Backsplice Junctions Research Articles

FL-circAS: an integrative resource and analysis for full-length sequences and alternative splicing of circular RNAs with nanopore sequencing.

Circular RNAs (circRNAs) are RNA molecules with a continuous loop structure characterized by back-splice junctions (BSJs). While analyses of short-read RNA sequencing have identified millions of BSJ events, it is inherently challenging to determine exact full-length sequences and alternatively spliced (AS) isoforms of circRNAs. Recent advances in nanopore long-read sequencing with circRNA enrichment bring an unprecedented opportunity for investigating the issues. Here, we developed FL-circAS (https://cosbi.ee.ncku.edu.tw/FL-circAS/), which collected such long-read sequencing data of 20 cell lines/tissues and thereby identified 884636 BSJs with 1853692 full-length circRNA isoforms in human and 115173 BSJs with 135617 full-length circRNA isoforms in mouse. FL-circAS also provides multiple circRNA features. For circRNA expression, FL-circAS calculates expression levels for each circRNA isoform, cell line/tissue specificity at both the BSJ and isoform levels, and AS entropy for each BSJ across samples. For circRNA biogenesis, FL-circAS identifies reverse complementary sequences and RNA binding protein (RBP) binding sites residing in flanking sequences of BSJs. For functional patterns, FL-circAS identifies potential microRNA/RBP binding sites and several types of evidence for circRNA translation on each full-length circRNA isoform. FL-circAS provides user-friendly interfaces for browsing, searching, analyzing, and downloading data, serving as the first resource for discovering full-length circRNAs at the isoform level.

Discovery and Comprehensive Characterization of Novel Circular RNAs of the Apoptosis-Related BOK Gene in Human Ovarian and Prostate Cancer Cells, Using Nanopore Sequencing.

CircRNAs have become a novel scientific research hotspot, and an increasing number of studies have shed light on their involvement in malignant progression. Prompted by the apparent scientific gap in circRNAs from apoptosis-related genes, such as BOK, we focused on the identification of novel BOK circRNAs in human ovarian and prostate cancer cells. Total RNA was extracted from ovarian and prostate cancer cell lines and reversely transcribed using random hexamer primers. A series of PCR assays utilizing gene-specific divergent primers were carried out. Next, third-generation sequencing based on nanopore technology followed by extensive bioinformatics analysis led to the discovery of 23 novel circRNAs. These novel circRNAs consist of both exonic and intronic regions of the BOK gene. Interestingly, the exons that form the back-splice junction were truncated in most circRNAs, and multiple back-splice sites were found for each BOK exon. Moreover, several BOK circRNAs are predicted to sponge microRNAs with a key role in reproductive cancers, while the presence of putative open reading frames indicates their translational potential. Overall, this study suggests that distinct alternative splicing events lead to the production of novel BOK circRNAs, which could come into play in the molecular landscape and clinical investigation of ovarian and prostate cancer.

Triple Amplification Strategy of CHA-PER-DNAzyme for Ultrasensitive Detection of circRNA Associated with Hepatocellular Carcinoma.

Biological and clinical studies have indicated that aberrant expression of circMTO1 served as a crucial biomarker for the diagnosis and prognosis of hepatocellular carcinoma (HCC) patients as well as a potential therapeutic target. However, the detection of circRNAs currently faces challenges such as homologous linear RNA interference and low-expression abundance of certain circRNAs. Therefore, we developed a triple amplification method based on catalytic hairpin assembly (CHA) activation by back-splice junction (BSJ), resulting in CHA products that triggered primer exchange reaction to generate DNAzyme. Subsequently, DNAzyme cleaved the fluorescent reporter chain, enabling ultrasensitive detection of hepatocellular carcinoma-associated circMTO1 through the output fluorescence signal. The catalytic hairpin opening sequence within CHA specifically targeted the BSJ sequence in circRNA, thereby avoiding false positive signals observed in circRNA assays due to the recognition of homologous linear RNA molecules. Moreover, this triple amplification method facilitated sensitive detection of circRNA and addressed the issue of low-abundance expression levels associated with circMTO1 in HCC samples. Notably, our newly designed assay for detecting circRNA exhibited a linear range from 1 fM to 100 nM with a detection limit of 0.265 fM. Furthermore, it demonstrated excellent and consistent performance even within complex systems. Our proposed method enabled the specific and sensitive detection of circMTO1 in various cancer cells and blood samples from HCC patients, providing an innovative approach for investigating the role of circRNA in tumorigenesis and development while promoting its clinical application.

