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Bing Chen, Shenglin Huang
Circular RNA (circRNA) is a type of covalently closed non-coding RNA that may regulate gene expression in eukaryotes. The recent application of high-throughput RNA sequencing and bioinformatics approaches has revealed a large number of circRNAs in human cells. Emerging evidence indicates that many circRNAs have cell-type specific expression and are linked to physiological development and various diseases. Specially, circRNAs can either serve as oncogenic stimuli or tumor suppressors in cancer. circRNAs have also been shown to be enriched and stable in extracellular fluid, indicating the potential of circRNAs as cancer biomarkers...
January 9, 2018: Cancer Letters
M Laura Idda, Rachel Munk, Kotb Abdelmohsen, Myriam Gorospe
Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the main cause of dementia among the elderly worldwide. Despite intense efforts to develop drugs for preventing and treating AD, no effective therapies are available as yet, posing a growing burden at the personal, medical, and socioeconomic levels. AD is characterized by the production and aggregation of amyloid β (Aβ) peptides derived from amyloid precursor protein (APP), the presence of hyperphosphorylated microtubule-associated protein Tau (MAPT), and chronic inflammation leading to neuronal loss...
January 12, 2018: Wiley Interdisciplinary Reviews. RNA
Yan Li, Bing Chen, Shenglin Huang
Circular RNAs from back-spliced exons (circRNAs) represent a novel class of widespread and endogenous RNAs in eukaryotes. circRNAs may bind to microRNAs (miRNAs) and inhibit the activity of miRNAs. Alternatively, miRNAs could also directly target circRNAs and regulate the expression of circRNAs. Here we describe the Argonaute2 (AGO2) RNA immunoprecipitation (RIP) and luciferase screening assays to identify the interaction between circRNAs and miRNAs. The AGO2 RIP assay evaluates the potential of the interaction between circRNAs and miRNAs...
2018: Methods in Molecular Biology
Xiao-Qin Zhang, Jian-Hua Yang
Circular RNAs (circRNAs) represent an abundant group of noncoding RNAs in eukaryotes and are emerging as important regulatory molecules in physiological and pathological processes. However, the precise mechanisms and functions of most of circRNAs remain largely unknown. In this chapter, we describe how to identify circRNA-microRNA interactions from Argonaute (AGO) cross-linking and immunoprecipitation followed by sequencing (CLIP-Seq) and RNA-Seq data using starBase platform and software. We developed three stand-alone computational software, including circSeeker, circAnno, and clipSearch, to identify and annotate circRNAs and their interactions with microRNAs (miRNAs)...
2018: Methods in Molecular Biology
Sonja Petkovic, Sabine Müller
Circular RNAs (circRNAs) have been discovered in all kingdoms of life. They are produced from introns as well as from exons. However, strongest interest is in circRNAs that are transcribed and spliced from exons of protein and noncoding genes in eukaryotic cells including humans. Therefore, synthesis and engineering of circRNAs as models for structure and function studies are strongly required. In vitro, methods for RNA synthesis and circularization are available. Chemical synthesis allows for preparation of RNAs incorporating nonnatural nucleotides in small RNA segments, whereas enzymatic synthesis is advantageous for production of long RNAs, however, without the possibility for site-specific modification...
2018: Methods in Molecular Biology
Deniz Bartsch, Anne Zirkel, Leo Kurian
A substantial proportion of the currently annotated genes in eukaryotes are proposed to function as RNA molecules (>200 bp) with no significant protein coding potential, currently classified as long noncoding RNAs (lncRNA). A distinct subgroup of lncRNAs is circular RNAs (circRNAs), which can be easily identified by unique junction reads, resulting from their biogenesis. CircRNAs are largely cytosolic and thought to either code for micro-peptides or facilitate gene regulation by sequestering microRNAs (miRNAs) or RNA-binding proteins (RBPs) from their targets...
2018: Methods in Molecular Biology
Thomas B Hansen
Circular RNAs (circRNAs) constitute a novel subset in the fascinating world of noncoding RNA, and they are found in practically all eukaryotes. Most of them exhibit low expression levels but some are extremely abundant. Typically, circRNAs are studied by RT-PCR-based assays, but for certain types of analyses this technique is not suitable. Circular RNA with repetitive exons (circular concatemers) has been observed by us and others when transiently expressing circRNAs in cells, however techniques and assays to study these species have not been established...
