RESUMEN
Most circular RNAs are produced from the back-splicing of exons of precursor mRNAs. Recent technological advances have in part overcome problems with their circular conformation and sequence overlap with linear cognate mRNAs, allowing a better understanding of their cellular roles. Depending on their localization and specific interactions with DNA, RNA, and proteins, circular RNAs can modulate transcription and splicing, regulate stability and translation of cytoplasmic mRNAs, interfere with signaling pathways, and serve as templates for translation in different biological and pathophysiological contexts. Emerging applications of RNA circles to interfere with cellular processes, modulate immune responses, and direct translation into proteins shed new light on biomedical research. In this review, we discuss approaches used in circular RNA studies and the current understanding of their regulatory roles and potential applications.
Asunto(s)
ARN Circular , ARN , Proteínas/metabolismo , ARN/metabolismo , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/metabolismoRESUMEN
Covalently closed, single-stranded circular RNAs can be produced from viral RNA genomes as well as from the processing of cellular housekeeping noncoding RNAs and precursor messenger RNAs. Recent transcriptomic studies have surprisingly uncovered that many protein-coding genes can be subjected to backsplicing, leading to widespread expression of a specific type of circular RNAs (circRNAs) in eukaryotic cells. Here, we discuss experimental strategies used to discover and characterize diverse circRNAs at both the genome and individual gene scales. We further highlight the current understanding of how circRNAs are generated and how the mature transcripts function. Some circRNAs act as noncoding RNAs to impact gene regulation by serving as decoys or competitors for microRNAs and proteins. Others form extensive networks of ribonucleoprotein complexes or encode functional peptides that are translated in response to certain cellular stresses. Overall, circRNAs have emerged as an important class of RNAmolecules in gene expression regulation that impact many physiological processes, including early development, immune responses, neurogenesis, and tumorigenesis.
Asunto(s)
MicroARNs , ARN Circular , Regulación de la Expresión Génica/genética , MicroARNs/genética , MicroARNs/metabolismo , ARN/genética , ARN/metabolismo , ARN Circular/genética , ARN no Traducido , ARN Viral , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMEN
Genes specifying long non-coding RNAs (lncRNAs) occupy a large fraction of the genomes of complex organisms. The term 'lncRNAs' encompasses RNA polymerase I (Pol I), Pol II and Pol III transcribed RNAs, and RNAs from processed introns. The various functions of lncRNAs and their many isoforms and interleaved relationships with other genes make lncRNA classification and annotation difficult. Most lncRNAs evolve more rapidly than protein-coding sequences, are cell type specific and regulate many aspects of cell differentiation and development and other physiological processes. Many lncRNAs associate with chromatin-modifying complexes, are transcribed from enhancers and nucleate phase separation of nuclear condensates and domains, indicating an intimate link between lncRNA expression and the spatial control of gene expression during development. lncRNAs also have important roles in the cytoplasm and beyond, including in the regulation of translation, metabolism and signalling. lncRNAs often have a modular structure and are rich in repeats, which are increasingly being shown to be relevant to their function. In this Consensus Statement, we address the definition and nomenclature of lncRNAs and their conservation, expression, phenotypic visibility, structure and functions. We also discuss research challenges and provide recommendations to advance the understanding of the roles of lncRNAs in development, cell biology and disease.
Asunto(s)
ARN Largo no Codificante , ARN Largo no Codificante/genética , Núcleo Celular/genética , Cromatina/genética , Secuencias Reguladoras de Ácidos Nucleicos , ARN Polimerasa II/genéticaRESUMEN
In eukaryotes, heterochromatin is generally located at the nuclear periphery. This study investigates the biological significance of perinuclear positioning for heterochromatin maintenance and gene silencing. We identify the nuclear rim protein Amo1NUPL2 as a factor required for the propagation of heterochromatin at endogenous and ectopic sites in the fission yeast genome. Amo1 associates with the Rix1PELP1-containing RNA processing complex RIXC and with the histone chaperone complex FACT. RIXC, which binds to heterochromatin protein Swi6HP1 across silenced chromosomal domains and to surrounding boundary elements, connects heterochromatin with Amo1 at the nuclear periphery. In turn, the Amo1-enriched subdomain is critical for Swi6 association with FACT that precludes histone turnover to promote gene silencing and preserve epigenetic stability of heterochromatin. In addition to uncovering conserved factors required for perinuclear positioning of heterochromatin, these analyses elucidate a mechanism by which a peripheral subdomain enforces stable gene repression and maintains heterochromatin in a heritable manner.
