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Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics., Competing Interests: Declaration of interests S.L. and P.A. are employees of AstraZeneca. F.A.-G. is employee of Cytena GmbH., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Renal fibrosis is a characteristic hallmark of chronic kidney disease (CKD) that ultimately results in renal failure, leaving patients with few therapeutic options. TGF-β is a master regulator of renal fibrosis and mediates progressive renal fibrosis via both canonical and noncanonical signaling pathways. In the canonical Smad signaling, Smad3 is a key mediator in tissue fibrosis and mediates renal fibrosis via a number of noncoding RNAs (ncRNAs). In this regard, targeting Smad3-dependent ncRNAs may offer a specific therapy for renal fibrosis. This review highlights the significance and innovation of TGF-β/Smad3-associated ncRNAs as biomarkers and therapeutic targets in renal fibrogenesis. In addition, the underlying mechanisms of these ncRNAs and their future perspectives in the treatment of renal fibrosis are discussed., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

G-quadruplexes (G4s) are stable, noncanonical structures formed in guanine (G)-rich sequences of DNA/RNA. G4 structures are reported to play a regulatory role in various cellular processes and, recently, a considerable number of studies have attributed new biological functions to these structures, especially in RNA. Noncoding RNA (ncRNA), which does not translate into a functional protein, is widely expressed and has been shown to play a key role in shaping cellular activity. There has been growing evidence of G4 formation in several ncRNA classes, and it has been identified as a key part for diverse biological functions and physio-pathological contexts in neurodegenerative diseases and cancer. This review discusses RNA G4s (rG4s) in ncRNA, focusing on the molecular mechanism underlying its function. This review also aims to highlight potential and emerging opportunities to identify and target the rG4s in ncRNA to understand its function and, ultimately, treat many diseases., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The brain constantly adapts to changes in the environment, a capability that underlies memory and behavior. Long-term adaptations require the remodeling of neural circuits that are mediated by activity-dependent alterations in gene expression. Over the last two decades, it has been shown that the expression of protein-coding genes is significantly regulated by a complex layer of non-coding RNA (ncRNA) interactions. The aim of this review is to summarize recent discoveries regarding the functional involvement of ncRNAs during different stages of neural circuit development, activity-dependent circuit remodeling, and circuit maladapations underlying neurological and neuropsychiatric disorders. In addition to the intensively studied microRNA (miRNA) family, we focus on more recently added ncRNA classes, such as long ncRNAs (lncRNAs) and circular RNAs (circRNAs), and discuss the complex regulatory interactions between these different RNAs. We conclude by discussing the potential relevance of ncRNAs for cell-type and -state-specific regulation in the context of memory formation, the evolution of human cognitive abilities, and the development of new diagnostic and therapeutic tools in brain disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The transcriptional termination of unstable non-coding RNAs (ncRNAs) is poorly understood compared to coding transcripts. We recently identified ZC3H4-WDR82 ("restrictor") as restricting human ncRNA transcription, but how it does this is unknown. Here, we show that ZC3H4 additionally associates with ARS2 and the nuclear exosome targeting complex. The domains of ZC3H4 that contact ARS2 and WDR82 are required for ncRNA restriction, suggesting their presence in a functional complex. Consistently, ZC3H4, WDR82, and ARS2 co-transcriptionally control an overlapping population of ncRNAs. ZC3H4 is proximal to the negative elongation factor, PNUTS, which we show enables restrictor function and is required to terminate the transcription of all major RNA polymerase II transcript classes. In contrast to short ncRNAs, longer protein-coding transcription is supported by U1 snRNA, which shields transcripts from restrictor and PNUTS at hundreds of genes. These data provide important insights into the mechanism and control of transcription by restrictor and PNUTS., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Characterizing influences of DNA methylation (DNAm) on non-coding RNAs (ncRNAs) is important to understand the mechanisms of gene regulation and cancer outcome. In our study, we describe the results of ncRNA quantitative trait methylation sites (ncQTM) analyses on 8,545 samples from The Cancer Genome Atlas (TCGA), 763 samples from the Clinical Proteomic Tumor Analysis Consortium (CPTAC), and 516 samples from Genotype-Tissue Expression (GTEx) to identify the significant associations between DNAm sites and ncRNAs (miRNA, long non-coding RNA [lncRNA], small nuclear RNA [snRNA], small nucleolar RNA [snoRNA], and rRNA) across 32 cancer types. With more than 22 billion tests, we identify 302,764 cis-ncQTMs (6.28% of all tested) and 79,841,728 trans-ncQTMs (1.15% of all tested). Most DNAm sites (70.6% on average) are in trans association, while only 25.2% DNAm sites are in cis association. Further, we develop a subtype named ncm cluster based on cancer-specific ncRNAs thatis associated with tumor microenvironment, clinical outcome, and biological pathways. To comprehensively describe the ncQTM patterns, we developed a database named Pancan-ncQTM (http://bigdata.njmu.edu.cn/Pancan-ncQTM/)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genome instability can drive aging in many organisms. The ribosomal RNA gene (rDNA) cluster is one of the most unstable regions in the genome and the stability of this region impacts replicative lifespan in budding yeast. To understand the underlying mechanism, we search for yeast mutants with stabler rDNA and longer lifespans than wild-type cells. We show that absence of a transcription elongation factor, Spt4, results in increased rDNA stability, reduced levels of non-coding RNA transcripts from the regulatory E-pro promoter in the rDNA, and extended replicative lifespan in a SIR2-dependent manner. Spt4-dependent lifespan restriction is abolished in the absence of non-coding RNA transcription at the E-pro locus. The amount of Spt4 increases and its function becomes more important as cells age. These findings suggest that Spt4 is a promising aging factor that accelerates cellular senescence through rDNA instability driven by non-coding RNA transcription., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

One snapshot of the peer-review process for "Somatic XIST activation and features of X chromosome inactivation in male human cancers" (Sadagopan et al., 2022)., (Copyright © 2022. Published by Elsevier Inc.)

