanisms. The bromodomain and extraterminal domain (BET) family of proteins consists of four conserved mammalian members (BRD2, BRD3, BRD4, and BRDT) that regulate the expression of many immunity-associated genes and pathways. In particular, in response to infection and sterile inflammation, abnormally expressed or dysfunctional BETs are involved in the activation of pattern recognition receptor (e.g., TLR, NLR, and CGAS) pathways, thereby linking chromatin machinery to innate immunity under disease or pathological conditions. Mechanistically, the BET family controls the transcription of a wide range of proinflammatory and immunoregulatory genes by recognizing acetylated histones (mainly H3 and H4) and recruiting transcription factors (e.g., RELA) and transcription elongation complex (e.g., P-TEFb) to the chromatin, thereby promoting the phosphorylation of RNA polymerase II and subsequent transcription initiation and elongation. This review covers the accumulating data about the roles of the BET family in innate immunity, and discusses the attractive prospect of manipulating the BET family as a new treatment for disease.

SARS-CoV-2: from its discovery to genome structure, transcription, and replication

Abstract: SARS-CoV-2 is an extremely contagious respiratory virus causing adult atypical pneumonia COVID-19 with severe acute respiratory syndrome (SARS). SARS-CoV-2 has a single-stranded, positive-sense RNA (+RNA) genome of ~ 29.9 kb and exhibits significant genetic shift from different isolates. After entering the susceptible cells expressing both ACE2 and TMPRSS2, the SARS-CoV-2 genome directly functions as an mRNA to translate two polyproteins from the ORF1a and ORF1b region, which are cleaved by two viral proteases into sixteen non-structural proteins (nsp1-16) to initiate viral genome replication and transcription. The SARS-CoV-2 genome also encodes four structural (S, E, M and N) and up to six accessory (3a, 6, 7a, 7b, 8, and 9b) proteins, but their translation requires newly synthesized individual subgenomic RNAs (sgRNA) in the infected cells. Synthesis of the full-length viral genomic RNA (gRNA) and sgRNAs are conducted inside double-membrane vesicles (DMVs) by the viral replication and transcription complex (RTC), which comprises nsp7, nsp8, nsp9, nsp12, nsp13 and a short RNA primer. To produce sgRNAs, RTC starts RNA synthesis from the highly structured gRNA 3' end and switches template at various transcription regulatory sequence (TRSB) sites along the gRNA body probably mediated by a long-distance RNA-RNA interaction. The TRS motif in the gRNA 5' leader (TRSL) is responsible for the RNA-RNA interaction with the TRSB upstream of each ORF and skipping of the viral genome in between them to produce individual sgRNAs. Abundance of individual sgRNAs and viral gRNA synthesized in the infected cells depend on the location and read-through efficiency of each TRSB. Although more studies are needed, the unprecedented COVID-19 pandemic has taught the world a painful lesson that is to invest and proactively prepare future emergence of other types of coronaviruses and any other possible biological horrors.

DNA G-quadruplex structures: more than simple roadblocks to transcription?

Abstract: It has been >20 years since the formation of G-quadruplex (G4) secondary structures in gene promoters was first linked to the regulation of gene expression. Since then, the development of small molecules to selectively target G4s and their cellular application have contributed to an improved understanding of how G4s regulate transcription. One model that arose from this work placed these non-canonical DNA structures as repressors of transcription by preventing polymerase processivity. Although a considerable number of studies have recently provided sufficient evidence to reconsider this simplistic model, there is still a misrepresentation of G4s as transcriptional roadblocks. In this review, we will challenge this model depicting G4s as simple 'off switches' for gene expression by articulating how their formation has the potential to alter gene expression at many different levels, acting as a key regulatory element perturbing the nature of epigenetic marks and chromatin architecture.

G-quadruplexes are transcription factor binding hubs in human chromatin.

Abstract: The binding of transcription factors (TF) to genomic targets is critical in the regulation of gene expression. Short, double-stranded DNA sequence motifs are routinely implicated in TF recruitment, but many questions remain on how binding site specificity is governed. Herein, we reveal a previously unappreciated role for DNA secondary structures as key features for TF recruitment. In a systematic, genome-wide study, we discover that endogenous G-quadruplex secondary structures (G4s) are prevalent TF binding sites in human chromatin. Certain TFs bind G4s with affinities comparable to double-stranded DNA targets. We demonstrate that, in a chromatin context, this binding interaction is competed out with a small molecule. Notably, endogenous G4s are prominent binding sites for a large number of TFs, particularly at promoters of highly expressed genes. Our results reveal a novel non-canonical mechanism for TF binding whereby G4s operate as common binding hubs for many different TFs to promote increased transcription.

