The GATAD2B-NuRD complex drives DNA:RNA hybrid-dependent chromatin boundary formation upon DNA damage.

Double-strand breaks (DSBs) are the most lethal form of DNA damage. Transcriptional activity at DSBs, as well as transcriptional repression around DSBs, are both required for efficient DNA repair. The chromatin landscape defines and coordinates these two opposing events. However, how the open and condensed chromatin architecture is regulated remains unclear. Here, we show that the GATAD2B-NuRD complex associates with DSBs in a transcription- and DNA:RNA hybrid-dependent manner, to promote histone deacetylation and chromatin condensation. This activity establishes a spatio-temporal boundary between open and closed chromatin, which is necessary for the correct termination of DNA end resection. The lack of the GATAD2B-NuRD complex leads to chromatin hyperrelaxation and extended DNA end resection, resulting in homologous recombination (HR) repair failure. Our results suggest that the GATAD2B-NuRD complex is a key coordinator of the dynamic interplay between transcription and the chromatin landscape, underscoring its biological significance in the RNA-dependent DNA damage response.

Tyrosine 1-phosphorylated RNA polymerase II transcribes PROMPTs to facilitate proximal promoter pausing and induce global transcriptional repression in response to DNA damage.

DNA damage triggers a complex transcriptional response that involves both activation and repression of gene expression. In this study, we investigated global changes in transcription in response to ionizing irradiation (IR), which induces double-strand breaks in DNA. We used mNET-seq to profile nascent transcripts bound to different phosphorylated forms of the RNA polymerase II (RNA Pol II) C-terminal domain (CTD). We found that IR leads to global transcriptional repression of protein-coding genes, accompanied by an increase in antisense transcripts near promoters, called PROMPTs, transcribed by RNA Pol II phosphorylated on tyrosine 1 (Y1P) residue of the CTD. These Y1P-transcribed PROMPTs are enriched for PRC2 binding sites and associated with RNA Pol II proximal promoter pausing. We show the interaction between Y1P RNA Pol II and PRC2, as well as PRC2 binding to PROMPTs. Inhibition of PROMPTs or depletion of PRC2 leads to loss of transcriptional repression. Our results reveal a novel function of Y1P-dependent PROMPTs in mediating PRC2 recruitment to chromatin and RNA Pol II promoter pausing in response to DNA damage.

Swiss army knives: non-canonical functions of nuclear Drosha and Dicer.

The RNase III enzymes Drosha and Dicer are essential for the production of small non-coding RNAs (ncRNAs). In canonical RNAi, microRNAs (miRNAs) regulate gene expression by post-transcriptional gene silencing. In non-canonical RNAi, nuclear RNAi factors generate small ncRNAs that are essential for transcriptional gene silencing. Recent evidence points to the existence of additional non-canonical nuclear RNAi functions in various organisms, including in genome maintenance and editing, as well as in DNA repair. Drosha and Dicer directly regulate gene expression and RNA metabolism at different stages, such as transcriptional initiation and termination, and the processing of various RNA species, including pre-mRNAs. Furthermore, Dicer isoforms were recently discovered and attributed with roles in apoptosis, development and disease.

TIRR regulates mRNA export and association with P-bodies in response to DNA damage.

To ensure the integrity of our genetic code, a coordinated network of signalling and repair proteins, known as the DNA damage response (DDR), detects and repairs DNA insults, the most toxic being double-strand breaks (DSBs). Tudor interacting repair regulator (TIRR) is a key factor in DSB repair, acting through its interaction with p53 binding protein 1 (53BP1). TIRR is also an RNA binding protein, yet its role in RNA regulation during the DDR remains elusive. Here, we show that TIRR selectively binds to a subset of messenger RNAs (mRNAs) in response to DNA damage. Upon DNA damage, TIRR interacts with the nuclear export protein Exportin-1 through a nuclear export signal. Furthermore, TIRR plays a crucial role in the modulation of RNA processing bodies (PBs). TIRR itself and TIRR-bound RNA co-localize with PBs, and TIRR depletion results in nuclear RNA retention and impaired PB formation. We also suggest a potential link between TIRR-regulated RNA export and efficient DDR. This work reveals intricate involvement of TIRR in orchestrating mRNA nuclear export and storage within PBs, emphasizing its significance in the regulation of RNA-mediated DDR.