Cardiovascular disease (CVD) has overtaken infectious illnesses as the leading cause of mortality and disability worldwide. The pathology that underpins CVD is atherosclerosis, characterized by chronic inflammation caused by the accumulation of plaques in the arteries. As our knowledge about the microenvironment of blood vessel walls deepens, there is an opportunity to fine-tune treatments to target the mechanisms driving atherosclerosis more directly. The application of non-coding RNAs (ncRNAs) as biomarkers or intervention targets is increasing. Although these ncRNAs play an important role in driving atherosclerosis and vascular dysfunction, the cellular and extracellular environments pose a challenge for targeted transmission and therapeutic regulation of ncRNAs. Specificity, delivery, and tolerance have hampered the clinical translation of ncRNA-based therapeutics. Nanomedicine is an emerging field that uses nanotechnology for targeted drug delivery and advanced imaging. Recently, nanoscale carriers have shown promising results and have introduced new possibilities for nucleic acid targeted drug delivery, particularly for atherosclerosis. In this review, we discuss the latest developments in nanoparticles to aid ncRNA-based drug development, particularly miRNA, and we analyze the current challenges in ncRNA targeted delivery. In particular, we highlight the emergence of various kinds of nanotherapeutic approaches based on ncRNAs, which can improve treatment options for atherosclerosis., Competing Interests: Declaration of interests The authors have no conflicts of interest to declare., (Copyright © 2022 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Exosomes have a crucial role in intercellular communication and mediate interactions between tumor cells and tumor-associated macrophages (TAMs). Exosome-encapsulated non-coding RNAs (ncRNAs) are involved in various physiological processes. Tumor-derived exosomal ncRNAs induce M2 macrophage polarization through signaling pathway activation, signal transduction, and transcriptional and post-transcriptional regulation. Conversely, TAM-derived exosomal ncRNAs promote tumor proliferation, metastasis, angiogenesis, chemoresistance, and immunosuppression. MicroRNAs induce gene silencing by directly targeting mRNAs, whereas lncRNAs and circRNAs act as miRNA sponges to indirectly regulate protein expressions. The role of ncRNAs in tumor-host interactions is ubiquitous. Current research is increasingly focused on the tumor microenvironment. On the basis of the "cancer-immunity cycle" hypothesis, we discuss the effects of exosomal ncRNAs on immune cells to induce T cell exhaustion, overexpression of programmed cell death ligands, and create a tumor immunosuppressive microenvironment. Furthermore, we discuss potential applications and prospects of exosomal ncRNAs as clinical biomarkers and drug delivery systems., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Linker histones are highly abundant chromatin-associated proteins with well-established structural roles in chromatin and as general transcriptional repressors. In addition, it has been long proposed that histone H1 exerts context-specific effects on gene expression. Here, we identify a function of histone H1 in chromatin structure and transcription using a range of genomic approaches. In the absence of histone H1, there is an increase in the transcription of non-coding RNAs, together with reduced levels of m6A modification leading to their accumulation on chromatin and causing replication-transcription conflicts. This strongly suggests that histone H1 prevents non-coding RNA transcription and regulates non-coding transcript turnover on chromatin. Accordingly, altering the m6A RNA methylation pathway rescues the replicative phenotype of H1 loss. This work unveils unexpected regulatory roles of histone H1 on non-coding RNA turnover and m6A deposition, highlighting the intimate relationship between chromatin conformation, RNA metabolism, and DNA replication to maintain genome performance., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The past decade has revolutionized our understanding of regulatory noncoding RNAs (ncRNAs). Among the most recently identified ncRNAs are downstream-of-gene (DoG)-containing transcripts that are produced by widespread transcriptional readthrough. The discovery of DoGs has set the stage for future studies to address many unanswered questions regarding the mechanisms that promote readthrough transcription, RNA processing, and the cellular functions of the unique transcripts. In this review, we summarize current findings regarding the biogenesis, function, and mechanisms regulating this exciting new class of RNA molecules., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The tumor microenvironment (TME) is a dynamic network of cellular organization that comprises diverse cell types and significantly contributes to cancer development. As pivotal immune stromal cells in the TME, macrophages are extensively heterogeneous and exert both antitumor and protumor functions. Exosomes are nanosized extracellular membranous vesicles with diameters between 30 and 150 nm. By transferring multiple bioactive substances such as proteins, lipids, and nucleic acids, exosomes play an important role in the communication between cells. Recently, growing evidence has demonstrated that non-coding RNAs (ncRNAs) are enriched in exosomes and that exosomal ncRNAs are involved in the crosstalk between cancer cells and macrophages. Furthermore, circulating exosomal ncRNAs can be detected in biofluids, serving as promising noninvasive biomarkers for the early diagnosis and prognostic prediction of cancer. Exosome-based therapies are emerging as potent strategies that can be utilized to alleviate tumor progression. Herein, the present knowledge of exosomal ncRNAs and their vital roles in regulating the interplay between cancer cells and macrophages, as well as their clinical applications are summarized., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Cortical expansion in primate brains relies on enlargement of germinal zones during a prolonged developmental period. Although most mammals have two cortical germinal zones, the ventricular zone (VZ) and subventricular zone (SVZ), gyrencephalic species display an additional germinal zone, the outer subventricular zone (oSVZ), which increases the number and diversity of neurons generated during corticogenesis. How the oSVZ emerged during evolution is poorly understood, but recent studies suggest a role for non-coding RNAs, which allow tight genetic program regulation during development. Here, using in vivo functional genetics, single-cell RNA sequencing, live imaging, and electrophysiology to assess progenitor and neuronal properties in mice, we identify two oSVZ-expressed microRNAs (miRNAs), miR-137 and miR-122, which regulate key cellular features of cortical expansion. miR-137 promotes basal progenitor self-replication and superficial layer neuron fate, whereas miR-122 decreases the pace of neuronal differentiation. These findings support a cell-type-specific role of miRNA-mediated gene expression in cortical expansion., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The role of chromatin-associated RNAi components in the nucleus of mammalian cells and in particular in the context of developmental programs remains to be elucidated. Here, we investigate the function of nuclear Argonaute 1 (Ago1) in gene expression regulation during skeletal muscle differentiation. We show that Ago1 is required for activation of the myogenic program by supporting chromatin modification mediated by developmental enhancer activation. Mechanistically, we demonstrate that Ago1 directly controls global H3K27 acetylation (H3K27ac) by regulating enhancer RNA (eRNA)-CREB-binding protein (CBP) acetyltransferase interaction, a key step in enhancer-driven gene activation. In particular, we show that Ago1 is specifically required for myogenic differentiation 1 (MyoD) and downstream myogenic gene activation, whereas its depletion leads to failure of CBP acetyltransferase activation and blocking of the myogenic program. Our work establishes a role of the mammalian enhancer-associated RNAi component Ago1 in epigenome regulation and activation of developmental programs., Competing Interests: Declaration of interests Authors declare no competing interests., (Copyright © 2021 King Abdullah University of Science and Technology. Published by Elsevier Inc. All rights reserved.)

Natural alleles that control multiple disease resistance (MDR) are valuable for crop breeding. However, only one MDR gene has been cloned in maize, and the molecular mechanisms of MDR remain unclear in maize. In this study, through map-based cloning we cloned a teosinte-derived allele of a resistance gene, Mexicana lesion mimic 1 (ZmMM1), which causes a lesion mimic phenotype and confers resistance to northern leaf blight (NLB), gray leaf spot (GLS), and southern corn rust (SCR) in maize. Strong MDR conferred by the teosinte allele is linked with polymorphisms in the 3' untranslated region of ZmMM1 that cause increased accumulation of ZmMM1 protein. ZmMM1 acts as a transcription repressor and negatively regulates the transcription of specific target genes, including ZmMM1-target gene 3 (ZmMT3), which functions as a negative regulator of plant immunity and associated cell death. The successful isolation of the ZmMM1 resistance gene will help not only in developing broad-spectrum and durable disease resistance but also in understanding the molecular mechanisms underlying MDR., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

Immunoglobulin heavy chain (IgH) locus-associated G-rich long noncoding RNA (SμGLT) is important for physiological and pathological B cell DNA recombination. We demonstrate that the METTL3 enzyme-catalyzed N 6 -methyladenosine (m 6 A) RNA modification drives recognition and 3' end processing of SμGLT by the RNA exosome, promoting class switch recombination (CSR) and suppressing chromosomal translocations. The recognition is driven by interaction of the MPP6 adaptor protein with nuclear m 6 A reader YTHDC1. MPP6 and YTHDC1 promote CSR by recruiting AID and the RNA exosome to actively transcribe SμGLT. Direct suppression of m 6 A modification of SμGLT or of m 6 A reader YTHDC1 reduces CSR. Moreover, METTL3, an essential gene for B cell development in the bone marrow and germinal center, suppresses IgH-associated aberrant DNA breaks and prevents genomic instability. Taken together, we propose coordinated and central roles for MPP6, m 6 A modification, and m 6 A reader proteins in controlling long noncoding RNA processing, DNA recombination, and development in B cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Nuclear chromosomes transcribe far more RNA than required to encode protein. Here we investigate whether non-coding RNA broadly contributes to cytological-scale chromosome territory architecture. We develop a procedure that depletes soluble proteins, chromatin, and most nuclear RNA from the nucleus but does not delocalize XIST, a known architectural RNA, from an insoluble chromosome "scaffold." RNA-seq analysis reveals that most RNA in the nuclear scaffold is repeat-rich, non-coding, and derived predominantly from introns of nascent transcripts. Insoluble, repeat-rich (C 0 T-1) RNA co-distributes with known scaffold proteins including scaffold attachment factor A (SAF-A), and distribution of these components inversely correlates with chromatin compaction in normal and experimentally manipulated nuclei. We further show that RNA is required for SAF-A to interact with chromatin and for enrichment of structurally embedded "scaffold attachment regions" prevalent in euchromatin. Collectively, the results indicate that long nascent transcripts contribute a dynamic structural role that promotes the open architecture of active chromosome territories., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

The majority of the mammalian genome is transcribed into non-coding RNAs, many of which co-evolve with RNA-binding proteins (RBPs) to function as biochemically defined and tractable ribonucleoproteins (RNPs). Here, we applied icSHAPE- a robust and versatile RNA structural probing pipeline- to endogenous RNPs purified from nuclei, providing base-resolution structural rationale for RNP activity and subcellular localization. Combining with genetic and biochemical reconstitutions, structural and functional alternations can be directly attributed to a given RBP without ambiguity. For complete details on the use and execution of this protocol, please refer to Chen et al. (2018)., Competing Interests: The authors declare no competing interests.

