A scalable and cost-efficient rRNA depletion approach to enrich RNAs for molecular biology investigations.

Transcriptomics analyses play pivotal roles in understanding the complex regulatory networks that govern cellular processes. The abundance of rRNAs, which account for 80%-90% of total RNA in eukaryotes, limits the detection and investigation of other transcripts. While mRNAs and long noncoding RNAs have poly(A) tails that are often used for positive selection, investigations of poly(A)- RNAs, such as circular RNAs, histone mRNAs, and small RNAs, typically require the removal of the abundant rRNAs for enrichment. Current approaches to deplete rRNAs for downstream molecular biology investigations are hampered by restrictive RNA input masses and high costs. To address these challenges, we developed rRNA Removal by RNaseH (rRRR), a method to efficiently deplete rRNAs from a wide range of human, mouse, and rat RNA inputs and of varying qualities at a cost 10- to 20-fold cheaper than other approaches. We used probe-based hybridization and enzymatic digestion to selectively target and remove rRNA molecules while preserving the integrity of non-rRNA transcripts. Comparison of rRRR to two commercially available approaches showed similar rRNA depletion efficiencies and comparable off-target effects. Our developed method provides researchers with a valuable tool for investigating gene expression and regulatory mechanisms across a wide range of biological systems at an affordable price that increases the accessibility for researchers to enter the field, ultimately advancing our understanding of cellular processes.

circCsnk1g3- and circAnkib1-regulated interferon responses in sarcoma promote tumorigenesis by shaping the immune microenvironment.

Exonic circular RNAs (circRNAs) produce predominantly non-coding RNA species that have been recently profiled in many tumors. However, their functional contribution to cancer progression is still poorly understood. Here, we identify the circRNAs expressed in soft tissue sarcoma cells and explore how the circRNAs regulate sarcoma growth in vivo. We show that circCsnk1g3 and circAnkib1 promote tumor growth by shaping a pro-tumorigenic microenvironment, possibly due to their capabilities to regulate tumor-promoting elements extrinsic to the tumor cells. Accordingly, circCsnk1g3 and circAnkib1 can control the expression of interferon-related genes and pro-inflammatory factors in the sarcoma cells, thus directing immune cell recruitment into the tumor mass, and hence their activation. Mechanistically, circRNAs may repress pro-inflammatory elements by buffering activation of the pathways mediated by RIG-I, the cytosolic viral RNA sensor. The current findings suggest that the targeting of specific circRNAs could augment the efficacy of tumor and immune response to mainstay therapies.

Inflammasome activation in infected macrophages drives COVID-19 pathology.

Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA and a sustained interferon (IFN) response, all of which are recapitulated and required for pathology in the SARS-CoV-2-infected MISTRG6-hACE2 humanized mouse model of COVID-19, which has a human immune system1-20. Blocking either viral replication with remdesivir21-23 or the downstream IFN-stimulated cascade with anti-IFNAR2 antibodies in vivo in the chronic stages of disease attenuates the overactive immune inflammatory response, especially inflammatory macrophages. Here we show that SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release interleukin 1 (IL-1) and IL-18, and undergo pyroptosis, thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and the accompanying inflammatory response are necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Notably, this blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 through the production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.

Inflammasome activation in infected macrophages drives COVID-19 pathology.

Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA, and sustained interferon (IFN) response all of which are recapitulated and required for pathology in the SARS-CoV-2 infected MISTRG6-hACE2 humanized mouse model of COVID-19 with a human immune system 1-20 . Blocking either viral replication with Remdesivir 21-23 or the downstream IFN stimulated cascade with anti-IFNAR2 in vivo in the chronic stages of disease attenuated the overactive immune-inflammatory response, especially inflammatory macrophages. Here, we show SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release IL-1 and IL-18 and undergo pyroptosis thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and its accompanying inflammatory response is necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Remarkably, this same blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 by production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.

Cellular origins of dsRNA, their recognition and consequences.