BET inhibition disrupts transcription but retains enhancer-promoter contact

Abstract: Enhancers are DNA sequences that enable complex temporal and tissue-specific regulation of genes in higher eukaryotes. Although it is not entirely clear how enhancer-promoter interactions can increase gene expression, this proximity has been observed in multiple systems at multiple loci and is thought to be essential for the maintenance of gene expression. Bromodomain and Extra-Terminal domain (BET) and Mediator proteins have been shown capable of forming phase condensates and are thought to be essential for super-enhancer function. Here, we show that targeting of cells with inhibitors of BET proteins or pharmacological degradation of BET protein Bromodomain-containing protein 4 (BRD4) has a strong impact on transcription but very little impact on enhancer-promoter interactions. Dissolving phase condensates reduces BRD4 and Mediator binding at enhancers and can also strongly affect gene transcription, without disrupting enhancer-promoter interactions. These results suggest that activation of transcription and maintenance of enhancer-promoter interactions are separable events. Our findings further indicate that enhancer-promoter interactions are not dependent on high levels of BRD4 and Mediator, and are likely maintained by a complex set of factors including additional activator complexes and, at some sites, CTCF and cohesin.

Promoter G-quadruplexes and transcription factors cooperate to shape the cell type-specific transcriptome.

Abstract: Cell identity is maintained by activation of cell-specific gene programs, regulated by epigenetic marks, transcription factors and chromatin organization. DNA G-quadruplex (G4)-folded regions in cells were reported to be associated with either increased or decreased transcriptional activity. By G4-ChIP-seq/RNA-seq analysis on liposarcoma cells we confirmed that G4s in promoters are invariably associated with high transcription levels in open chromatin. Comparing G4 presence, location and transcript levels in liposarcoma cells to available data on keratinocytes, we showed that the same promoter sequences of the same genes in the two cell lines had different G4-folding state: high transcript levels consistently associated with G4-folding. Transcription factors AP-1 and SP1, whose binding sites were the most significantly represented in G4-folded sequences, coimmunoprecipitated with their G4-folded promoters. Thus, G4s and their associated transcription factors cooperate to determine cell-specific transcriptional programs, making G4s to strongly emerge as new epigenetic regulators of the transcription machinery.

Long noncoding RNA MALAT1 and Regulation of the Antioxidant Defense System in Diabetic Retinopathy

Abstract: The retina experiences increased oxidative stress in diabetes, and the transcriptional activity of Nrf2, which is critical in regulating many antioxidant genes, is decreased. The nuclear movement/transcriptional activity of Nrf2 is mediated by its intracellular inhibitor Keap1, and retinal Keap1 levels are increased in diabetes. Gene expression is also regulated by long noncoding RNAs (LncRNAs). Our aim was to investigate the role of LncRNA MALAT1 in the regulation of Keap1-Nrf2-antioxidant defense in diabetic retinopathy. LncRNA MALAT1 expression (quantitative real-time PCR, immunofluorescence, and RNA sequencing), its interactions with Keap1 (FACS), Keap1-Nrf2 interactions, and transcription of the antioxidant response genes (immunofluorescence and nuclear RNA sequencing) were investigated in retinal endothelial cells exposed to high glucose. Glucose increased LncRNA MALAT1 levels by increasing Sp1 transcription factor binding at its promoter. Downregulation of LncRNA MALAT1 by its siRNA prevented glucose-induced increase in Keap1 and facilitated Nrf2 nuclear translocation and antioxidant gene transcription. Retinal microvessels from streptozotocin-induced diabetic mice and human donors with diabetic retinopathy also presented similar increases in LncRNA MALAT1 and its interactions with Keap1 and decreases in Nrf2-mediated antioxidant defense genes. Thus, LncRNA MALAT1, via Keap1-Nrf2, regulates antioxidant defense in diabetic retinopathy. Inhibition of LncRNA MALAT1 has potential to protect the retina from oxidative damage and to prevent or slow down diabetic retinopathy.

Systematic analysis of binding of transcription factors to noncoding variants

Abstract: Many sequence variants have been linked to complex human traits and diseases1, but deciphering their biological functions remains challenging, as most of them reside in noncoding DNA. Here we have systematically assessed the binding of 270 human transcription factors to 95,886 noncoding variants in the human genome using an ultra-high-throughput multiplex protein-DNA binding assay, termed single-nucleotide polymorphism evaluation by systematic evolution of ligands by exponential enrichment (SNP-SELEX). The resulting 828 million measurements of transcription factor-DNA interactions enable estimation of the relative affinity of these transcription factors to each variant in vitro and evaluation of the current methods to predict the effects of noncoding variants on transcription factor binding. We show that the position weight matrices of most transcription factors lack sufficient predictive power, whereas the support vector machine combined with the gapped k-mer representation show much improved performance, when assessed on results from independent SNP-SELEX experiments involving a new set of 61,020 sequence variants. We report highly predictive models for 94 human transcription factors and demonstrate their utility in genome-wide association studies and understanding of the molecular pathways involved in diverse human traits and diseases.