Asunto(s)
Epigénesis Genética/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Represión Epigenética/genética , Silenciador del Gen , Herencia , Histonas/genética , Histonas/metabolismo , Metilación , Proteínas Nucleares/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
Long noncoding RNAs (lncRNAs) evolve more rapidly than mRNAs. Whether conserved lncRNAs undergo conserved processing, localization, and function remains unexplored. We report differing subcellular localization of lncRNAs in human and mouse embryonic stem cells (ESCs). A significantly higher fraction of lncRNAs is localized in the cytoplasm of hESCs than in mESCs. This turns out to be important for hESC pluripotency. FAST is a positionally conserved lncRNA but is not conserved in its processing and localization. In hESCs, cytoplasm-localized hFAST binds to the WD40 domain of the E3 ubiquitin ligase ß-TrCP and blocks its interaction with phosphorylated ß-catenin to prevent degradation, leading to activated WNT signaling, required for pluripotency. In contrast, mFast is nuclear retained in mESCs, and its processing is suppressed by the splicing factor PPIE, which is highly expressed in mESCs but not hESCs. These findings reveal that lncRNA processing and localization are previously under-appreciated contributors to the rapid evolution of function.
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Espacio Intracelular/genética , ARN Largo no Codificante/metabolismo , Células Madre/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Células Cultivadas , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Humanos , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Empalme del ARN/genética , ARN Largo no Codificante/genética , ARN Mensajero/metabolismo , Transducción de Señal/genética , Células Madre/patologíaRESUMEN
Circular RNAs (circRNAs) produced from back-splicing of exons of pre-mRNAs are widely expressed, but current understanding of their functions is limited. These RNAs are stable in general and are thought to have unique structural conformations distinct from their linear RNA cognates. Here, we show that endogenous circRNAs tend to form 16-26 bp imperfect RNA duplexes and act as inhibitors of double-stranded RNA (dsRNA)-activated protein kinase (PKR) related to innate immunity. Upon poly(I:C) stimulation or viral infection, circRNAs are globally degraded by RNase L, a process required for PKR activation in early cellular innate immune responses. Augmented PKR phosphorylation and circRNA reduction are found in peripheral blood mononuclear cells (PBMCs) derived from patients with autoimmune disease systemic lupus erythematosus (SLE). Importantly, overexpression of the dsRNA-containing circRNA in PBMCs or T cells derived from SLE can alleviate the aberrant PKR activation cascade, thus providing a connection between circRNAs and SLE.
Asunto(s)
ARN Circular/metabolismo , ARN Circular/fisiología , eIF-2 Quinasa/metabolismo , Adolescente , Adulto , Enfermedades Autoinmunes/genética , Línea Celular , Endorribonucleasas/metabolismo , Femenino , Humanos , Inmunidad Innata/genética , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Sistémico/genética , Persona de Mediana Edad , Fosforilación , ARN/metabolismo , Empalme del ARN/genética , Estabilidad del ARN/fisiología , ARN Circular/genética , ARN Bicatenario/metabolismo , Virosis/metabolismo , eIF-2 Quinasa/inmunologíaRESUMEN
Evidence accumulated over the past decade shows that long non-coding RNAs (lncRNAs) are widely expressed and have key roles in gene regulation. Recent studies have begun to unravel how the biogenesis of lncRNAs is distinct from that of mRNAs and is linked with their specific subcellular localizations and functions. Depending on their localization and their specific interactions with DNA, RNA and proteins, lncRNAs can modulate chromatin function, regulate the assembly and function of membraneless nuclear bodies, alter the stability and translation of cytoplasmic mRNAs and interfere with signalling pathways. Many of these functions ultimately affect gene expression in diverse biological and physiopathological contexts, such as in neuronal disorders, immune responses and cancer. Tissue-specific and condition-specific expression patterns suggest that lncRNAs are potential biomarkers and provide a rationale to target them clinically. In this Review, we discuss the mechanisms of lncRNA biogenesis, localization and functions in transcriptional, post-transcriptional and other modes of gene regulation, and their potential therapeutic applications.