Double-stranded RNA (dsRNA) is associated with most viral infections - it either constitutes the viral genome (in the case of dsRNA viruses) or is generated in host cells during viral replication. Hence, nearly all organisms have the capability of recognizing dsRNA and mounting a response, the primary aim of which is to mitigate the potential infection. In vertebrates, a set of innate immune receptors for dsRNA induce a multitude of cell-intrinsic and cell-extrinsic immune responses upon dsRNA recognition. Notably, recent studies showed that vertebrate cells can accumulate self-derived dsRNAs or dsRNA-like species upon dysregulation of several cellular processes, activating the very same immune pathways as in infected cells. On the one hand, such aberrant immune activation in the absence of infection can lead to pathogenesis of immune disorders, such as Aicardi-Goutières syndrome. On the other hand, the same innate immune reaction can be induced in a controlled setting for a therapeutic benefit, as occurs in immunotherapies. In this Review, we describe mechanisms by which immunostimulatory dsRNAs are generated in mammalian cells, either by viruses or by the host cells, and how cells respond to them, with the focus on recent developments regarding the role of cellular dsRNAs in immune modulation.

The design and synthesis of circular RNAs.

Circular RNAs (circRNAs) are a novel class of RNAs distinguished by their single-stranded, covalently-closed topology. Although initially perceived as rare byproducts of aberrant splicing, circRNAs are now recognized as ubiquitously expressed and functionally significant. These discoveries have led to a growing need for ways to model circRNAs in living cells to advance our understanding of their biogenesis, regulation, and function, and to adopt them as new technologies for application within research and medicine. In this review, we provide an updated summary of approaches used to produce circRNAs in vitro and in vivo, the latter of which has grown considerably in recent years. Given increased interest in the unique functions carried out by individual circRNAs, we further dedicate a section on how to customize synthesized circRNAs for specific biological roles. We focus on the most common applications, including designing circRNAs for protein delivery, to target miRNAs and proteins, to act as fluorescent reporters, and to modulate cellular immunity.

Emerging roles of circular RNAs in innate immunity.

The proper function of the innate immune system depends on an intricate network of regulation that promotes effective responses to pathogens while avoiding autoimmunity. Circular RNAs (circRNAs), a class of RNAs that lack 5' and 3' ends, have emerged as key actors in these networks. Recent studies have demonstrated that endogenous circRNAs in eukaryotes regulate the activation of innate immune proteins and cells through diverse modes of action. Some DNA viruses also encode circRNAs, and foreign circRNAs have been found to stimulate an innate immune response. This review summarizes recent investigations that reveal the critical roles that circRNAs play in innate immunity and points to future areas of study in this emerging field.

One Ring to Rule Them All: Mitochondrial Circular RNAs Control Mitochondrial Function.

Circular RNAs (circRNAs) have emerged as key regulators of a wide variety of biological processes, but the roles of mitochondrial circRNAs are largely unknown. In this issue of Cell, Zhao et al. (2020) reveal that mitochondrial DNA-encoded circRNAs interact with ATP synthase subunit ß (ATP5B) to inhibit the output of mitochondrial reactive oxygen species and the activation of liver fibroblasts, which regulate the pathogenesis of liver disease.

Circular RNAs in Immune Response and Viral Infection.

Circular RNAs (circRNAs) are a diverse class of RNAs with varying sizes, cellular abundance, and biological functions. Investigations from the past decade have revealed that circRNAs are ubiquitously found in eukaryotes and have defined the different biological roles of circRNAs to illuminate this previously unrecognized class of molecules. In the context of the immune system, immune responses and immune-related diseases alter circRNA expression. More recently, several oncogenic double-stranded DNA viruses have been found to encode circRNAs. In this review, we summarize the current understanding of circRNAs and their emerging functions in immune regulation and autoimmune disorders, and discuss the identification and potential roles of viral circRNAs during infections. Finally, we present promising areas for future investigations in the nascent field of circRNAs.

N6-Methyladenosine Modification Controls Circular RNA Immunity.