While the role of transcription factors and coactivators in controlling enhancer activity and chromatin structure linked to gene expression is well established, the involvement of corepressors is not. Using inflammatory macrophage activation as a model, we investigate here a corepressor complex containing GPS2 and SMRT both genome-wide and at the Ccl2 locus, encoding the chemokine CCL2 (MCP-1). We report that corepressors co-occupy candidate enhancers along with the coactivators CBP (H3K27 acetylase) and MED1 (mediator) but act antagonistically by repressing eRNA transcription-coupled H3K27 acetylation. Genome editing, transcriptional interference, and cistrome analysis reveals that apparently related enhancer and silencer elements control Ccl2 transcription in opposite ways. 4C-seq indicates that corepressor depletion or inflammatory signaling functions mechanistically similarly to trigger enhancer activation. In ob/ob mice, adipose tissue macrophage-selective depletion of the Ccl2 enhancer-transcribed eRNA reduces metaflammation. Thus, the identified corepressor-eRNA-chemokine pathway operates in vivo and suggests therapeutic opportunities by targeting eRNAs in immuno-metabolic diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The lymphatic vascular system acts as the major transportation highway of tissue fluids, and its activation or impairment is associated with a wide range of diseases. There has been increasing interest in understanding the mechanisms that control lymphatic vessel formation (lymphangiogenesis) and function in development and disease. Here, we discuss recent insights into new players whose identification has contributed to deciphering the lymphatic regulatory code. We reveal how lymphatic endothelial cells, the building blocks of lymphatic vessels, utilize their transcriptional, post-transcriptional, and epigenetic portfolio to commit to and maintain their vascular lineage identity and function, with a particular focus on development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Transcription through noncoding regions of the genome is pervasive. How these transcription events regulate gene expression remains poorly understood. Here, we report that, in S. cerevisiae, the levels of transcription through a noncoding region, IRT2, located upstream in the promoter of the inducer of meiosis, IME1, regulate opposing chromatin and transcription states. At low levels, the act of IRT2 transcription promotes histone exchange, delivering acetylated histone H3 lysine 56 to chromatin locally. The subsequent open chromatin state directs transcription factor recruitment and induces downstream transcription to repress the IME1 promoter and meiotic entry. Conversely, increasing transcription turns IRT2 into a repressor by promoting transcription-coupled chromatin assembly. The two opposing functions of IRT2 transcription shape a regulatory circuit, which ensures a robust cell-type-specific control of IME1 expression and yeast meiosis. Our data illustrate how intergenic transcription levels are key to controlling local chromatin state, gene expression, and cell fate outcomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA (ncRNA). The RT uses the ncRNA as template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. We report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the ncRNA, and an effector protein. We examined multiple retron systems and show that they confer defense against a broad range of phages via abortive infection. Focusing on retron Ec48, we show evidence that it "guards" RecBCD, a complex with central anti-phage functions in bacteria. Inhibition of RecBCD by phage proteins activates the retron, leading to abortive infection and cell death. Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed., Competing Interests: Declaration of Interests R.S. is a scientific cofounder and consultant of BiomX Ltd., Pantheon Ltd., and EcoPhage., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The xbp-1 mRNA encodes the XBP-1 transcription factor, a critical part of the unfolded protein response. Here we report that an RNA fragment produced from xbp-1 mRNA cleavage is a biologically active non-coding RNA (ncRNA) essential for axon regeneration in Caenorhabditis elegans. We show that the xbp-1 ncRNA acts independently of the protein-coding function of the xbp-1 transcript as part of a dual output xbp-1 mRNA stress response axis. Structural analysis indicates that the function of the xbp-1 ncRNA depends on a single RNA stem; this stem forms only in the cleaved xbp-1 ncRNA fragment. Disruption of this stem abolishes the non-coding, but not the coding, function of the endogenous xbp-1 transcript. Thus, cleavage of the xbp-1 mRNA bifurcates it into a coding and a non-coding pathway; modulation of the two pathways may allow neurons to fine-tune their response to injury and other stresses., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A large share of the non-coding transcriptome in yeast is controlled by the Nrd1-Nab3-Sen1 (NNS) complex, which promotes transcription termination of non-coding RNA (ncRNA) genes, and by the nuclear exosome, which limits the steady-state levels of the transcripts produced. How unconstrained ncRNA levels affect RNA metabolism and gene expression are long-standing and important questions. Here, we show that degradation of ncRNAs by the exosome is required for freeing Nrd1 and Nab3 from the released transcript after termination. In exosome mutants, these factors are sequestered by ncRNAs and cannot be efficiently recycled to sites of transcription, inducing termination defects at NNS targets. ncRNA-dependent, genome-wide termination defects can be recapitulated by the expression of a degradation-resistant, circular RNA containing a natural NNS target in exosome-proficient cells. Our results have important implications for the mechanism of termination, the general impact of ncRNAs abundance, and the importance of nuclear ncRNA degradation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020. Published by Elsevier Inc.)