Asunto(s)
Regulación de la Expresión Génica , Enfermedades del Sistema Inmune/patología , Neoplasias/patología , Trastornos del Neurodesarrollo/patología , ARN Largo no Codificante/genética , Animales , Humanos , Enfermedades del Sistema Inmune/genética , Neoplasias/genética , Trastornos del Neurodesarrollo/genética , Transducción de SeñalRESUMEN
Many protein-coding genes in higher eukaryotes can produce circular RNAs (circRNAs) through back-splicing of exons. CircRNAs differ from mRNAs in their production, structure and turnover and thereby have unique cellular functions and potential biomedical applications. In this Review, I discuss recent progress in our understanding of the biogenesis of circRNAs and the regulation of their abundance and of their biological functions, including in transcription and splicing, sequestering or scaffolding of macromolecules to interfere with microRNA activities or signalling pathways, and serving as templates for translation. I further discuss the emerging roles of circRNAs in regulating immune responses and cell proliferation, and the possibilities of applying circRNA technologies in biomedical research.
Asunto(s)
ARN Circular/genética , ARN Circular/metabolismo , ARN Circular/fisiología , Empalme Alternativo/genética , Animales , Exones/genética , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Humanos , MicroARNs/metabolismo , ARN/genética , Empalme del ARN/genética , ARN Mensajero/metabolismoRESUMEN
Dysregulated rRNA synthesis by RNA polymerase I (Pol I) is associated with uncontrolled cell proliferation. Here, we report a box H/ACA small nucleolar RNA (snoRNA)-ended long noncoding RNA (lncRNA) that enhances pre-rRNA transcription (SLERT). SLERT requires box H/ACA snoRNAs at both ends for its biogenesis and translocation to the nucleolus. Deletion of SLERT impairs pre-rRNA transcription and rRNA production, leading to decreased tumorigenesis. Mechanistically, SLERT interacts with DEAD-box RNA helicase DDX21 via a 143-nt non-snoRNA sequence. Super-resolution images reveal that DDX21 forms ring-shaped structures surrounding multiple Pol I complexes and suppresses pre-rRNA transcription. Binding by SLERT allosterically alters individual DDX21 molecules, loosens the DDX21 ring, and evicts DDX21 suppression on Pol I transcription. Together, our results reveal an important control of ribosome biogenesis by SLERT lncRNA and its regulatory role in DDX21 ring-shaped arrangements acting on Pol I complexes.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , ARN Polimerasa I/metabolismo , Precursores del ARN/genética , ARN Largo no Codificante/metabolismo , Sitio Alostérico , Animales , Carcinogénesis , Línea Celular , Línea Celular Tumoral , ARN Helicasas DEAD-box/química , Femenino , Técnicas de Inactivación de Genes , Humanos , Ratones , Ratones Desnudos , Precursores del ARN/metabolismo , Transcripción GenéticaRESUMEN
Circular RNAs (circRNAs) are upregulated during neurogenesis. Where and how circRNAs are localized and what roles they play during this process have remained elusive. Comparing the nuclear and cytoplasmic circRNAs between H9 cells and H9-derived forebrain (FB) neurons, we identify that a subset of adenosine (A)-rich circRNAs are restricted in H9 nuclei but exported to cytosols upon differentiation. Such a subcellular relocation of circRNAs is modulated by the poly(A)-binding protein PABPC1. In the H9 nucleus, newly produced (A)-rich circRNAs are bound by PABPC1 and trapped by the nuclear basket protein TPR to prevent their export. Modulating (A)-rich motifs in circRNAs alters their subcellular localization, and introducing (A)-rich circRNAs in H9 cytosols results in mRNA translation suppression. Moreover, decreased nuclear PABPC1 upon neuronal differentiation enables the export of (A)-rich circRNAs, including circRTN4(2,3), which is required for neurite outgrowth. These findings uncover subcellular localization features of circRNAs, linking their processing and function during neurogenesis.