Dicer dependent tRNA derived small RNAs promote nascent RNA silencing.

In mammalian cells, small non-coding RNAs (sncRNAs) negatively regulate gene expression in a pathway known as RNA interference (RNAi). RNAi can be categorized into post-transcriptional gene silencing (PTGS), which involves the cleavage of target messenger RNA (mRNA) or inhibition of translation in the cytoplasm, and transcriptional gene silencing (TGS), which is mediated by the establishment of repressive epigenetic marks at target loci. Transfer RNAs (tRNAs), which are essential for translation, can be processed into small ncRNAs, termed tRNA-derived small RNAs (tsRNAs). The biogenesis of tsRNAs and their role in gene expression regulation has not yet been fully understood. Here, we show that Dicer dependent tsRNAs promote gene silencing through a mechanism distinct from PTGS and TGS. tsRNAs can lead to downregulation of target genes by targeting introns via nascent RNA silencing (NRS) in nuclei. Furthermore, we show that Ago2 slicer activity is required for this mechanism. Synthetic tsRNAs can significantly reduce expression of a target gene at both RNA and protein levels. Target genes regulated by NRS are associated with various diseases, which further underpins its biological significance. Finally, we show that NRS is evolutionarily conserved and has the potential to be explored as a novel synthetic sRNA based therapeutic.

An old friend with a new face: tRNA-derived small RNAs with big regulatory potential in cancer biology.

Transfer RNAs (tRNAs) are small non-coding RNAs (sncRNAs) essential for protein translation. Emerging evidence suggests that tRNAs can also be processed into smaller fragments, tRNA-derived small RNAs (tsRNAs), a novel class of sncRNAs with powerful applications and high biological relevance to cancer. tsRNAs biogenesis is heterogeneous and involves different ribonucleases, such as Angiogenin and Dicer. For many years, tsRNAs were thought to be just degradation products. However, accumulating evidence shows their roles in gene expression: either directly via destabilising the mRNA or the ribosomal machinery, or indirectly via regulating the expression of ribosomal components. Furthermore, tsRNAs participate in various biological processes linked to cancer, including apoptosis, cell cycle, immune response, and retroviral insertion into the human genome. It is emerging that tsRNAs have significant therapeutic potential. Endogenous tsRNAs can be used as cancer biomarkers, while synthetic tsRNAs and antisense oligonucleotides can be employed to regulate gene expression. In this review, we are recapitulating the regulatory roles of tsRNAs, with a focus on cancer biology.

Cohesin complex promotes transcriptional termination between convergent genes in S. pombe.

Transcription analyses reported in these studies reveal that convergent genes in S. pombe generate overlapping transcripts in the G1 phase of the cell cycle. We show that this double-strand (ds) RNA induces localized RNAi (Dicer and RITS) dependent transient heterochromatin structures including histone H3 lysine 9 trimethylation marks and Swi6 association. Consequently cohesin is recruited to these chromosomal positions through interaction with Swi6. In G2, localized cohesin is further concentrated into the intergenic regions of the convergent genes tested. This results in a block to further dsRNA formation by promoting gene-proximal transcription termination between the convergent genes. Cohesin release at mitosis leads to a new G1 phase with repeated dsRNA formation, transient heterochromatin, and cohesin recruitment. Our results uncover a hitherto unanticipated role for cohesin and further suggest a widespread role for the selective formation of dsRNA, heterochromatin, and subsequent cohesin recruitment in regulated transcriptional termination.

Tyrosine kinase c-Abl couples RNA polymerase II transcription to DNA double-strand breaks.

DNA is constantly exposed to endogenous and exogenous damage. Various types of DNA repair counteract highly toxic DNA double-strand breaks (DSBs) to maintain genome stability. Recent findings suggest that the human DNA damage response (DDR) utilizes small RNA species, which are produced as long non-coding (nc)RNA precursors and promote recognition of DSBs. However, regulatory principles that control production of such transcripts remain largely elusive. Here we show that the Abelson tyrosine kinase c-Abl/ABL1 causes formation of RNA polymerase II (RNAPII) foci, predominantly phosphorylated at carboxy-terminal domain (CTD) residue Tyr1, at DSBs. CTD Tyr1-phosphorylated RNAPII (CTD Y1P) synthetizes strand-specific, damage-responsive transcripts (DARTs), which trigger formation of double-stranded (ds)RNA intermediates via DNA-RNA hybrid intermediates to promote recruitment of p53-binding protein 1 (53BP1) and Mediator of DNA damage checkpoint 1 (MDC1) to endogenous DSBs. Interference with transcription, c-Abl activity, DNA-RNA hybrid formation or dsRNA processing impairs CTD Y1P foci formation, attenuates DART synthesis and delays recruitment of DDR factors and DSB signalling. Collectively, our data provide novel insight in RNA-dependent DDR by coupling DSB-induced c-Abl activity on RNAPII to generate DARTs for consequent DSB recognition.