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Regulation of translation during human development is poorly understood, and its dysregulation is associated with Rett syndrome (RTT). To discover shifts in mRNA ribosomal engagement (RE) during human neurodevelopment, we use parallel translating ribosome affinity purification sequencing (TRAP-seq) and RNA sequencing (RNA-seq) on control and RTT human induced pluripotent stem cells, neural progenitor cells, and cortical neurons. We find that 30% of transcribed genes are translationally regulated, including key gene sets (neurodevelopment, transcription and translation factors, and glycolysis). Approximately 35% of abundant intergenic long noncoding RNAs (lncRNAs) are ribosome engaged. Neurons translate mRNAs more efficiently and have longer 3' UTRs, and RE correlates with elements for RNA-binding proteins. RTT neurons have reduced global translation and compromised mTOR signaling, and >2,100 genes are translationally dysregulated. NEDD4L E3-ubiquitin ligase is translationally impaired, ubiquitinated protein levels are reduced, and protein targets accumulate in RTT neurons. Overall, the dynamic translatome in neurodevelopment is disturbed in RTT and provides insight into altered ubiquitination that may have therapeutic implications., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Glial cell-derived neurotrophic factor (GDNF) has a potent action in promoting the survival of dopamine (DA) neurons. Several studies indicate that increasing GDNF levels may be beneficial for the treatment of Parkinson's disease (PD) by reducing neurodegeneration of DA neurons. Despite a plethora of preclinical studies showing GDNF efficacy in PD animal models, its application in humans remains questionable for its poor efficacy and side effects due to its uncontrolled, ectopic expression. Here we took advantage of SINEUPs, a new class of antisense long non-coding RNA, that promote translation of partially overlapping sense protein-coding mRNAs with no effects on their mRNA levels. By synthesizing a SINEUP targeting Gdnf mRNA, we were able to increase endogenous GDNF protein levels by about 2-fold. Adeno-associated virus (AAV)9-mediated delivery in the striatum of wild-type (WT) mice led to an increase of endogenous GDNF protein for at least 6 months and the potentiation of the DA system's functions while showing no side effects. Furthermore, SINEUP-GDNF was able to ameliorate motor deficits and neurodegeneration of DA neurons in a PD neurochemical mouse model. Our data indicate that SINEUP-GDNF could represent a new strategy to increase endogenous GDNF protein levels in a more physiological manner for therapeutic treatments of PD., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

How adult neurons coordinate lipid metabolism to regenerate axons remains elusive. We found that depleting neuronal lipin1, a key enzyme controlling the balanced synthesis of glycerolipids through the glycerol phosphate pathway, enhanced axon regeneration after optic nerve injury. Axotomy elevated lipin1 in retinal ganglion cells, which contributed to regeneration failure in the CNS by favorably producing triglyceride (TG) storage lipids rather than phospholipid (PL) membrane lipids in neurons. Regrowth induced by lipin1 depletion required TG hydrolysis and PL synthesis. Decreasing TG synthesis by deleting neuronal diglyceride acyltransferases (DGATs) and enhancing PL synthesis through the Kennedy pathway promoted axon regeneration. In addition, peripheral neurons adopted this mechanism for their spontaneous axon regeneration. Our study reveals a critical role of lipin1 and DGATs as intrinsic regulators of glycerolipid metabolism in neurons and indicates that directing neuronal lipid synthesis away from TG synthesis and toward PL synthesis may promote axon regeneration., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

For decades, research into cancer biology focused on the involvement of protein-coding genes. Only recently was it discovered that an entire class of molecules, termed non-coding RNA (ncRNA), plays key regulatory roles in shaping cellular activity. An explosion of studies into ncRNA biology has since shown that they represent a diverse and prevalent group of RNAs, including both oncogenic molecules and those that work in a tumor suppressive manner. As a result, hundreds of cancer-focused clinical trials involving ncRNAs as novel biomarkers or therapies have begun and these are likely just the beginning., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Interferon gamma (IFN-γ), critical for host defense and tumor surveillance, requires tight control of its expression. Multiple cis-regulatory elements exist around Ifng along with a non-coding transcript, Ifng-as1 (also termed NeST). Here, we describe two genetic models generated to dissect the molecular functions of this locus and its RNA product. DNA deletion within the Ifng-as1 locus disrupted chromatin organization of the extended Ifng locus, impaired Ifng response, and compromised host defense. Insertion of a polyA signal ablated the Ifng-as1 full-length transcript and impaired host defense, while allowing proper chromatin structure. Transient knockdown of Ifng-as1 also reduced IFN-γ production. In humans, discordant expression of IFNG and IFNG-AS1 was evident in memory T cells, with high expression of this long non-coding RNA (lncRNA) and low expression of the cytokine. These results establish Ifng-as1 as an important regulator of Ifng expression, as a DNA element and transcribed RNA, involved in dynamic and cell state-specific responses to infection., (Published by Elsevier Inc.)