Asunto(s)
Transporte Activo de Núcleo Celular , Adenosina , Núcleo Celular , Neurogénesis , Neuronas , Proteína I de Unión a Poli(A) , ARN Circular , ARN , ARN Circular/metabolismo , ARN Circular/genética , Neuronas/metabolismo , Adenosina/metabolismo , Núcleo Celular/metabolismo , Humanos , Proteína I de Unión a Poli(A)/metabolismo , Proteína I de Unión a Poli(A)/genética , Animales , ARN/metabolismo , ARN/genética , Línea Celular , Diferenciación Celular , Citoplasma/metabolismo , Prosencéfalo/metabolismoRESUMEN
Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing RNA circles as PKR inhibitors. Here, we report that RNA circles synthesized by permuted self-splicing thymidylate synthase (td) introns from T4 bacteriophage or by Anabaena pre-tRNA group I intron could induce an immune response. Autocatalytic splicing introduces â¼74 nt td or â¼186 nt Anabaena extraneous fragments that can distort the folding status of original circular RNAs or form structures themselves to provoke innate immune responses. In contrast, synthesized RNA circles produced by T4 RNA ligase without extraneous fragments exhibit minimized immunogenicity. Importantly, directly ligated circular RNAs that form short dsRNA regions efficiently suppress PKR activation 103- to 106-fold higher than reported chemical compounds C16 and 2-AP, highlighting the future use of circular RNAs as potent inhibitors for diseases related to PKR overreaction.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , ARN Circular/farmacología , eIF-2 Quinasa/antagonistas & inhibidores , Células A549 , Bacteriófago T4/enzimología , Bacteriófago T4/genética , Células HEK293 , Células HeLa , Humanos , Inmunidad Innata/efectos de los fármacos , Intrones , Conformación de Ácido Nucleico , Inhibidores de Proteínas Quinasas/inmunología , ARN Ligasa (ATP)/genética , ARN Ligasa (ATP)/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Circular/genética , ARN Circular/inmunología , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
Microexons are frequently underestimated in transcriptome analyses. Two studies published in Cell and Genome Research now independently report the identification of hundreds of microexons. Alternative splicing of some microexons is regulated by neuronal-specific RNA-binding proteins and modifies the function of proteins involved in neurogenesis, with misregulation linked to autism.
RESUMEN
Exon circularization has been identified from many loci in mammals, but the detailed mechanism of its biogenesis has remained elusive. By using genome-wide approaches and circular RNA recapitulation, we demonstrate that exon circularization is dependent on flanking intronic complementary sequences. Such sequences and their distribution exhibit rapid evolutionary changes, showing that exon circularization is evolutionarily dynamic. Strikingly, exon circularization efficiency can be regulated by competition between RNA pairing across flanking introns or within individual introns. Importantly, alternative formation of inverted repeated Alu pairs and the competition between them can lead to alternative circularization, resulting in multiple circular RNA transcripts produced from a single gene. Collectively, exon circularization mediated by complementary sequences in human introns and the potential to generate alternative circularization products extend the complexity of mammalian posttranscriptional regulation.
Asunto(s)
Empalme Alternativo , Exones , Genoma Humano , Elementos Alu , Animales , Secuencia de Bases , Células Madre Embrionarias/metabolismo , Evolución Molecular , Humanos , Intrones , Mamíferos/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Alineación de SecuenciaRESUMEN
The nucleolus is the most prominent membraneless condensate in the nucleus. It comprises hundreds of proteins with distinct roles in the rapid transcription of ribosomal RNA (rRNA) and efficient processing within units comprising a fibrillar centre and a dense fibrillar component and ribosome assembly in a granular component1. The precise localization of most nucleolar proteins and whether their specific localization contributes to the radial flux of pre-rRNA processing have remained unknown owing to insufficient resolution in imaging studies2-5. Therefore, how these nucleolar proteins are functionally coordinated with stepwise pre-rRNA processing requires further investigation. Here we screened 200 candidate nucleolar proteins using high-resolution live-cell microscopy and identified 12 proteins that are enriched towards the periphery of the dense fibrillar component (PDFC). Among these proteins, unhealthy ribosome biogenesis 1 (URB1) is a static, nucleolar protein that ensures 3' end pre-rRNA anchoring and folding for U8 small nucleolar RNA recognition and the subsequent removal of the 3' external transcribed spacer (ETS) at the dense fibrillar component-PDFC boundary. URB1 depletion leads to a disrupted PDFC, uncontrolled pre-rRNA movement, altered pre-rRNA conformation and retention of the 3' ETS. These aberrant 3' ETS-attached pre-rRNA intermediates activate exosome-dependent nucleolar surveillance, resulting in decreased 28S rRNA production, head malformations in zebrafish and delayed embryonic development in mice. This study provides insight into functional sub-nucleolar organization and identifies a physiologically essential step in rRNA maturation that requires the static protein URB1 in the phase-separated nucleolus.
