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Despite the numerous sequencing methods available, the diversity in RNA size and chemical modification makes it difficult to capture all RNAs in a cell. We developed a method that combines quasi-random priming with template switching to construct sequencing libraries from RNA molecules of any length and with any type of 3' modifications, allowing for the sequencing of virtually all RNA species. Our ligation-independent detection of all types of RNA (LIDAR) is a simple, effective tool to identify and quantify all classes of coding and non-coding RNAs. With LIDAR, we comprehensively characterized the transcriptomes of mouse embryonic stem cells, neural progenitor cells, mouse tissues, and sperm. LIDAR detected a much larger variety of tRNA-derived RNAs (tDRs) compared with traditional ligation-dependent sequencing methods and uncovered tDRs with blocked 3' ends that had previously escaped detection. Therefore, LIDAR can capture all RNAs in a sample and uncover RNA species with potential regulatory functions.
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Circular RNA (circRNA) molecules have critical functions during brain development and in brain-related disorders. Here, we identified and validated a circRNA, circHTT(2,3,4,5,6), stemming from the Huntington's disease (HD) gene locus that is most abundant in the central nervous system (CNS). We uncovered its evolutionary conservation in diverse mammalian species, and a correlation between circHTT(2,3,4,5,6) levels and the length of the CAG-repeat tract in exon-1 of HTT in human and mouse HD model systems. The mouse orthologue, circHtt(2,3,4,5,6), is expressed during embryogenesis, increases during nervous system development, and is aberrantly upregulated in the presence of the expanded CAG tract. While an IRES-like motif was predicted in circH TT (2,3,4,5,6), the circRNA does not appear to be translated in adult mouse brain tissue. Nonetheless, a modest, but consistent fraction of circHtt(2,3,4,5,6) associates with the 40S ribosomal subunit, suggesting a possible role in the regulation of protein translation. Finally, circHtt(2,3,4,5,6) overexpression experiments in HD-relevant STHdh striatal cells revealed its ability to modulate CAG expansion-driven cellular defects in cell-to-substrate adhesion, thus uncovering an unconventional modifier of HD pathology.
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The Malat1 (metastasis-associated lung adenocarcinoma transcript 1) long noncoding RNA is highly and broadly expressed in mammalian tissues, accumulating in the nucleus where it modulates expression and pre-mRNA processing of many protein-coding genes. In this issue of Genes & Development, Xiao and colleagues (doi:10.1101/gad.351557.124) report that a significant fraction of Malat1 transcripts in cultured mouse neurons are surprisingly exported from the nucleus. These transcripts are packaged with Staufen proteins in RNA granules and traffic down the lengths of neurites. They then can be released in a stimulus-dependent manner to be locally translated into a microprotein that alters neuronal gene expression patterns.
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Núcleo Celular , Neurônios , Biossíntese de Proteínas , RNA Longo não Codificante , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neurônios/metabolismo , Camundongos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
The current study aimed to elucidate the regulatory mechanisms of the circRNA hsa_circ_0139697 (circSTAG2(16-25)) in BCa and to consider the opportunity of using circSTAG2(16-25) isolated from BCa patient urine as a marker for disease development prediction. The selection of this circRNA was determined by the special role of its parental gene STAG2 in BCa biology. The circRNA hsa_circ_0139697 was chosen from 25 STAG2 circRNAs due to its differential expression in the urine of BCa patients and healthy volunteers. Higher levels of circSTAG2(16-25) were detected in urine samples obtained from patients with recurrent tumors. A higher expression of circSTAG2(16-25) was also detected in more tumorigenic BCa cell lines. The overexpression of circSTAG2(16-25) in BCa cells induced the elevation of proliferation, motility, and invasion. To study the mechanisms of circSTAG2(16-25) activity, we confirmed that circSTAG2(16-25) can bind miR-145-5p in vitro as was predicted by bioinformatic search. miR-145-5p was shown to suppress some genes that promoted BCa progression. One of these genes, TAGLN2, encodes the protein Transgelin 2, which plays a role in BCa cell motility and invasion. Therefore, the possible mechanism of action of circSTAG2(16-25) could be sponging the tumor suppressor miR-145-5p, which results in activation of TAGLN2. In addition, circSTAG2(16-25) might be considered as a potential biomarker for recurrence prediction.