2018: Methods in Molecular Biology
Xiaolin Wang, Ge Shan
Circular RNAs (circRNAs) are recognized as a special species of transcripts in metazoans with increasing studies, and northern blotting is a direct way to confirm the existence and to evaluate the size of individual circRNAs. Northern blotting probes can be radioactive isotope (32P) labeled, which is not environment-friendly and sometimes inconvenient to use. Here, we describe a nonradioactive northern blot protocol with digoxigenin-labeled probe to detect circRNA.
2018: Methods in Molecular Biology
Tim Schneider, Silke Schreiner, Christian Preußer, Albrecht Bindereif, Oliver Rossbach
Northern blotting enables the specific detection and characterization of RNA molecules. Recently, circular RNAs (circRNAs) were described as a new class of cell type-specific noncoding RNAs. With the discovery of many novel circRNAs on the basis of high-throughput sequencing and bioinformatics, a solid biochemical approach is required to directly detect and validate specific circRNA species. Here we give a detailed overview of how different Northern blot methods can be employed to validate specific circRNAs...
2018: Methods in Molecular Biology
Yun Yang, Zefeng Wang
Human transcriptome contains a large number of circular RNAs (circRNAs) that are mainly produced by back splicing of pre-mRNA. Here we describe a minigene reporter system containing a single exon encoding split GFP in reverse order, which can be efficiently back spliced to produce a circRNA encoding intact GFP gene. This simple reporter system can be adopted to study how different cis-elements and trans-factors affect circRNA production, and also can serve as a reliable system to measure the activity of IRES-mediated translation...
2018: Methods in Molecular Biology
Dawei Liu, Vanessa Conn, Gregory J Goodall, Simon J Conn
Circular RNAs (circRNAs) constitute an emerging class of widespread, abundant, and evolutionarily conserved noncoding RNA. They play important and diverse roles in cell development, growth, and tumorigenesis, but functions of the majority of circRNAs remain enigmatic. In order to investigate circRNA function it is necessary to manipulate its expression. While various standard approaches exist for circRNA knockdown, here we present cloning vectors for simplifying the laborious process of cloning circRNAs to achieve high-efficiency overexpression in mammalian cell lines...
2018: Methods in Molecular Biology
Christine Kocks, Anastasiya Boltengagen, Monika Piwecka, Agnieszka Rybak-Wolf, Nikolaus Rajewsky
Individual mRNA molecules can be imaged in fixed cells by hybridization with multiple, singly labeled oligonucleotide probes, followed by computational identification of fluorescent signals. This approach, called single-molecule RNA fluorescence in situ hybridization (smRNA FISH), allows subcellular localization and absolute quantification of RNA molecules in individual cells. Here, we describe a simple smRNA FISH protocol for two-color imaging of a circular RNA, CDR1as, simultaneously with an unrelated messenger RNA...
2018: Methods in Molecular Biology
Anne Zirkel, Argyris Papantonis
Fluorescence in situ hybridization (FISH) coupled to high-resolution microscopy is a powerful method for analyzing the subcellular localization of RNA. However, the detection of circular RNAs (circRNAs) using microscopy is challenging because the only feature of a circRNA that can be used for the probe design is its junction. Circular RNAs are expressed at varying levels, and for their efficient monitoring by FISH, background fluorescence levels need to be kept low. Here, we describe a FISH protocol coupled to high-precision localizations using a single fluorescently labeled probe spanning the circRNA junction; this allows circRNA detection in mammalian cells with high signal-to-noise ratios...
2018: Methods in Molecular Biology
Amaresh C Panda, Dawood B Dudekula, Kotb Abdelmohsen, Myriam Gorospe
Circular RNAs (circRNAs) are generated through nonlinear back splicing, during which the 5' and 3' ends are covalently joined. Consequently, the lack of free ends makes them very stable compared to their counterpart linear RNAs. By selectively interacting with microRNAs and RNA-binding proteins (RBPs), circRNAs have been shown to influence gene expression programs. We designed a web tool, CircInteractome, in order to (1) explore potential interactions of circRNAs with RBPs, (2) design specific divergent primers to detect circRNAs, (3) study tissue- and cell-specific circRNAs, (4) identify gene-specific circRNAs, (5) explore potential miRNAs interacting with circRNAs, and (6) design specific siRNAs to silence circRNAs...