Gene expression profiles of mRNA, lncRNA, miRNA, and circRNA and their clinical implications in chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease with complex pathophysiology and therapeutic strategies. Moreover, the molecular mechanisms underlying the pathogenesis of CRSwNP are incompletely understood. This study aimed to investigate the transcriptomic characteristics, ceRNA networks, and whether these molecular markers play a role in the occurrence and development of CRSwNP. Following RNA sequencing, a ceRNA network was predicted and constructed based on the sequencing results and multiple databases. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and disease ontology (DO) were applied to analyze the potential mechanisms in relation to the pathogenesis of CRSwNP. CIBERSORT was used to evaluate the immune cell infiltration levels in CRSwNP. The candidate genes of differentially expressed (DE) mRNA, DE-lncRNA, DE-miRNA, and DE-circRNA were verified by RT-qPCR, and the back-splice junction of circRNA was verified using Sanger sequencing. The clinical significance of differentially expressed genes was analyzed with correlation test and receiver operating characteristic curve. We identified 716 DE-mRNA, 230 DE-lncRNA, 42 DE-miRNA, and 46 DE-circRNA, and GO and KEGG enrichment analyses indicated that they were involved in multiple biological pathways, predominantly those associated with immunity and inflammation. DO analysis revealed CRSwNP is associated with diseases such as gastroesophageal reflux and allergic reactions. High expression of circ_0021727 was significantly and positively correlated with several important clinical indicators, and the area under the curve was 0.741. This study provides transcriptomic characteristics, which are potential biomarkers or therapeutic targets for the diagnosis and treatment of CRSwNP.

Full-length circular RNA profiling by nanopore sequencing with CIRI-long.

Circular RNAs (circRNAs) have important roles in regulating developmental processes and disease progression. As most circRNA sequences are highly similar to their cognate linear transcripts, the current short-read sequencing-based methods rely on the back-spliced junction signal for distinguishing circular and linear reads, which does not allow circRNAs' full-length structure to be effectively reconstructed. Here we describe a long-read sequencing-based protocol, CIRI-long, for the detection of full-length circular RNAs. The CIRI-long protocol combines rolling circular reverse transcription and nanopore sequencing to capture full-length circRNA sequences. After poly(A) tailing, RNase R treatment, and size selection of polymerase chain reaction products, CIRI-long achieves an increased percentage (6%) of circular reads in the constructed library, which is 20-fold higher compared with previous Illumina-based strategies. This method can be applied in cell lines or tissue samples, enabling accurate detection of full-length circRNAs in the range of 100-3,000 bp. The entire protocol can be completed in 1 d, and can be scaled up for large-scale analysis using the nanopore barcoding kit and PromethION sequencing device. CIRI-long can serve as an effective and user-friendly protocol for characterizing full-length circRNAs, generating direct and convincing evidence for the existence of detected circRNAs. The analytical pipeline offers convenient functions for identification of full-length circRNA isoforms and integration of multiple datasets. The assembled full-length transcripts and their splicing patterns provide indispensable information to explore the biological function of circRNAs.