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A subset of circular RNAs (circRNAs) and linear RNAs have been proposed to 'sponge' or block microRNA activity. Additionally, certain RNAs induce microRNA destruction through the process of Target RNA-Directed MicroRNA Degradation (TDMD), but whether both linear and circular transcripts are equivalent in driving TDMD is unknown. Here, we studied whether circular/linear topology of endogenous and artificial RNA targets affects TDMD. Consistent with previous knowledge that Cdr1as (ciRS-7) circular RNA protects miR-7 from Cyrano-mediated TDMD, we demonstrate that depletion of Cdr1as reduces miR-7 abundance. In contrast, overexpression of an artificial linear version of Cdr1as drives miR-7 degradation. Using plasmids that express a circRNA with minimal co-expressed cognate linear RNA, we show differential effects on TDMD that cannot be attributed to the nucleotide sequence, as the TDMD properties of a sequence often differ when in a circular versus linear form. By analysing RNA sequencing data of a neuron differentiation system, we further detect potential effects of circRNAs on microRNA stability. Our results support the view that RNA circularity influences TDMD, either enhancing or inhibiting it on specific microRNAs.
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MicroRNAs , Estabilidade de RNA , RNA Circular , MicroRNAs/genética , MicroRNAs/metabolismo , RNA/genética , RNA/metabolismo , RNA Circular/metabolismo , Humanos , Animais , CamundongosRESUMO
Thousands of eukaryotic protein-coding genes can be alternatively spliced to yield linear mRNAs and circular RNAs (circRNAs). Some circRNAs accumulate to higher levels than their cognate linear mRNAs, but the vast majority are expressed at low levels. Hence, for most circRNAs, only a handful of sequencing reads, if any, that span the backsplicing junction are observed in standard RNA-seq libraries. It thus has become common to use the 3'-5' exonuclease ribonuclease R (RNase R) to selectively degrade linear RNAs when aiming to prove transcript circularity or biochemically enrich circRNAs. However, RNase R fails to degrade linear RNAs with structured 3' ends or internal G-quadruplex structures. To overcome these shortcomings, we describe an improved protocol for circRNA purification from total RNA that employs a poly(A) tailing step prior to RNase R digestion, which is performed in a Li+ containing buffer (rather than K+) to destabilize G-quadruplexes. This biochemical method enables higher enrichment (two- to threefold) of circRNAs to be obtained compared to standard RNase R protocols due to more efficient removal of linear RNAs. By then performing quantitative RT-PCR (RT-qPCR) or generating RNA-seq libraries, the expression of individual circRNAs can be examined or the entire set of expressed circRNAs defined using established annotation algorithms. We describe step-by-step methods for annotating circRNAs using the CIRI2 and CIRCexplorer2 algorithms. In total, this overall approach can be used to enrich for circRNAs from any total RNA sample, thereby enabling one to quickly identify and validate circRNAs of interest for functional studies.
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Exorribonucleases , RNA Circular , RNA , RNA Mensageiro , RNA/genética , Exonucleases , DigestãoRESUMO
Numerous components of the transcription machinery, including RNA polymerase II (Pol II), accumulate in regions of high local concentration known as clusters, which are thought to facilitate transcription. Using the histone locus of Drosophila nurse cells as a model, we find that Pol II forms long-lived, transcriptionally poised clusters distinct from liquid droplets, which contain unbound and paused Pol II. Depletion of the Integrator complex endonuclease module, but not its phosphatase module or Pol II pausing factors disperses these Pol II clusters. Consequently, histone transcription fails to reach peak levels during S-phase and aberrantly continues throughout the cell cycle. We propose that Pol II clustering is a regulatory step occurring near promoters that limits rapid gene activation to defined times. One Sentence Summary: Using the Drosophila histone locus as a model, we show that clustered RNA polymerase II is poised for synchronous activation.
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Circular RNAs (circRNAs) are a class of single-stranded, covalently closed RNA that contain a unique back-splice junction (bsj) sequence created by the ligation of their 5' and 3' ends via spliceosome-catalyzed back-splicing. A key step in illuminating the cellular roles of specific circRNAs is via increasing their expression. This is frequently done by transfecting cells with plasmid DNA containing cloned exons from which the circRNA is transcribed, flanked by sequences that promote back-splicing. We observed that commonly used plasmids lead to the production of circRNAs with molecular scars at the circRNA bsj. Stepwise redesign of the cloning vector corrected this problem, ensuring bona fide circRNAs are produced with their natural bsj at high efficiency. The fidelity of circRNAs produced from this new construct was validated by RNA sequencing and also functionally validated. To increase the utility of this modified resource for expressing circRNA, we developed an expanded set of vectors incorporating this design that (i) enables selection with a variety of antibiotics and fluorescent proteins, (ii) employs a range of promoters varying in promoter strength and (iii) generated a complementary set of lentiviral plasmids for difficult-to-transfect cells. These resources provide a novel and versatile toolkit for high-efficiency and scarless overexpression of circular RNAs that fulfill a critical need for the investigation of circRNA function.