2018: Methods in Molecular Biology
Daphne A Cooper, Mariela Cortés-López, Pedro Miura
The genome-wide expression patterns of circular RNAs (circRNAs) are of increasing interest for their potential roles in normal cellular homeostasis, development, and disease. Thousands of circRNAs have been annotated from various species in recent years. Analysis of publically available or user-generated rRNA-depleted total RNA-seq data can be performed to uncover new circRNA expression trends. Here we provide a primer for profiling circRNAs from RNA-seq datasets. The description is tailored for the wet lab scientist with limited or no experience in analyzing RNA-seq data...
2018: Methods in Molecular Biology
Tobias Jakobi, Christoph Dieterich
Circular RNAs (circRNAs) have been first described as "scrambled exons" in the 1990s. CircRNAs originate from back splicing or exon skipping of linear RNA templates and have continuously gained attention in recent years due to the availability of high-throughput whole-transcriptome sequencing methods. Numerous manuscripts describe thousands of circRNAs throughout uni- and multicellular eukaryote species and demonstrated that they are conserved, stable, and abundant in specific tissues or conditions. This manuscript provides a walk-through of our bioinformatics toolbox, which covers all aspects of in silico circRNA analysis, starting from raw sequencing data and back-splicing junction discovery to circRNA quantitation and reconstruction of internal the circRNA structure...
2018: Methods in Molecular Biology
Yi Zheng, Fangqing Zhao
Recent studies have shown that circular RNAs (circRNAs) are a novel class of abundant, stable, and ubiquitous noncoding RNA molecules in eukaryotic organisms. Comprehensive detection and reconstruction of circRNAs from high-throughput transcriptome data is an initial step to study their biogenesis and function. Several tools have been developed to deal with this issue, but they require many steps and are difficult to use. To solve this problem, we provide a protocol for researchers to detect and reconstruct circRNA by employing CIRI2, CIRI-AS, and CIRI-full...
2018: Methods in Molecular Biology
Fan Yang, Ping Zhu, Jintao Guo, Xiang Liu, Sheng Wang, Guoxin Wang, Wen Liu, Shupeng Wang, Nan Ge
Circular RNAs (circRNAs) are a class of noncoding RNAs with continuous, covalently closed circular structures. Investigators have shown previously that circRNAs are regulators of gene expression in mammals. These tissue-specific transcripts are produced primarily by exonic or intronic sequences of housekeeping genes. Several biosynthetic models have been proposed for circRNAs, and consensus is lacking. CircRNAs are widely expressed in the cytoplasm and highly conserved, what is more, unlike other noncoding RNAs, circRNAs are relatively stable...
December 2017: Journal of Thoracic Disease
Zhi-Bin Zhou, Di Du, Gao-Xiang Huang, Aimin Chen, Lei Zhu
Osteoarthritis (OA) is the most common joint disease and is mainly characterized by degradation of the articular cartilage. Recently, circular RNAs (circRNAs), novel noncoding RNAs with different biological functions and pathological implications, have been reported to be closely associated with various diseases. Growing evidence indicates that circRNAs act as competing endogenous RNAs (ceRNAs) that bind with microRNAs (miRNAs) and regulate their downstream functions. Here, we identified a new circRNA, circRNA_Atp9b, and further investigated its function in OA using a well-established mouse chondrocyte model...
January 3, 2018: Gene
Mariela Cortés-López, Matthew R Gruner, Daphne A Cooper, Hannah N Gruner, Alexandru-Ioan Voda, Alexander M van der Linden, Pedro Miura
BACKGROUND: Circular RNAs (CircRNAs) are a newly appreciated class of RNAs that lack free 5' and 3' ends, are expressed by the thousands in diverse forms of life, and are mostly of enigmatic function. Ostensibly due to their resistance to exonucleases, circRNAs are known to be exceptionally stable. Previous work in Drosophila and mice have shown that circRNAs increase during aging in neural tissues. RESULTS: Here, we examined the global profile of circRNAs in C...
January 3, 2018: BMC Genomics
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