Nuclear re-localization of Dicer in primary mouse embryonic fibroblast nuclei following DNA damage.

Dicer is a key component of RNA interference (RNAi) and well-known for its role in biogenesis of micro (mi)RNA in the cytoplasm. Increasing evidence suggests that mammalian Dicer is also present and active in the nucleus. We have previously shown that phosphorylated human Dicer associates with chromatin in response to DNA damage and processes double-stranded (ds)RNA in the nucleus. However, a recent study by Much et al. investigated endogenously tagged HA-Dicer both in primary mouse embryonic fibroblasts (PMEFs) as well as adult homozygous viable and fertile HA-Dicer mice under physiological conditions and concluded that murine Dicer is exclusively cytoplasmic. The authors challenged several findings, reporting functions of Dicer in mammalian nuclei. We have re-investigated this issue by applying subcellular fractionation, super-resolution microscopy followed by 3D reconstitution, and phospho-Dicer-specific antibodies using the same HA-Dicer PMEF cell line. Our data show that a small fraction of the murine HA-Dicer pool, approximately 5%, localises in the nucleus and is phosphorylated upon DNA damage. We propose that Dicer localisation is dynamic and not exclusively cytoplasmic, particularly in cells exposed to DNA damage.

Subcellular RNA profiling links splicing and nuclear DICER1 to alternative cleavage and polyadenylation.

Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we used a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape APA profiles in different subcellular locations. Here, we show that APA isoforms with shorter 3' UTRs tend to be overrepresented in the cytoplasm and appear to be cell-type-specific events. Nuclear retention of longer APA isoforms occurs and is partly a result of incomplete splicing contributing to the observed cytoplasmic bias of transcripts with shorter 3' UTRs. We demonstrate that the endoribonuclease III, DICER1, contributes to the establishment of subcellular APA profiles not only by expected cytoplasmic miRNA-mediated destabilization of APA mRNA isoforms, but also by affecting polyadenylation site choice.

Autoregulation of convergent RNAi genes in fission yeast.

RNAi plays a central role in the regulation of eukaryotic genes. In Schizosaccharomyces pombe fission yeast, RNAi involves the formation of siRNA from dsRNA that acts to establish and maintain heterochromatin over centromeres, telomeres, and mating loci. We showed previously that transient heterochromatin also forms over S. pombe convergent genes (CGs). Remarkably, most RNAi genes are themselves convergent. We demonstrate here that transient heterochromatin formed by the RNAi pathway over RNAi CGs leads to their autoregulation in G1-S. Furthermore, the switching of RNAi gene orientation from convergent to tandem causes loss of their G1-S down-regulation. Surprisingly, yeast mutants with tandemized dcr1, ago1, or clr4 genes display aberrant centromeric heterochromatin, which results in abnormal cell morphology. Our results emphasize the significance of gene orientation for correct RNAi gene expression, and suggest a role for cell cycle-dependent formation of RNAi CG heterochromatin in cellular integrity.

Transcription facilitates sister chromatid cohesion on chromosomal arms.

Cohesin is a multi-subunit protein complex essential for sister chromatid cohesion, gene expression and DNA damage repair. Although structurally well studied, the underlying determinant of cohesion establishment on chromosomal arms remains enigmatic. Here, we show two populations of functionally distinct cohesin on chromosomal arms using a combination of genomics and single-locus specific DNA-FISH analysis. Chromatin bound cohesin at the loading sites co-localizes with Pds5 and Eso1 resulting in stable cohesion. In contrast, cohesin independent of its loader is unable to maintain cohesion and associates with chromatin in a dynamic manner. Cohesive sites coincide with highly expressed genes and transcription inhibition leads to destabilization of cohesin on chromatin. Furthermore, induction of transcription results in de novo recruitment of cohesive cohesin. Our data suggest that transcription facilitates cohesin loading onto chromosomal arms and is a key determinant of cohesive sites in fission yeast.