The human ether-a-go-go-related gene KCNH2 encodes the voltage-gated potassium channel underlying I Kr , a current critical for the repolarization phase of the cardiac action potential. Mutations in KCNH2 that cause a reduction of the repolarizing current can result in cardiac arrhythmias associated with long-QT syndrome. Here, we investigate the regulation of KCNH2 and identify multiple active enhancers. A transcribed enhancer ∼85 kbp downstream of Kcnh2 physically contacts the promoters of two Kcnh2 isoforms in a cardiac-specific manner in vivo. Knockdown of its ncRNA transcript results in reduced expression of Kcnh2b and two neighboring mRNAs, Nos3 and Abcb8, in vitro. Genomic deletion of the enhancer, including the ncRNA transcription start site, from the mouse genome causes a modest downregulation of both Kcnh2a and Kcnh2b in the ventricles. These findings establish that the regulation of Kcnh2a and Kcnh2b is governed by a complex regulatory landscape that involves multiple partially redundantly acting enhancers., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Mediator is a large protein complex required for basal and regulated expression of most RNA polymerase II (RNAP II)-transcribed genes, in part due to its interaction with and phosphorylation of the conserved C-terminal domain (CTD) of Rpb1 [1, 2]. Mediator has been implicated in many aspects of gene expression including chromatin looping [3], higher-order chromatin folding [4], mRNA processing [5] and export [6], and transcriptional memory [7]. Mediator is thought to have played a major role during eukaryotic diversification [8, 9], although its function remains unknown in evolutionarily deep branching eukaryotes lacking canonical CTD heptad repeats. We used the ciliate protozoan Tetrahymena thermophila as a model organism whose genome encodes a highly divergent Rpb1 lacking canonical CTD heptad repeats. We endogenously tagged the Med31 subunit of the Mediator complex and performed affinity purification coupled with mass spectrometry (AP-MS) to identify Mediator subunits. We found that Med31 physically interacts with a large number of proteins (>20), several of which share similarities to canonical Mediator subunits in yeast and humans as well as Tetrahymena-specific proteins. Furthermore, Med31 ChIP-seq analysis suggested a global role for Mediator in transcription regulation. We demonstrated that MED31 knockdown in growing Tetrahymena results in the ectopic expression of developmental genes important for programmed DNA rearrangements. In addition, indirect immunofluorescence revealed Med31 localization in meiotic micronuclei, implicating Mediator in RNAPII-dependent ncRNA transcription. Our results reveal structural and functional insights and implicate Mediator as an ancient cellular machinery for transcription regulation with a possible involvement in global transcription of ncRNAs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

To preserve genome integrity, eukaryotic cells use small RNA-directed mechanisms to repress transposable elements (TEs). Paradoxically, in order to silence TEs, precursors of the small RNAs must be transcribed from TEs. However, it is still poorly understood how these precursors are transcribed from TEs under silenced conditions. In the otherwise transcriptionally silent germline micronucleus (MIC) of Tetrahymena, a burst of non-coding RNA (ncRNA) transcription occurs during meiosis. The transcripts are processed into small RNAs that serve to identify TE-related sequences for elimination. The Mediator complex (Med) has an evolutionarily conserved role for transcription by bridging gene-specific transcription factors and RNA polymerase II. Here, we report that three Med-associated factors, Emit1, Emit2, and Rib1, are required for the biogenesis of small ncRNAs. Med localizes to the MIC only during meiosis, and both Med localization and MIC ncRNA transcription require Emit1 and Emit2. In the MIC, Med occupies TE-rich pericentromeric and telomeric regions in a Rib1-dependent manner. Rib1 is dispensable for ncRNA transcription but is required for the accumulation of double-stranded ncRNAs. Nuclear and sub-nuclear localization of the three Med-associated proteins is interdependent. Hence, Emit1 and Emit2 act coordinately to import Med into the MIC, and Rib1 recruits Med to specific chromosomal locations to quantitatively or qualitatively promote the biogenesis of functional ncRNA. Our results underscore that the transcription machinery can be regulated by a set of specialized Med-associated proteins to temporally transcribe TE-related sequences from a silent genome for small RNA biogenesis and genome defense., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The Nrd1-Nab3-Sen1 (NNS) complex carries out the transcription termination of non-coding RNAs (ncRNAs) by RNA polymerase II (Pol II) in yeast, although the detailed interactions among its subunits remain obscure. Here we have identified three sequence motifs in Sen1 that mediate direct interactions with the Pol II CTD interaction domain (CID) of Nrd1, determined the crystal structures of these Nrd1 interaction motifs (NIMs) bound to the CID, and characterized the interactions in vitro and in yeast. Removal of all three NIMs abolishes NNS complex formation and gives rise to ncRNA termination defects., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cellular RNAs often colocalize with cytoplasmic, membrane-less ribonucleoprotein (RNP) granules enriched for RNA-processing enzymes, termed processing bodies (PBs). Here we track the dynamic localization of individual miRNAs, mRNAs, and long non-coding RNAs (lncRNAs) to PBs using intracellular single-molecule fluorescence microscopy. We find that unused miRNAs stably bind to PBs, whereas functional miRNAs, repressed mRNAs, and lncRNAs both transiently and stably localize within either the core or periphery of PBs, albeit to different extents. Consequently, translation potential and 3' versus 5' placement of miRNA target sites significantly affect the PB localization dynamics of mRNAs. Using computational modeling and supporting experimental approaches, we show that partitioning in the PB phase attenuates mRNA silencing, suggesting that physiological mRNA turnover occurs predominantly outside of PBs. Instead, our data support a PB role in sequestering unused miRNAs for surveillance and provide a framework for investigating the dynamic assembly of RNP granules by phase separation at single-molecule resolution., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The cancer transcriptome