Abstract 3743: circPMS1 is a pro-metastatic circular RNA in melanoma

Abstract Deregulated gene expression is a major driver of melanoma metastasis, a process that results in the majority of melanoma-related deaths. Importantly, these alterations are not limited to protein-coding mRNAs, but also include noncoding RNAs. Despite many recent studies implicating circular RNAs (circRNAs) in cancer development, it is unknown if their deregulation contributes to melanoma metastasis. To identify circRNAs with putative roles in melanomagenesis, we performed RNA sequencing on a panel of melanoma and melanocyte cell lines. 21 differentially expressed circRNAs were validated by qPCR and Sanger sequencing of the backsplice junction. Further analyses of the ratio of the circular and cognate linear RNA transcripts and expression in a larger panel of cell lines identified circPMS1 as a circRNA that is upregulated in melanoma through enhanced backsplicing of the linear PMS1 mRNA. Using transposon-mediated delivery, we examined the effects of stable circPMS1 overexpression on melanoma cells. While proliferation and focus formation were not impacted, migration and invasion were enhanced by circPMS1. Moreover, tail vein injection of circPMS1 overexpressing melanoma cells increased lung metastasis. In addition to this transplant model, we generated the first genetically engineered mouse model of melanoma overexpressing a pro-tumorigenic circRNA. These mice are currently being analyzed. Mechanistically, we find that circularization is required for the pro-migratory effects of the circPMS1 overexpression construct. Additionally, circPMS1 harbors the canonical PMS1 start codon, a predicted ORF spanning over the backsplice junction, and a predicted IRES sequence. Indeed, inserting a flag-tag in the overexpression construct revealed circPMS1 to be translated into a truncated 25kD PMS1 protein as well as a 20kD protein likely arising from an in-frame downstream start codon. Importantly, these truncated circPMS1 proteins are endogenously expressed and increased in melanoma compared to melanocytes while the full-length PMS1 protein is evenly expressed. These findings suggest that circPMS1 promotes melanoma metastasis, at least in part, by coding for truncated circPMS1 proteins. Further work will establish the functions of the circPMS1-encoded proteins and their roles in driving melanoma progression and metastasis. Citation Format: Nicol Mecozzi, Olga Vera, Florian Karreth. circPMS1 is a pro-metastatic circular RNA in melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3743.

Abstract P5-03-13: Circular RNAs express heterogeneously across different breast cancer subtypes and correlate with invasive disease-free survival

Abstract Background: Circular RNAs (circRNAs) are a large class of RNAs derived from back splicing and subsequent circularization of precursor mRNAs. Due to their circular structures, circRNAs are protected from exonuclease-induced degradation and are thereby comparatively more stable than linear RNAs. circRNAs have been implicated in the progression of multiple types of cancers but few studies have systematically examined how circRNAs associate with different subtypes of breast cancer and prognosis. Methods: We conducted a nested case-control study of prospectively ascertained participants in the Chicago Multiethnic Breast Cancer Cohort (ChiMEC), in which patients without recurrence were matched with patients with recurrence on time to recurrence, age of diagnosis, tumor stage, and clinical subtype. We performed whole exome capture RNA sequencing on tumor samples that passed quality control. We then used CIRIquant to identify circRNAs by aligning back-splice junction (BSJ) reads to pseudo-circular reference sequences and retained circRNAs that had a BSJ read count of 2 or greater in at least 5 patients. After normalization using the trimmed mean of M-values method, we used edgeR to model circRNA expression via a negative binomial distribution and performed differential expression (DE) analyses of circRNAs by ER and HER2 status, as well as between Black (self-reported) and White patients. Furthermore, we conducted survival analyses for each circRNA using Cox proportional hazards models to assess how the expression of each circRNA associated with survival outcomes of invasive disease-free survival (IDFS) and overall survival, while adjusting for age at diagnosis, stage, and HER2 status. Results: A total of 123 of 126 sequenced patients were included in the analysis, including 56 Black patients, 59 White patients, and 8 patients from other racial groups. The mean age of diagnosis was 51.9 years of age (SD 13.2) with 68% ER+, 48% PR+, and 30% HER2+ patients. We identified 16,927 high-confidence circRNAs. In the crude DE analysis, we found 489 circRNAs differentially expressed between patients with ER+ compared to patients with ER- tumors and 33 circRNAs between HER2+ vs. HER2- at a false discovery rate of 0.05. After adjusting for race and grade, we discovered 187 circRNAs differentially expressed by ER status and 38 by HER2 status. In the DE analysis by race, we found 88 circRNAs that were differentially expressed between Blacks and Whites. After adjusting for grade, ER, PR, and HER2 status, 14 circRNAs remained significantly different between racial groups. After a median of follow up of 8 years, 41 patients died, 41 patients had invasive recurrent diseases, and 2 patients had second primary breast cancers, for a total of 57 events in the IDFS analysis. Because of the matching study design to limit the impact of known prognostic factors, none of known prognostic factors (stage, ER, PR, HER2, grade, and race) were statistically associated with IDFS. In the survival analyses, we discovered two circRNAs (hsa-GSK3B_0001 and hsa-CMPK1_0006) that met the Bonferroni threshold for significance for their associations with IDFS but did not detect any circRNAs that were significantly associated with overall survival after correction for multiple testing. Discussion: This preliminary study demonstrates that multiple candidate circRNAs were differentially expressed between BC subtypes and racial groups, and several circRNAs were associated with IDFS. Future studies are warranted to validate our findings and cement the portability of these circRNAs as prognostic biomarkers across populations. Citation Format: James L. Li, Toshio F. Yoshimatsu, Julian C. McClellan, Fangyuan Zhao, Yonglan Zheng, Olufunmilayo I. Olopade, Dezheng Huo. Circular RNAs express heterogeneously across different breast cancer subtypes and correlate with invasive disease-free survival [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-03-13.