Genome-wide analysis of poly(A) site selection in Schizosaccharomyces pombe.

Polyadenylation of pre-mRNAs, a critical step in eukaryotic gene expression, is mediated by cis elements collectively called the polyadenylation signal. Genome-wide analysis of such polyadenylation signals was missing in fission yeast, even though it is an important model organism. We demonstrate that the canonical AATAAA motif is the most frequent and functional polyadenylation signal in Schizosaccharomyces pombe. Using analysis of RNA-Seq data sets from cells grown under various physiological conditions, we identify 3' UTRs for nearly 90% of the yeast genes. Heterogeneity of cleavage sites is common, as is alternative polyadenylation within and between conditions. We validated the computationally identified sequence elements likely to promote polyadenylation by functional assays, including qRT-PCR and 3'RACE analysis. The biological importance of the AATAAA motif is underlined by functional analysis of the genes containing it. Furthermore, it has been shown that convergent genes require trans elements, like cohesin for efficient transcription termination. Here we show that convergent genes lacking cohesin (on chromosome 2) are generally associated with longer overlapping mRNA transcripts. Our bioinformatic and experimental genome-wide results are summarized and can be accessed and customized in a user-friendly database Pomb(A).

The ARID1A-METTL3-m6A axis ensures effective RNase H1-mediated resolution of R-loops and genome stability.

R-loops are three-stranded structures that can pose threats to genome stability. RNase H1 precisely recognizes R-loops to drive their resolution within the genome, but the underlying mechanism is unclear. Here, we report that ARID1A recognizes R-loops with high affinity in an ATM-dependent manner. ARID1A recruits METTL3 and METTL14 to the R-loop, leading to the m6A methylation of R-loop RNA. This m6A modification facilitates the recruitment of RNase H1 to the R-loop, driving its resolution and promoting DNA end resection at DSBs, thereby ensuring genome stability. Depletion of ARID1A, METTL3, or METTL14 leads to R-loop accumulation and reduced cell survival upon exposure to cytotoxic agents. Therefore, ARID1A, METTL3, and METTL14 function in a coordinated, temporal order at DSB sites to recruit RNase H1 and to ensure efficient R-loop resolution. Given the association of high ARID1A levels with resistance to genotoxic therapies in patients, these findings open avenues for exploring potential therapeutic strategies for cancers with ARID1A abnormalities.

Understanding non-coding DNA regions in yeast.

Non-coding transcripts play an important role in gene expression regulation in all species, including budding and fission yeast. Such regulatory transcripts include intergenic ncRNA (non-coding RNA), 5' and 3' UTRs, introns and antisense transcripts. In the present review, we discuss advantages and limitations of recently developed sequencing techniques, such as ESTs, DNA microarrays, RNA-Seq (RNA sequencing), DRS (direct RNA sequencing) and TIF-Seq (transcript isoform sequencing). We provide an overview of methods applied in yeast and how each of them has contributed to our knowledge of gene expression regulation and transcription.

Detecting RNA-protein proximity at DNA double-strand breaks using combined fluorescence in situ hybridization with proximity ligation assay.

RNA transcribed at DNA double-strand breaks (DSBs) contributes to accurate DNA repair. Here, using the repair factors 53BP1 and TIRR as examples, we combine the fluorescence in situ hybridization (FISH) and proximity ligation assay (PLA) techniques to determine protein proximity to DSB-transcribed RNA. In this FISH-PLA protocol, we detail steps for designing DNA probes and image analysis using CellProfiler™ software. This approach has many potential applications for the study of the RNA-binding proteins and nascent RNA interactions. For complete details on the use and execution of this protocol, please refer to Ketley et al. (2022).1.

The phosphorylated trimeric SOSS1 complex and RNA polymerase II trigger liquid-liquid phase separation at double-strand breaks.

