CtIP-dependent nascent RNA expression flanking DNA breaks guides the choice of DNA repair pathway

The RNA world is changing our views about sensing and resolution of DNA damage. Here, we developed single-molecule DNA/RNA analysis approaches to visualize how nascent RNA facilitates the repair of DNA double-strand breaks (DSBs). RNA polymerase II (RNAPII) is crucial for DSB resolution in human cells. DSB-flanking, RNAPII-generated nascent RNA forms RNA:DNA hybrids, guiding the upstream DNA repair steps towards favouring the error-free Homologous Recombination (HR) pathway over Non-Homologous End Joining. Specific RNAPII inhibitor, THZ1, impairs recruitment of essential HR proteins to DSBs, implicating nascent RNA in DNA end resection, initiation and execution of HR repair. We further propose that resection factor CtIP interacts with and re-activates RNAPII when paused by the RNA:DNA hybrids, collectively promoting faithful repair of chromosome breaks to maintain genomic integrity.

Structural Basis for Control of Bacterial RNA Polymerase Pausing by a Riboswitch and its Ligand

Folding of nascent transcripts can be modulated by the proximal RNA polymerase (RNAP) that carries out their transcription, and vice versa. A pause of RNAP during transcription of a preQ1 riboswitch (que-ePEC) is stabilized by a previously characterized template consensus sequence and the ligand-free conformation of the nascent RNA. Ligand binding to the riboswitch induces RNAP pause release and downstream transcription termination, however, the mechanism by which riboswitch folding modulates pausing is unclear. Here, we report single-particle cryo-electron microscopy reconstructions of que-ePEC in ligand-free and ligand-bound states. In the absence of preQ1, the RNA transcript is in an unexpected hyper-translocated state, preventing downstream nucleotide incorporation. Strikingly, upon ligand binding the riboswitch rotates around its helical axis, expanding the surrounding RNAP exit channel and repositioning the transcript for elongation. Our study reveals the tight coupling by which small nascent RNA structures and their ligands can functionally regulate the macromolecular transcription machinery.

Landscape of transcription termination in Arabidopsis revealed by single-molecule nascent RNA sequencing

Background The dynamic process of transcription termination produces transient RNA intermediates that are difficult to distinguish from each other via short-read sequencing methods. Results Here, we use single-molecule nascent RNA sequencing to characterize the various forms of transient RNAs during termination at genome-wide scale in wildtype Arabidopsis and in atxrn3, fpa, and met1 mutants. Our data reveal a wide range of termination windows among genes, ranging from ~ 50 nt to over 1000 nt. We also observe efficient termination before downstream tRNA genes, suggesting that chromatin structure around the promoter region of tRNA genes may block pol II elongation. 5′ Cleaved readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. Consistent with previous reports, we also observe long chimeric transcripts with cryptic splicing in fpa mutant; but loss of CG DNA methylation has no obvious impact on termination in the met1 mutant. Conclusions Our method is applicable to establish a comprehensive termination landscape in a broad range of species.

Rapid Analysis and Prediction of Drug Resistance in Leukemia By Novel Nascent RNA-Driven Flow Imaging Technologies and Biomarkers