Proangiogenic role of circRNA-007371 in liver fibrosis.

Circular RNAs (circRNAs) are crucially involved in cancers as competing endogenous RNA (ceRNA) or microRNA (miRNA) sponges. However, the function and mechanism of circRNAs in liver fibrosis remain unknown and are the focus of this study. Murine fibrotic models were induced by thioacetamide (TAA) or carbon tetrachloride (CCl4 ). Increased angiogenesis is accompanied by liver fibrosis in TAA- and CCl4 -induced murine fibrotic livers. circRNA microarray and argonaute 2 (AGO2)-RNA immunoprecipitation (RIP) sequencing (AGO2-RIP sequencing) were performed in murine livers to screen for functional circRNAs. Compared to control livers, 86 differentially expressed circRNAs were obtained in TAA-induced murine fibrotic livers using circRNA microarray. In addition, 551 circRNAs were explored by AGO2-RIP sequencing of murine fibrotic livers. The circRNA-007371 was then selected and verified for back-spliced junction, resistance to RNase R, and loop formation. In vitro, murine hemangioendothelioma endothelial (EOMA) cells were transfected with circRNA-007371 overexpressing plasmid or empty plasmid. circRNA-007371 overexpression promoted tube formation, migration, and cell proliferation of EOMA cells. RNA sequencing and miRNA sequencing were then performed to explore the mechanism of the proangiogenic effects of circRNA-007371. circRNA-007371 promotes liver fibrosis via miRNA sponges or ceRNA mechanisms. Stag1, the parent gene of circRNA-007371, may play a significant role in proangiogenic progression. In conclusion, circRNA-007371 enhances angiogenesis via a miRNA sponge mechanism in liver fibrosis. The antiangiogenic effect of circRNA-007371 inhibition may provide a new strategy for treating patients with liver cirrhosis.

Assessing the impacts of various factors on circular RNA reliability.

Circular RNAs (circRNAs) are non-polyadenylated RNAs with a continuous loop structure characterized by a non-colinear back-splice junction (BSJ). Although millions of circRNA candidates have been identified, it remains a major challenge for determining circRNA reliability because of various types of false positives. Here, we systematically assess the impacts of numerous factors related to circRNA identification, conservation, biogenesis, and function on circRNA reliability by comparisons of circRNA expression from mock and the corresponding colinear/polyadenylated RNA-depleted datasets based on three different RNA treatment approaches. Eight important indicators of circRNA reliability are determined. The relative contribution to variability explained analyses reveal that the relative importance of these factors in affecting circRNA reliability in descending order is the conservation level of circRNA, full-length circular sequences, supporting BSJ read count, both BSJ donor and acceptor splice sites at the same colinear transcript isoforms, both BSJ donor and acceptor splice sites at the annotated exon boundaries, BSJs detected by multiple tools, supporting functional features, and both BSJ donor and acceptor splice sites undergoing alternative splicing. This study thus provides a useful guideline and an important resource for selecting high-confidence circRNAs for further investigations.