Double-strand breaks (DSBs) are the most severe type of DNA damage. Previously, we demonstrated that RNA polymerase II (RNAPII) phosphorylated at the tyrosine 1 (Y1P) residue of its C-terminal domain (CTD) generates RNAs at DSBs. However, the regulation of transcription at DSBs remains enigmatic. Here, we show that the damage-activated tyrosine kinase c-Abl phosphorylates hSSB1, enabling its interaction with Y1P RNAPII at DSBs. Furthermore, the trimeric SOSS1 complex, consisting of hSSB1, INTS3, and c9orf80, binds to Y1P RNAPII in response to DNA damage in an R-loop-dependent manner. Specifically, hSSB1, as a part of the trimeric SOSS1 complex, exhibits a strong affinity for R-loops, even in the presence of replication protein A (RPA). Our in vitro and in vivo data reveal that the SOSS1 complex and RNAPII form dynamic liquid-like repair compartments at DSBs. Depletion of the SOSS1 complex impairs DNA repair, underscoring its biological role in the R-loop-dependent DNA damage response.

DNA damage response, a double-edged sword for vascular aging.

Vascular aging is a major risk factor for age-related cardiovascular diseases, which have high rates of morbidity and mortality. It is characterized by changes in the blood vessels, such as macroscopically increased vascular diameter and intima-medial thickness, chronic inflammation, vascular calcification, arterial stiffening, and atherosclerosis. DNA damage and the subsequent various DNA damage response (DDR) pathways are important causative factors of vascular aging. Deficient DDR, which may result in the accumulation of unrepaired damaged DNA or mutations, can lead to vascular aging. On the other hand, over-activation of some DDR proteins, such as poly (ADP ribose) polymerase (PARP) and ataxia telangiectasia mutated (ATM), also can enhance the process of vascular aging, suggesting that DDR can have both positive and negative effects on vascular aging. Despite the evidence reviewed in this paper, the role of DDR in vascular aging and potential therapeutic targets remain poorly understood and require further investigation.

Small vault RNA1-2 modulates expression of cell membrane proteins through nascent RNA silencing.

Gene expression can be regulated by transcriptional or post-transcriptional gene silencing. Recently, we described nuclear nascent RNA silencing that is mediated by Dicer-dependent tRNA-derived small RNA molecules. In addition to tRNA, RNA polymerase III also transcribes vault RNA, a component of the ribonucleoprotein complex vault. Here, we show that Dicer-dependent small vault RNA1-2 (svtRNA1-2) associates with Argonaute 2 (Ago2). Although endogenous vtRNA1-2 is present mostly in the cytoplasm, svtRNA1-2 localises predominantly in the nucleus. Furthermore, in Ago2 and Dicer knockdown cells, a subset of genes that are up-regulated at the nascent level were predicted to be targeted by svtRNA1-2 in the intronic region. Genomic deletion of vtRNA1-2 results in impaired cellular proliferation and the up-regulation of genes associated with cell membrane physiology and cell adhesion. Silencing activity of svtRNA1-2 molecules is dependent on seed-plus-complementary-paired hybridisation features and the presence of a 5-nucleotide loop protrusion on target RNAs. Our data reveal a role of Dicer-dependent svtRNA1-2, possessing unique molecular features, in modulation of the expression of membrane-associated proteins at the nascent RNA level.

The Methylation Game: Epigenetic and Epitranscriptomic Dynamics of 5-Methylcytosine.

DNA and RNA methylation dynamics have been linked to a variety of cellular processes such as development, differentiation, and the maintenance of genome integrity. The correct deposition and removal of methylated cytosine and its oxidized analogues is pivotal for cellular homeostasis, rapid responses to exogenous stimuli, and regulated gene expression. Uncoordinated expression of DNA/RNA methyltransferases and demethylase enzymes has been linked to genome instability and consequently to cancer progression. Furthermore, accumulating evidence indicates that post-transcriptional DNA/RNA modifications are important features in DNA/RNA function, regulating the timely recruitment of modification-specific reader proteins. Understanding the biological processes that lead to tumorigenesis or somatic reprogramming has attracted a lot of attention from the scientific community. This work has revealed extensive crosstalk between epigenetic and epitranscriptomic pathways, adding a new layer of complexity to our understanding of cellular programming and responses to environmental cues. One of the key modifications, m5C, has been identified as a contributor to regulation of the DNA damage response (DDR). However, the various mechanisms of dynamic m5C deposition and removal, and the role m5C plays within the cell, remains to be fully understood.