Asunto(s)
Nucléolo Celular , Exosomas , Precursores del ARN , Procesamiento Postranscripcional del ARN , ARN Ribosómico , Pez Cebra , Animales , Ratones , Nucléolo Celular/metabolismo , Desarrollo Embrionario , Exosomas/metabolismo , Cabeza/anomalías , Microscopía , Proteínas Nucleares/metabolismo , Precursores del ARN/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Ribosómico 28S/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Chen et al. (2021) have identified many internal ribosome entry site-like elements that can potentially drive circRNA translation. Dozens of such element-containing circRNAs-encoded peptides are validated, among which a circFGFR1-encoded protein acts as an antagonist of FGFR1.
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Sitios Internos de Entrada al Ribosoma , ARN Circular , Regulación de la Expresión GénicaRESUMEN
Circular RNAs (circRNAs) are produced from precursor mRNA (pre-mRNA) back-splicing of thousands of genes in eukaryotes. Although circRNAs are generally expressed at low levels, recent findings have shed new light on their cell type-specific and tissue-specific expression and on the regulation of their biogenesis. Furthermore, the data indicate that circRNAs shape gene expression by titrating microRNAs, regulating transcription and interfering with splicing, thus effectively expanding the diversity and complexity of eukaryotic transcriptomes.
Asunto(s)
ARN/genética , ARN/metabolismo , Animales , Regulación de la Expresión Génica , Humanos , MicroARNs/metabolismo , Modelos Moleculares , Especificidad de Órganos , Empalme del ARN , ARN CircularRESUMEN
Dynamic imaging of genomic loci is key for understanding gene regulation, but methods for imaging genomes, in particular non-repetitive DNAs, are limited. We developed CRISPRdelight, a DNA-labeling system based on endonuclease-deficient CRISPR-Cas12a (dCas12a), with an engineered CRISPR array to track DNA location and motion. CRISPRdelight enables robust imaging of all examined 12 non-repetitive genomic loci in different cell lines. We revealed the confined movement of the CCAT1 locus (chr8q24) at the nuclear periphery for repressed expression and active motion in the interior nucleus for transcription. We uncovered the selective repositioning of HSP gene loci to nuclear speckles, including a remarkable relocation of HSPH1 (chr13q12) for elevated transcription during stresses. Combining CRISPR-dCas12a and RNA aptamers allowed multiplex imaging of four types of satellite DNA loci with a single array, revealing their spatial proximity to the nucleolus-associated domain. CRISPRdelight is a user-friendly and robust system for imaging and tracking genomic dynamics and regulation.
RESUMEN
Plants have evolved complex mechanisms to adapt to harsh environmental conditions. Rice (Oryza sativa) is a staple food crop that is sensitive to low temperatures. However, its cold stress responses remain poorly understood, thus limiting possibilities for crop engineering to achieve greater cold tolerance. In this study, we constructed a rice pan-transcriptome and characterized its transcriptional regulatory landscape in response to cold stress. We performed Iso-Seq and RNA-Seq of 11 rice cultivars subjected to a time-course cold treatment. Our analyses revealed that alternative splicing-regulated gene expression plays a significant role in the cold stress response. Moreover, we identified CATALASE C (OsCATC) and Os03g0701200 as candidate genes for engineering enhanced cold tolerance. Importantly, we uncovered central roles for the 2 serine-arginine-rich proteins OsRS33 and OsRS2Z38 in cold tolerance. Our analysis of cold tolerance and resequencing data from a diverse collection of 165 rice cultivars suggested that OsRS2Z38 may be a key selection gene in japonica domestication for cold adaptation, associated with the adaptive evolution of rice. This study systematically investigated the distribution, dynamic changes, and regulatory mechanisms of alternative splicing in rice under cold stress. Overall, our work generates a rich resource with broad implications for understanding the genetic basis of cold response mechanisms in plants.