Identification of potential proteins translated from circular RNA splice variants

Circular RNAs (circRNAs) are covalently closed RNA molecules generated from precursor RNAs by the head-to-tail backsplicing of exons. Hundreds of studies demonstrated that circRNAs are ubiquitously expressed and regulate cellular events by modulating microRNA (miRNA) and RNA-binding protein (RBP) activities. A few circRNAs are also known to translate into functional polypeptides regulating cellular physiology. All these functions primarily depend on the full-length sequence of the circRNAs. CircRNA backsplice junction sequence is the key to identifying circRNAs and their full-length mature sequence. However, some multi-exonic circRNAs exist in different isoforms sharing identical backsplice junction sequences and are termed circRNA splice variants. Here, we analyzed the previously published HeLa cell RNA-seq datasets to identify circRNA splice variants using the de novo module of the CIRCexplorer2 circRNA annotation pipeline. A subset of circRNAs with splice variants was validated by the circRNA-rolling circle amplification (circRNA-RCA) method. Interestingly, several validated circRNAs were predicted to translate into proteins by the riboCIRC database. Furthermore, polyribosome fractionation followed by quantitative PCR confirmed the association of a subset of circRNAs with polyribosome supporting their protein-coding potential. Finally, bioinformatics analysis of proteins derived from splice variants of circCORO1C and circASPH suggested altered protein sequences and structures that could affect their physiological functions. Together, our study identified novel circRNA splice variants and their potential translation into protein isoforms which may regulate various physiological processes.

CircRNA-sponging: a pipeline for extensive analysis of circRNA expression and their role in miRNA sponging.

Circular RNAs (circRNAs) are long noncoding RNAs (lncRNAs) often associated with diseases and considered potential biomarkers for diagnosis and treatment. Among other functions, circRNAs have been shown to act as microRNA (miRNA) sponges, preventing the role of miRNAs that repress their targets. However, there is no pipeline to systematically assess the sponging potential of circRNAs. We developed circRNA-sponging, a nextflow pipeline that (i) identifies circRNAs via backsplicing junctions detected in RNA-seq data, (ii) quantifies their expression values in relation to their linear counterparts spliced from the same gene, (iii) performs differential expression analysis, (iv) identifies and quantifies miRNA expression from miRNA-sequencing (miRNA-seq) data, (v) predicts miRNA binding sites on circRNAs, (vi) systematically investigates potential circRNA-miRNA sponging events, (vii) creates a network of competing endogenous RNAs and (viii) identifies potential circRNA biomarkers. We showed the functionality of the circRNA-sponging pipeline using RNA sequencing data from brain tissues, where we identified two distinct types of circRNAs characterized by a specific ratio of the number of the binding site to the length of the transcript. The circRNA-sponging pipeline is the first end-to-end pipeline to identify circRNAs and their sponging systematically with raw total RNA-seq and miRNA-seq files, allowing us to better indicate the functional impact of circRNAs as a routine aspect in transcriptomic research. https://github.com/biomedbigdata/circRNA-sponging. Supplementary data are available at Bioinformatics Advances online.

Discovery of fusion circular RNAs in leukemia with KMT2A::AFF1 rearrangements by the new software CircFusion.

Chromosomal translocations in cancer genomes, key players in many types of cancers, generate chimeric proteins that drive oncogenesis. Genomes with chromosomal rearrangements can also produce fusion circular RNAs (f-circRNAs) by backsplicing of chimeric transcripts, as first shown in leukemias with PML::RARα and KMT2A::MLLT3 translocations and later in solid cancers. F-circRNAs contribute to the oncogenic processes and reinforce the oncogenic activity of chimeric proteins. In leukemia with KMT2A::AFF1 (MLL::AF4) fusions, we previously reported specific alterations of circRNA expression, but nothing was known about f-circRNAs. Due to the presence of two chimeric sequences, fusion and backsplice junctions, the identification of f-circRNAs with available tools is challenging, possibly resulting in the underestimation of this RNA species, especially when the breakpoint is not known. We developed CircFusion, a new software tool to detect linear fusion transcripts and f-circRNAs from RNA-seq data, both in samples for which the breakpoints are known and when the information about the joined exons is missing. CircFusion can detect linear and circular chimeric transcripts deriving from the main and reciprocal translocations also in the presence of multiple breakpoints, which are common in malignant cells. Benchmarking tests on simulated and real datasets of cancer samples with previously experimentally determined f-circRNAs showed that CircFusion provides reliable predictions and outperforms available methods for f-circRNA detection. We discovered and validated novel f-circRNAs in acute leukemia harboring KMT2A::AFF1 rearrangements, leading the way to future functional studies aimed to unveil their role in this malignancy.