Background: Drug selectivity and resistance is a major obstacle to successful cancer therapy, including traditional chemotherapies (Vasan, N. et al. Nature 2019), immunotherapy (Hu-Lieskovan, S. et al. Future Oncol, 2021), and epigenetic therapy (Saliba, A.N. et al. Cancer Drug Resist,2021). However, no reliable technologies and biomarkers have been developed to analyze and predict drug resistance in clinical settings. Nascent RNAs composed of mostly non-coding RNAs undergo extensive modifications at co- and post-transcriptional levels. Nascent RNAs, and their associated modifications and modifying proteins (RMPs), such as RNA 5-methylcytosine (RNA:m 5C) and RNA cytosine methyltransferases (RCMTs), regulate almost all essential bioprocesses, including chromatin remodeling, DNA transcription, RNA processing/splicing and protein translation. Largely due to technology limitation, the role of nascent RNAs and RNA epigenetics remain largely unknown. Our previous publication first demonstrated distinct cell lineage-associated, RCMTs/BRD4-mediated, drug (azacitidine)-resistant transcriptionally active chromatin structures (TAC) at nascent (newly synthesized) RNAs in leukemia cells (Cheng, J.X. et al. Nat Commun. 2018). The goal of this study is to develop novel nascent RNA/TAC-driven technologies and biomarkers that enable us to rapidly analyze and predict drug resistance in clinical settings. Results: Our experimental data demonstrated that TAC and RNA epigenetics regulate the resistance to venetoclax, a selective inhibitor of the anti-apoptotic protein BCL2, in leukemia cells. There is a significant, lineage-associated, increase in specific RCMTs, such as NSUN2 and NSUN1/NOP2, in venetoclax-resistant leukemia cells. Knockdown of NSUN2 and/or NSUN1 overcome venetoclax resistance in these leukemia cells. Our data also demonstrated distinct patterns of drug- and lineage-specific RNA synthesis dynamics in drug-sensitive vs. -resistant leukemia cells. In drug-sensitive leukemia cells, dinaciclib, a potent a potent, selective small molecule inhibitor of CDKs inhibiting CDK1, CDK2, CDK5 and CDK9 at nano-molar concentrations (Parry, D. et al. Mol.Cancer Ther, 2010), completely inhibit nascent RNA synthesis within 3-5 min, while venetoclax and azacitidine partially inhibit nascent RNA synthesis within 15 min and 240 min, respectively. Such drug-induced inhibition of nascent RNA synthesis is completely independent of apoptosis and program cell death. In contrast, no drug-induced inhibition of nascent RNA synthesis is observed in drug-resistant leukemia cells. Based on our data, we have developed a novel multifactorial system that targets the unique drug- and lineage-specific features of nascent RNA synthesis, TAC and RNA epigenetics for rapid analysis and prediction of drug resistance in clinical settings. Conclusion: Our data demonstrated distinct drug- and lineage-specific patterns of RNA synthesis in drug-sensitive vs. -resistant leukemia cells, which enabled us to develop novel nascent RNA/TAC-drive technologies and biomarkers for rapid analysis and prediction of anticancer drug resistance. Disclosures No relevant conflicts of interest to declare.

Transcriptional Regulation of MECP2E1-E2 Isoforms and BDNF by Metformin and Simvastatin through Analyzing Nascent RNA Synthesis in a Human Brain Cell Line

Methyl CpG binding protein 2 (MeCP2) is the main DNA methyl-binding protein in the brain that binds to 5-methylcytosine and 5-hydroxymethyl cytosine. MECP2 gene mutations are the main origin of Rett Syndrome (RTT), a neurodevelopmental disorder in young females. The disease has no existing cure, however, metabolic drugs such as metformin and statins have recently emerged as potential therapeutic candidates. In addition, induced MECP2-BDNF homeostasis regulation has been suggested as a therapy avenue. Here, we analyzed nascent RNA synthesis versus steady state total cellular RNA to study the transcriptional effects of metformin (an anti-diabetic drug) on MECP2 isoforms (E1 and E2) and BNDF in a human brain cell line. Additionally, we investigated the impact of simvastatin (a cholesterol lowering drug) on transcriptional regulation of MECP2E1/E2-BDNF. Metformin was capable of post-transcriptionally inducing BDNF and/or MECP2E1, while transcriptionally inhibiting MECP2E2. In contrast simvastatin significantly inhibited BDNF transcription without significantly impacting MECP2E2 transcripts. Further analysis of ribosomal RNA transcripts confirmed that the drug neither individually nor in combination affected these fundamentally important transcripts. Experimental analysis was completed in conditions of the presence or absence of serum starvation that showed minimal impact for serum deprival, although significant inhibition of steady state MECP2E1 by simvastatin was only detected in non-serum starved cells. Taken together, our results suggest that metformin controls MECP2E1/E2-BDNF transcriptionally and/or post-transcriptionally, and that simvastatin is a potent transcriptional inhibitor of BDNF. The transcriptional effect of these drugs on MECP2E1/E2-BDNF were not additive under these tested conditions, however, either drug may have potential application for related disorders.

Quantifying RNA Synthesis at Rate-Limiting Steps of Transcription Using Nascent RNA-Sequencing Data

Nascent RNA-sequencing tracks transcription at nucleotide resolution. The genomic distribution of engaged transcription complexes, in turn, uncovers functional genomic regions. Here, we provide data-analytical steps to 1) identify transcribed regulatory elements de novo genome-wide, 2) quantify engaged transcription complexes at enhancers, promoter-proximal regions, divergent transcripts, gene bodies and termination windows, and 3) measure distribution of transcription machineries and regulatory proteins across functional genomic regions. This protocol follows RNA synthesis and genome-regulation in mammals, as demonstrated in human K562 erythroleukemia cells.