Nuclear m 6 A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos

Abstract: N6-methyladenosine (m6A) on chromosome-associated regulatory RNAs (carRNAs), including repeat RNAs, plays important roles in tuning the chromatin state and transcription, but the intrinsic mechanism remains unclear. Here, we report that YTHDC1 plays indispensable roles in the self-renewal and differentiation potency of mouse embryonic stem cells (ESCs), which highly depends on the m6A-binding ability. Ythdc1 is required for sufficient rRNA synthesis and repression of the 2-cell (2C) transcriptional program in ESCs, which recapitulates the transcriptome regulation by the LINE1 scaffold. Detailed analyses revealed that YTHDC1 recognizes m6A on LINE1 RNAs in the nucleus and regulates the formation of the LINE1-NCL partnership and the chromatin recruitment of KAP1. Moreover, the establishment of H3K9me3 on 2C-related retrotransposons is interrupted in Ythdc1-depleted ESCs and inner cell mass (ICM) cells, which consequently increases the transcriptional activities. Our study reveals a role of m6A in regulating the RNA scaffold, providing a new model for the RNA-chromatin cross-talk.

Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ.

Abstract: Factor-dependent transcription termination mechanisms are poorly understood. We determined a series of cryo-electron microscopy structures portraying the hexameric adenosine triphosphatase (ATPase) ρ on a pathway to terminating NusA/NusG-modified elongation complexes. An open ρ ring contacts NusA, NusG, and multiple regions of RNA polymerase, trapping and locally unwinding proximal upstream DNA. NusA wedges into the ρ ring, initially sequestering RNA. Upon deflection of distal upstream DNA over the RNA polymerase zinc-binding domain, NusA rotates underneath one capping ρ subunit, which subsequently captures RNA. After detachment of NusG and clamp opening, RNA polymerase loses its grip on the RNA:DNA hybrid and is inactivated. Our structural and functional analyses suggest that ρ, and other termination factors across life, may use analogous strategies to allosterically trap transcription complexes in a moribund state.

The roles of hnRNP A2/B1 in RNA biology and disease.

Abstract: The RNA-binding protein hnRNPA2/B1 is a member of the hnRNPs family and is widely expressed in various tissues. hnRNPA2/B1 recognizes and binds specific RNA substrates and DNA motifs and is involved in the transcription, splicing processing, transport, stability, and translation regulation of a variety of RNA molecules and in regulating the expression of a large number of genes. hnRNPA2/B1 is also involved in telomere maintenance and DNA repair, while its expression changes and mutations are involved in the development of various tumors and neurodegenerative and autoimmune diseases. This paper reviews the role and mechanism of hnRNPA2/B1 in RNA metabolism, tumors, and neurodegenerative and autoimmune diseases. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA in Disease and Development > RNA in Disease.

The long noncoding RNA MdLNC499 bridges MdWRKY1 and MdERF109 function to regulate early-stage light-induced anthocyanin accumulation in apple fruit.

Abstract: Anthocyanin pigments contribute to plant coloration and are valuable sources of antioxidants in the human diet as components of fruits and vegetables. Their production is known to be induced by light in apple fruit (Malus domestica); however, the underlying molecular mechanism responsible for early-stage light-induced anthocyanin biosynthesis remains unclear. Here, we identified an ethylene response factor (ERF) protein, ERF109, involved in light-induced anthocyanin biosynthesis and found that it promotes coloration by directly binding to anthocyanin-related gene promoters. Promoter::β-glucuronidase reporter analysis and Hi-C sequencing showed that a long noncoding RNA, MdLNC499, located nearby MdERF109, induces the expression of MdERF109. A W-box cis-element in the MdLNC499 promoter was found to be regulated by a transcription factor, MdWRKY1. Transient expression in apple fruit and stable transformation of apple calli allowed us to reconstruct a MdWRKY1-MdLNC499-MdERF109 transcriptional cascade in which MdWRKY1 is activated by light to increase the transcription of MdLNC499, which in turn induces MdERF109. The MdERF109 protein induces the expression of anthocyanin-related genes and the accumulation of anthocyanins in the early stages of apple coloration. Our results provide a platform for better understanding the various regulatory mechanisms involved in light-induced apple fruit coloration.