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The metazoan-specific Integrator complex catalyzes 3' end processing of small nuclear RNAs (snRNAs) and premature termination that attenuates the transcription of many protein-coding genes. Integrator has RNA endonuclease and protein phosphatase activities, but it remains unclear if both are required for complex function. Here, we show IntS6 (Integrator subunit 6) over-expression blocks Integrator function at a subset of Drosophila protein-coding genes, although having no effect on snRNAs or attenuation of other loci. Over-expressed IntS6 titrates protein phosphatase 2A (PP2A) subunits, thereby only affecting gene loci where phosphatase activity is necessary for Integrator function. IntS6 functions analogous to a PP2A regulatory B subunit as over-expression of canonical B subunits, which do not bind Integrator, is also sufficient to inhibit Integrator activity. These results show that the phosphatase module is critical at only a subset of Integrator-regulated genes and point to PP2A recruitment as a tunable step that modulates transcription termination efficiency.
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Proteínas de Drosophila , Terminação da Transcrição Genética , Animais , RNA , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Nuclear Pequeno/genética , Fatores de Transcrição/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogasterRESUMO
Despite the numerous sequencing methods available, the vast diversity in size and chemical modifications of RNA molecules makes the capture of the full spectrum of cellular RNAs a difficult task. By combining quasi-random hexamer priming with a custom template switching strategy, we developed a method to construct sequencing libraries from RNA molecules of any length and with any type of 3' terminal modification, allowing the sequencing and analysis of virtually all RNA species. Ligation-independent detection of all types of RNA (LIDAR) is a simple, effective tool to comprehensively characterize changes in small non-coding RNAs and mRNAs simultaneously, with performance comparable to separate dedicated methods. With LIDAR, we comprehensively characterized the coding and non-coding transcriptome of mouse embryonic stem cells, neural progenitor cells, and sperm. LIDAR detected a much larger variety of tRNA-derived RNAs (tDRs) compared to traditional ligation-dependent sequencing methods, and uncovered the presence of tDRs with blocked 3' ends that had previously escaped detection. Our findings highlight the potential of LIDAR to systematically detect all RNAs in a sample and uncover new RNA species with potential regulatory functions.
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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.
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RNA Longo não Codificante , RNA Longo não Codificante/genética , Núcleo Celular/genética , Cromatina/genética , Sequências Reguladoras de Ácido Nucleico , RNA Polimerase II/genéticaRESUMO
Ribosomal RNAs (rRNAs) are the most abundant cellular RNAs, and their synthesis from rDNA repeats by RNA polymerase I accounts for the bulk of all transcription. Despite substantial variation in rRNA transcription rates across cell types, little is known about cell-type-specific factors that bind rDNA and regulate rRNA transcription to meet tissue-specific needs. Using hematopoiesis as a model system, we mapped about 2,200 ChIP-seq datasets for 250 transcription factors (TFs) and chromatin proteins to human and mouse rDNA and identified robust binding of multiple TF families to canonical TF motifs on rDNA. Using a 47S-FISH-Flow assay developed for nascent rRNA quantification, we demonstrated that targeted degradation of C/EBP alpha (CEBPA), a critical hematopoietic TF with conserved rDNA binding, caused rapid reduction in rRNA transcription due to reduced RNA Pol I occupancy. Our work identifies numerous potential rRNA regulators and provides a template for dissection of TF roles in rRNA transcription.
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RNA Polimerase I , Fatores de Transcrição , Humanos , Camundongos , Animais , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , RNA Ribossômico/genética , Transcrição Gênica , DNA Ribossômico/genética , RNA , CromatinaRESUMO
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.
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MicroRNAs , RNA Circular , Regulação da Expressão Gênica/genética , MicroRNAs/genética , MicroRNAs/metabolismo , RNA/genética , RNA/metabolismo , RNA Circular/genética , RNA não Traduzido , RNA Viral , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMO
CRISPR/Cas13 effectors have garnered increasing attention as easily customizable tools for detecting and depleting RNAs of interest. Near perfect complementarity between a target RNA and the Cas13-associated guide RNA is required for activation of Cas13 ribonuclease activity. Nonetheless, the specificity of Cas13 effectors in eukaryotic cells has been debated as the Cas13 nuclease domains can be exposed on the enzyme surface, providing the potential for promiscuous cleavage of nearby RNAs (so-called collateral damage). Here, using co-transfection assays in Drosophila and human cells, we found that the off-target effects of RxCas13d, a commonly used Cas13 effector, can be as strong as the level of on-target RNA knockdown. The extent of off-target effects is positively correlated with target RNA expression levels, and collateral damage can be observed even after reducing RxCas13d/guide RNA levels. The PspCas13b effector showed improved specificity and, unlike RxCas13d, can be used to deplete a Drosophila circular RNA without affecting the expression of the associated linear RNA. PspCas13b nonetheless still can have off-target effects and we notably found that the extent of off-target effects for Cas13 effectors differs depending on the cell type and target RNA examined. In total, these results highlight the need for caution when designing and interpreting Cas13-based knockdown experiments.