Circular RNAs (circRNAs) are prevalent in eukaryotic cells and viral genomes. Mammalian cells possess innate immunity to detect foreign circRNAs, but the molecular basis of self versus foreign identity in circRNA immunity is unknown. Here, we show that N6-methyladenosine (m6A) RNA modification on human circRNAs inhibits innate immunity. Foreign circRNAs are potent adjuvants to induce antigen-specific T cell activation, antibody production, and anti-tumor immunity in vivo, and m6A modification abrogates immune gene activation and adjuvant activity. m6A reader YTHDF2 sequesters m6A-circRNA and is essential for suppression of innate immunity. Unmodified circRNA, but not m6A-modified circRNA, directly activates RNA pattern recognition receptor RIG-I in the presence of lysine-63-linked polyubiquitin chain to cause filamentation of the adaptor protein MAVS and activation of the downstream transcription factor IRF3. CircRNA immunity has considerable parallel to prokaryotic DNA restriction modification system that transforms nucleic acid chemical modification into organismal innate immunity.

Promoter of lncRNA Gene PVT1 Is a Tumor-Suppressor DNA Boundary Element.

Noncoding mutations in cancer genomes are frequent but challenging to interpret. PVT1 encodes an oncogenic lncRNA, but recurrent translocations and deletions in human cancers suggest alternative mechanisms. Here, we show that the PVT1 promoter has a tumor-suppressor function that is independent of PVT1 lncRNA. CRISPR interference of PVT1 promoter enhances breast cancer cell competition and growth in vivo. The promoters of the PVT1 and the MYC oncogenes, located 55 kb apart on chromosome 8q24, compete for engagement with four intragenic enhancers in the PVT1 locus, thereby allowing the PVT1 promoter to regulate pause release of MYC transcription. PVT1 undergoes developmentally regulated monoallelic expression, and the PVT1 promoter inhibits MYC expression only from the same chromosome via promoter competition. Cancer genome sequencing identifies recurrent mutations encompassing the human PVT1 promoter, and genome editing verified that PVT1 promoter mutation promotes cancer cell growth. These results highlight regulatory sequences of lncRNA genes as potential disease-associated DNA elements.

LncRNA Seduction of GOT2 Goes Viral.

Mechanisms of viral infection are active areas of investigation. In a recent issue of Science, Wang et al. (2017) reveal an additional function of a host-encoded long non-coding RNA (lncRNA) in regulating viral expression by binding a host metabolic enzyme to enhance its catalytic activity.

The conserved RNA helicase YTHDC2 regulates the transition from proliferation to differentiation in the germline.

The switch from mitosis to meiosis is the key event marking onset of differentiation in the germline stem cell lineage. In Drosophila, the translational repressor Bgcn is required for spermatogonia to stop mitosis and transition to meiotic prophase and the spermatocyte state. Here we show that the mammalian Bgcn homolog YTHDC2 facilitates a clean switch from mitosis to meiosis in mouse germ cells, revealing a conserved role for YTHDC2 in this critical cell fate transition. YTHDC2-deficient male germ cells enter meiosis but have a mixed identity, maintaining expression of Cyclin A2 and failing to properly express many meiotic markers. Instead of continuing through meiotic prophase, the cells attempt an abnormal mitotic-like division and die. YTHDC2 binds multiple transcripts including Ccna2 and other mitotic transcripts, binds specific piRNA precursors, and interacts with RNA granule components, suggesting that proper progression of germ cells through meiosis is licensed by YTHDC2 through post-transcriptional regulation.

m6A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways.