is remarkably complex, including low-abundance transcripts, many not polyadenylated. To fully characterize the transcriptome of localized prostate cancer, we performed ultra-deep total RNA-seq on 144 tumors with rich clinical annotation. This revealed a linear transcriptomic subtype associated with the aggressive intraductal carcinoma sub-histology and a fusion profile that differentiates localized from metastatic disease. Analysis of back-splicing events showed widespread RNA circularization, with the average tumor expressing 7,232 circular RNAs (circRNAs). The degree of circRNA production was correlated to disease progression in multiple patient cohorts. Loss-of-function screening identified 11.3% of highly abundant circRNAs as essential for cell proliferation; for ∼90% of these, their parental linear transcripts were not essential. Individual circRNAs can have distinct functions, with circCSNK1G3 promoting cell growth by interacting with miR-181. These data advocate for adoption of ultra-deep RNA-seq without poly-A selection to interrogate both linear and circular transcriptomes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The fidelity of transcription initiation is essential for accurate gene expression, but the determinants of start site selection are not fully understood. Rap1 and other general regulatory factors (GRFs) control the expression of many genes in yeast. We show that depletion of these factors induces widespread ectopic transcription initiation within promoters. This generates many novel non-coding RNAs and transcript isoforms with diverse stability, drastically altering the coding potential of the transcriptome. Ectopic transcription initiation strongly correlates with altered nucleosome positioning. We provide evidence that Rap1 can suppress ectopic initiation by a "place-holder" mechanism whereby it physically occludes inappropriate sites for pre-initiation complex formation. These results reveal an essential role for GRFs in the fidelity of transcription initiation and in the suppression of pervasive transcription, profoundly redefining current models for their function. They have important implications for the mechanism of transcription initiation and the control of gene expression., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Many active eukaryotic gene promoters exhibit divergent noncoding transcription, but the mechanisms restricting expression of these transcripts are not well understood. Here, we demonstrate how a sequence-specific transcription factor represses divergent noncoding transcription at highly expressed genes in yeast. We find that depletion of the transcription factor Rap1 induces noncoding transcription in a large fraction of Rap1-regulated gene promoters. Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction. We further provide evidence that Rap1 acts independently of previously described chromatin-based mechanisms to repress cryptic or divergent transcription. Finally, we show that divergent transcription in the absence of Rap1 is elicited by the RSC chromatin remodeler. We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality toward productive transcription., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Long non-coding RNAs (lncRNAs) can often function in the regulation of gene expression during development; however, their generality as essential regulators in developmental processes and organismal phenotypes remains unclear. Here, we performed a tailored investigation of lncRNA expression and function during Drosophila embryogenesis, interrogating multiple stages, tissue specificity, nuclear localization, and genetic backgrounds. Our results almost double the number of annotated lncRNAs expressed at these embryonic stages. lncRNA levels are generally positively correlated with those of their neighboring genes, with little evidence of transcriptional interference. Using fluorescent in situ hybridization, we report the spatiotemporal expression of 15 new lncRNAs, revealing very dynamic tissue-specific patterns. Despite this, deletion of selected lncRNA genes had no obvious developmental defects or effects on viability under standard and stressed conditions. However, two lncRNA deletions resulted in modest expression changes of a small number of genes, suggesting that they fine-tune expression of non-essential genes. Several lncRNAs have strain-specific expression, indicating that they are not fixed within the population. This intra-species variation across genetic backgrounds may thereby be a useful tool to distinguish rapidly evolving lncRNAs with as yet non-essential roles., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Protein kinase RNA-activated (PKR) induces immune response by sensing viral double-stranded RNAs (dsRNAs). However, growing evidence suggests that PKR can also be activated by endogenously expressed dsRNAs. Here, we capture these dsRNAs by formaldehyde-mediated crosslinking and immunoprecipitation sequencing and find that various noncoding RNAs interact with PKR. Surprisingly, the majority of the PKR-interacting RNA repertoire is occupied by mitochondrial RNAs (mtRNAs). MtRNAs can form intermolecular dsRNAs owing to bidirectional transcription of the mitochondrial genome and regulate PKR and eIF2α phosphorylation to control cell signaling and translation. Moreover, PKR activation by mtRNAs is counteracted by PKR phosphatases, disruption of which causes apoptosis from PKR overactivation even in uninfected cells. Our work unveils dynamic regulation of PKR even without infection and establishes PKR as a sensor for nuclear and mitochondrial signaling cues in regulating cellular metabolism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Amyloid bodies (A-bodies) are inducible membrane-less nuclear compartments composed of heterogeneous proteins that adopt an amyloid-like state. A-bodies are seeded by noncoding RNA derived from stimuli-specific loci of the rDNA intergenic spacer (rIGSRNA). This raises the question of how rIGSRNA recruits a large population of diverse proteins to confer A-body identity. Here, we show that long low-complexity dinucleotide repeats operate as the architectural determinants of rIGSRNA. On stimulus, clusters of rIGSRNA with simple cytosine/uracil (CU) or adenosine/guanine (AG) repeats spanning hundreds of nucleotides accumulate in the nucleolar area. The low-complexity sequences facilitate charge-based interactions with short cationic peptides to produce multiple nucleolar liquid-like foci. Local concentration of proteins with fibrillation propensity in these