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Sistemas CRISPR-Cas , RNA Guia de Cinetoplastídeos , Animais , Drosophila/genética , Células Eucarióticas , RNA/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
The human transcriptome contains many types of noncoding RNAs, which rival the number of protein-coding species. From long noncoding RNAs (lncRNAs) that are over 200 nucleotides long to piwi-interacting RNAs (piRNAs) of only 20 nucleotides, noncoding RNAs play important roles in regulating transcription, epigenetic modifications, translation, and cell signaling. Roles for noncoding RNAs in disease mechanisms are also being uncovered, and several species have been identified as potential drug targets. On May 11-14, 2021, the Keystone eSymposium "Noncoding RNAs: Biology and Applications" brought together researchers working in RNA biology, structure, and technologies to accelerate both the understanding of RNA basic biology and the translation of those findings into clinical applications.
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Congressos como Assunto/tendências , Epigênese Genética/genética , Marcação de Genes/tendências , RNA não Traduzido/administração & dosagem , RNA não Traduzido/genética , Relatório de Pesquisa , Animais , Sistemas de Liberação de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/tendências , Marcação de Genes/métodos , Humanos , MicroRNAs/administração & dosagem , MicroRNAs/genética , RNA Longo não Codificante/administração & dosagem , RNA Longo não Codificante/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Pequeno RNA não Traduzido/administração & dosagem , Pequeno RNA não Traduzido/genética , Transdução de Sinais/genéticaRESUMO
Circular RNAs with covalently linked ends are generated from many eukaryotic protein-coding genes when the pre-mRNA splicing machinery backsplices. These mature transcripts are resistant to digestion by exonucleases and typically have much longer half-lives than their associated linear mRNAs. Circular RNAs thus have great promise as sensitive biomarkers, including for detection of transcriptional activity. Here, we show that circular RNAs can serve as markers of readthrough transcription events in Drosophila and human cells, thereby revealing mechanistic insights into RNA polymerase II transcription termination as well as pre-mRNA 3' end processing. We describe methods that take advantage of plasmids that generate a circular RNA when an upstream polyadenylation signal fails to be used and/or RNA polymerase II fails to terminate. As a proof-of-principle, we show that RNAi-mediated depletion of well-established transcription termination factors, including the RNA endonuclease Cpsf73, results in increased circular RNA output from these plasmids in Drosophila and human cells. This method is generalizable as a circular RNA can be easily encoded downstream of any genomic region of interest. Circular RNA biomarkers, therefore, have great promise for identifying novel cellular factors and conditions that impact transcription termination processes.
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Poliadenilação , RNA Circular , Biomarcadores , Poliadenilação/genética , RNA/genética , RNA/metabolismo , Splicing de RNA/genética , RNA Circular/genéticaRESUMO
Pre-mRNAs from thousands of eukaryotic genes can be non-canonically spliced to generate circular RNAs (circRNAs) that have covalently linked ends. Most mature circular RNAs are expressed at low levels, but some have known physiological functions and/or accumulate to higher levels than their associated linear mRNAs. These observations have sparked great interest into this class of previously underappreciated RNAs and prompted the development of new experimental approaches to study them, especially methods to measure or modulate circular RNA expression levels. Nonetheless, each of these approaches has caveats and potential pitfalls that must be controlled for when designing experiments and interpreting results. Here, we provide practical advice, tips, and suggested guidelines for performing robust identification, validation, and functional characterization of circular RNAs. Beyond promoting rigor and reproducibility, these suggestions should help bring clarity to the field, especially how circular RNAs function and whether these transcripts may sponge microRNAs/proteins or serve as templates for translation.
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Precursores de RNA , RNA Circular , RNA/genética , RNA/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , Reprodutibilidade dos TestesRESUMO
Circular RNAs (circRNAs) are highly stable RNA molecules that are attractive templates for expression of therapeutic proteins and non-coding RNAs. In eukaryotes, circRNAs are primarily generated by the spliceosome through backsplicing. Here, we interrogate different molecular elements including intron type and length, Alu repeats, internal ribosome entry sites (IRESs), and exon length essential for circRNA formation and exploit this information to engineer robust backsplicing and circRNA expression. Specifically, we leverage the finding that the downstream intron can tolerate large inserts without affecting splicing to achieve tandem expression of backspliced circRNAs and tRNA intronic circRNAs from the same template. Further, truncation of selected intronic regions markedly increased circRNA formation in different cell types in vitro as well as AAV-mediated circRNA expression in cardiac and skeletal muscle tissue in vivo. We also observed that different IRES elements and exon length influenced circRNA expression and translation, revealing an exonic contribution to splicing, as evidenced by different RNA species produced. Taken together, these data provide new insight into improving the design and expression of synthetic circRNAs. When combined with AAV capsid and promoter technologies, the backsplicing introns and IRES elements constituting this modular platform significantly expand the gene expression toolkit.