N6-methyladenosine (m6A) is the most common and abundant messenger RNA modification, modulated by 'writers', 'erasers' and 'readers' of this mark. In vitro data have shown that m6A influences all fundamental aspects of mRNA metabolism, mainly mRNA stability, to determine stem cell fates. However, its in vivo physiological function in mammals and adult mammalian cells is still unknown. Here we show that the deletion of m6A 'writer' protein METTL3 in mouse T cells disrupts T cell homeostasis and differentiation. In a lymphopaenic mouse adoptive transfer model, naive Mettl3-deficient T cells failed to undergo homeostatic expansion and remained in the naive state for up to 12 weeks, thereby preventing colitis. Consistent with these observations, the mRNAs of SOCS family genes encoding the STAT signalling inhibitory proteins SOCS1, SOCS3 and CISH were marked by m6A, exhibited slower mRNA decay and showed increased mRNAs and levels of protein expression in Mettl3-deficient naive T cells. This increased SOCS family activity consequently inhibited IL-7-mediated STAT5 activation and T cell homeostatic proliferation and differentiation. We also found that m6A has important roles for inducible degradation of Socs mRNAs in response to IL-7 signalling in order to reprogram naive T cells for proliferation and differentiation. Our study elucidates for the first time, to our knowledge, the in vivo biological role of m6A modification in T-cell-mediated pathogenesis and reveals a novel mechanism of T cell homeostasis and signal-dependent induction of mRNA degradation.

Gene regulation in the immune system by long noncoding RNAs.

Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression in the immune system. Studies have shown that lncRNAs are expressed in a highly lineage-specific manner and control the differentiation and function of innate and adaptive cell types. In this Review, we focus on mechanisms used by lncRNAs to regulate genes encoding products involved in the immune response, including direct interactions with chromatin, RNA and proteins. In addition, we address new areas of lncRNA biology, such as the functions of enhancer RNAs, circular RNAs and chemical modifications to RNA in cellular processes. We emphasize critical gaps in knowledge and future prospects for the roles of lncRNAs in the immune system and autoimmune disease.

Sensing Self and Foreign Circular RNAs by Intron Identity.

Circular RNAs (circRNAs) are single-stranded RNAs that are joined head to tail with largely unknown functions. Here we show that transfection of purified in vitro generated circRNA into mammalian cells led to potent induction of innate immunity genes and confers protection against viral infection. The nucleic acid sensor RIG-I is necessary to sense foreign circRNA, and RIG-I and foreign circRNA co-aggregate in cytoplasmic foci. CircRNA activation of innate immunity is independent of a 5' triphosphate, double-stranded RNA structure, or the primary sequence of the foreign circRNA. Instead, self-nonself discrimination depends on the intron that programs the circRNA. Use of a human intron to express a foreign circRNA sequence abrogates immune activation, and mature human circRNA is associated with diverse RNA binding proteins reflecting its endogenous splicing and biogenesis. These results reveal innate immune sensing of circRNA and highlight introns-the predominant output of mammalian transcription-as arbiters of self-nonself identity.

Discovery and biological characterization of geranylated RNA in bacteria.

A general MS-based screen for unusually hydrophobic cellular small molecule-RNA conjugates revealed geranylated RNA in Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa and Salmonella enterica var. Typhimurium. The geranyl group is conjugated to the sulfur atom in two 5-methylaminomethyl-2-thiouridine nucleotides. These geranylated nucleotides occur in the first anticodon position of tRNA(Glu)(UUC), tRNA(Lys)(UUU) and tRNA(Gln)(UUG) at a frequency of up to 6.7% (~400 geranylated nucleotides per cell). RNA geranylation can be increased or abolished by mutation or deletion of the selU (ybbB) gene in E. coli, and purified SelU protein in the presence of geranyl pyrophosphate and tRNA can produce geranylated tRNA. The presence or absence of the geranyl group in tRNA(Glu)(UUC), tRNA(Lys)(UUU) and tRNA(Gln)(UUG) affects codon bias and frameshifting during translation. These RNAs represent the first reported examples of oligoisoprenylated cellular nucleic acids.

LC/MS analysis of cellular RNA reveals NAD-linked RNA.

We developed a general method to detect cellular small molecule-RNA conjugates that does not rely on the reactivity of the small molecule. This technique revealed NAD-linked RNA in Escherichia coli and Streptomyces venezuelae. Subsequent characterization showed that NAD is a 5' modification of RNA, cannot be installed in vitro through aberrant transcriptional initiation, is only found among smaller cellular RNAs and is present at a surprisingly high abundance of approximately 3,000 copies per cell.