nucleolar foci induces the formation of an amyloidogenic liquid phase that seeds A-bodies. These results demonstrate the physiological importance of low-complexity RNA and repetitive regions of the genome often dismissed as "junk" DNA., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genome-wide association studies (GWAS) have identified rs11672691 at 19q13 associated with aggressive prostate cancer (PCa). Here, we independently confirmed the finding in a cohort of 2,738 PCa patients and discovered the biological mechanism underlying this association. We found an association of the aggressive PCa-associated allele G of rs11672691 with elevated transcript levels of two biologically plausible candidate genes, PCAT19 and CEACAM21, implicated in PCa cell growth and tumor progression. Mechanistically, rs11672691 resides in an enhancer element and alters the binding site of HOXA2, a novel oncogenic transcription factor with prognostic potential in PCa. Remarkably, CRISPR/Cas9-mediated single-nucleotide editing showed the direct effect of rs11672691 on PCAT19 and CEACAM21 expression and PCa cellular aggressive phenotype. Clinical data demonstrated synergistic effects of rs11672691 genotype and PCAT19/CEACAM21 gene expression on PCa prognosis. These results provide a plausible mechanism for rs11672691 associated with aggressive PCa and thus lay the ground work for translating this finding to the clinic., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Animal reproduction responds to nutritional status. During starvation, Drosophila and Caenorhabditis elegans enter a period of reproductive diapause with increase apoptosis, while maintaining a stable pool of germline stem cells (GSCs). How GSCs are protected is not understood. Here, we show that a sisRNA/miRNA axis maintains ovarian GSCs during starvation in Drosophila. Starvation induces the expression of an ovary-enriched sisRNA sisR-2, which negatively regulates GSC maintenance via a fatty acid metabolism gene dFAR1. sisR-2 promotes the expression of bantam, which in turn inhibits the activity of sisR-2, forming a negative feedback loop. Therefore, bantam acts as a buffer to counteract sisR-2 activity to prevent GSC loss during starvation. We propose that the sisR-2/bantam axis confers robustness to GSCs in Drosophila., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The enhancer regions of the myogenic master regulator MyoD give rise to at least two enhancer RNAs. Core enhancer eRNA ( CE eRNA) regulates transcription of the adjacent MyoD gene, whereas DRR eRNA affects expression of Myogenin in trans. We found that DRR eRNA is recruited at the Myogenin locus, where it colocalizes with Myogenin nascent transcripts. DRR eRNA associates with the cohesin complex, and this association correlates with its transactivating properties. Despite being expressed in undifferentiated cells, cohesin is not loaded on Myogenin until the cells start expressing DRR eRNA, which is then required for cohesin chromatin recruitment and maintenance. Functionally, depletion of either cohesin or DRR eRNA reduces chromatin accessibility, prevents Myogenin activation, and hinders muscle cell differentiation. Thus, DRR eRNA ensures spatially appropriate cohesin loading in trans to regulate gene expression., (Published by Elsevier Inc.)

Many non-coding transcripts (ncRNA) generated by RNA polymerase II in S. cerevisiae are terminated by the Nrd1-Nab3-Sen1 complex. However, Sen1 helicase levels are surprisingly low compared with Nrd1 and Nab3, raising questions regarding how ncRNA can be terminated in an efficient and timely manner. We show that Sen1 levels increase during the S and G2 phases of the cell cycle, leading to increased termination activity of NNS. Overexpression of Sen1 or failure to modulate its abundance by ubiquitin-proteasome-mediated degradation greatly decreases cell fitness. Sen1 toxicity is suppressed by mutations in other termination factors, and NET-seq analysis shows that its overexpression leads to a decrease in ncRNA production and altered mRNA termination. We conclude that Sen1 levels are carefully regulated to prevent aberrant termination. We suggest that ncRNA levels and coding gene transcription termination are modulated by Sen1 to fulfill critical cell cycle-specific functions., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Covalent nucleotide modifications in noncoding RNAs affect a plethora of biological processes, and new functions continue to be discovered even for well-known modifying enzymes. To systematically compare the functions of a large set of noncoding RNA modifications in gene regulation, we carried out ribosome profiling in budding yeast to characterize 57 nonessential genes involved in tRNA modification. Deletion mutants exhibited a range of translational phenotypes, with enzymes known to modify anticodons, or non-tRNA substrates such as rRNA, exhibiting the most dramatic translational perturbations. Our data build on prior reports documenting translational upregulation of the nutrient-responsive transcription factor Gcn4 in response to numerous tRNA perturbations, and identify many additional translationally regulated mRNAs throughout the yeast genome. Our data also uncover unexpected roles for tRNA-modifying enzymes in regulation of TY retroelements, and in rRNA 2'-O-methylation. This dataset should provide a rich resource for discovery of additional links between tRNA modifications and gene regulation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Noncoding RNAs (ncRNAs) regulate gene expression in all organisms. Bacterial 6S RNAs globally regulate transcription by binding RNA polymerase (RNAP) holoenzyme and competing with promoter DNA. Escherichia coli (Eco) 6S RNA interacts specifically with the housekeeping σ 70 -holoenzyme (Eσ 70 ) and plays a key role in the transcriptional reprogramming upon shifts between exponential and stationary phase. Inhibition is relieved upon 6S RNA-templated RNA synthesis. We report here the 3.8 Å resolution structure of a complex between 6S RNA and Eσ 70 determined by single-particle cryo-electron microscopy and validation of the structure using footprinting and crosslinking approaches. Duplex RNA segments have A-form C3' endo sugar puckers but widened major groove widths, giving the RNA an overall architecture that mimics B-form promoter DNA. Our results help explain the specificity of Eco 6S RNA for Eσ 70 and show how an ncRNA can mimic B-form DNA to directly regulate transcription by the DNA-dependent RNAP., (Copyright © 2017 Elsevier Inc. All rights reserved.)