LCAT3, a novel m6A-regulated long non-coding RNA, plays an oncogenic role in lung cancer via binding with FUBP1 to activate c-MYC

Abstract: Background Long non-coding RNAs (lncRNAs) are important epigenetic regulators, which play critical roles in diverse physiological and pathological processes. However, the regulatory mechanism of lncRNAs in lung carcinogenesis remains elusive. Here, we characterized a novel oncogenic lncRNA, designated as Lung Cancer Associated Transcript 3 (LCAT3). Methods We predicted and validated LCAT3 by analyzing RNA-sequencing (RNA-seq) data of lung cancer tissues from TCGA. Methylated RNA immunoprecipitation was performed to assess m6A modification on LCAT3. The LCAT3-FUBP1-MYC axis was assessed by dual-luciferase reporter, RNA immunoprecipitation and Chromatin immunoprecipitation assays. Signaling pathways altered by LCAT3 knockdown were identified using RNA-seq. Furthermore, the mechanism of LCAT3 was investigated using loss-of-function and gain-of-function assays in vivo and in vitro. Results LCAT3 was found to be up-regulated in lung adenocarcinomas (LUAD), and its over-expression was associated with the poor prognosis of LUAD patients. LCAT3 upregulation is attributable to N6-methyladenosine (m6A) modification mediated by methyltransferase like 3 (METTL3), leading to LCAT3 stabilization. Biologically, loss-of-function assays revealed that LCAT3 knockdown significantly suppressed lung cancer cell proliferation, migration and invasion in vitro, and inhibited tumor growth and metastasis in vivo. LCAT3 knockdown induced cell cycle arrest at the G1 phase. Mechanistically, LCAT3 recruited Far Upstream Element Binding Protein 1 (FUBP1) to the MYC far-upstream element (FUSE) sequence, thereby activating MYC transcription to promote proliferation, survival, invasion and metastasis of lung cancer cells. Conclusions Taken together, we identified and characterized LCAT3 as a novel oncogenic lncRNA in the lung, and validated the LCAT3-FUBP1-MYC axis as a potential therapeutic target for LUAD.

RNA transcripts stimulate homologous recombination by forming DR-loops.

Abstract: Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) in the S and G2 phases of the cell cycle1–3. Several HR proteins are preferentially recruited to DSBs at transcriptionally active loci4–10, but how transcription promotes HR is poorly understood. Here we develop an assay to assess the effect of local transcription on HR. Using this assay, we find that transcription stimulates HR to a substantial extent. Tethering RNA transcripts to the vicinity of DSBs recapitulates the effects of local transcription, which suggests that transcription enhances HR through RNA transcripts. Tethered RNA transcripts stimulate HR in a sequence- and orientation-dependent manner, indicating that they function by forming DNA–RNA hybrids. In contrast to most HR proteins, RAD51-associated protein 1 (RAD51AP1) only promotes HR when local transcription is active. RAD51AP1 drives the formation of R-loops in vitro and is required for tethered RNAs to stimulate HR in cells. Notably, RAD51AP1 is necessary for the DSB-induced formation of DNA–RNA hybrids in donor DNA, linking R-loops to D-loops. In vitro, RAD51AP1-generated R-loops enhance the RAD51-mediated formation of D-loops locally and give rise to intermediates that we term ‘DR-loops’, which contain both DNA–DNA and DNA–RNA hybrids and favour RAD51 function. Thus, at DSBs in transcribed regions, RAD51AP1 promotes the invasion of RNA transcripts into donor DNA, and stimulates HR through the formation of DR-loops. RNA transcripts stimulate homologous recombination through the formation of DR-loops, intermediate structures that contain both DNA–DNA and DNA–RNA hybrids.

Structures of the human Mediator and Mediator-bound preinitiation complex.

Abstract: The 1.3-megadalton transcription factor IID (TFIID) is required for preinitiation complex (PIC) assembly and RNA polymerase II (Pol II)-mediated transcription initiation on almost all genes. The 26-subunit Mediator stimulates transcription and cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the Pol II C-terminal domain (CTD). We determined the structures of human Mediator in the Tail module-extended (at near-atomic resolution) and Tail-bent conformations and structures of TFIID-based PIC-Mediator (76 polypeptides, ~4.1 megadaltons) in four distinct conformations. PIC-Mediator assembly induces concerted reorganization (Head-tilting and Middle-down) of Mediator and creates a Head-Middle sandwich, which stabilizes two CTD segments and brings CTD to CDK7 for phosphorylation; this suggests a CTD-gating mechanism favorable for phosphorylation. The TFIID-based PIC architecture modulates Mediator organization and TFIIH stabilization, underscoring the importance of TFIID in orchestrating PIC-Mediator assembly.