CYCLeR-a novel tool for the full isoform assembly and quantification of circRNAs.

Splicing is one key mechanism determining the state of any eukaryotic cell. Apart from linear splice variants, circular splice variants (circRNAs) can arise via non-canonical splicing involving a back-splice junction (BSJ). Most existing methods only identify circRNAs via the corresponding BSJ, but do not aim to estimate their full sequence identity or to identify different, alternatively spliced circular isoforms arising from the same BSJ. We here present CYCLeR, the first computational method for identifying the full sequence identity of new and alternatively spliced circRNAs and their abundances while simultaneously co-estimating the abundances of known linear splicing isoforms. We show that CYCLeR significantly outperforms existing methods in terms of Fscore and quantification of transcripts in simulated data. In a in a comparative study with long-read data, we also show the advantages of CYCLeR compared to existing methods. When analysing Drosophila melanogaster data, CYCLeR uncovers biological patterns of circRNA expression that other methods fail to observe.

Tetrahedron supported CRISPR/Cas13a cleavage for electrochemical detection of circular RNA in bladder cancer

As a diagnostic biomarker, the detection of circular RNA (circRNA) is vital for the early screening of bladder cancer. Usually, the low abundance of circRNA in clinic samples results in the necessarily complicated extraction before detection. In this work, a tetrahedron supported CRISPR/Cas13a cleavage has been explored for direct electrochemical detection of circRNA in urine from bladder cancer. CRISPR/Cas13a system has been reasonably designed to recognize the characteristic back-splice junction site of circRNA. The activated CRISPR/Cas13a by circRNA can cleave uracil bases composed of DNA tetrahedron immobilized on the surface of gold electrode, resulting in the breakage of DNA tetrahedron and the release of electrochemical active molecule methylene blue. By virtue of highly catalytic efficiency of CRISPR/Cas13a and rigid structure of tetrahedron, the developed electrochemical biosensor can directly detect circRNA in 25 μL urine sample with the lowest detection limit of 0.089 fM and linear detection range from 10 fM to 50 nM in less than 10 min, so as to avoid complicated extraction process and benefit for on-site detection.

Integrated analysis of microRNAs, circular RNAs, long non-coding RNAs, and mRNAs revealed competing endogenous RNA networks involved in brown adipose tissue whitening in rabbits

BackgroundThe brown adipose tissue (BAT) is a target for treating obesity. BAT losses thermogenic capacity and gains a “white adipose tissue-like” phenotype (“BAT whitening”) under thermoneutral environments, which is a potential factor causing a low curative effect in BAT-related obesity treatments. Circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) can act as competing endogenous RNAs (ceRNA) to mRNAs and function in various processes by sponging shared microRNAs (miRNAs). However, the roles of circRNA- and lncRNA-related ceRNA networks in regulating BAT whitening remain litter known.ResultsIn this study, BATs were collected from rabbits at day0 (D0), D15, D85, and 2 years (Y2). MiRNA-seq was performed to investigate miRNA changes during BAT whitening. Then, a combined analysis of circRNA-seq and whole-transcriptome sequencing was used for circRNA assembly and quantification during BAT whitening. Our data showed that 1187 miRNAs and 6204 circRNAs were expressed in the samples, and many of which were identified as significantly changed during BAT whitening. Target prediction showed that D0-selective miRNAs were significantly enriched in the Ras, MAPK, and PI3K-Akt signaling pathways, and Y2-selective miRNAs were predicted to be involved in cell proliferation. The cyclization of several circRNAs could form novel response elements of key thermogenesis miRNAs at the back-splicing junction (BSJ) sites, and in combination with a dual-luciferase reporter assay confirmed the binding between the BSJ site of novel_circ_0013792 and ocu-miR-378-5p. CircRNAs and lncRNAs have high cooperativity in sponging miRNAs during BAT whitening. Both circRNA-miRNA-mRNA and lncRNA-miRNA-mRNA triple networks were significantly involved in immune response-associated biological processes. The D15-selective circRNA might promote BAT whitening by increasing the expression of IDH2. The Y2-selective circRNA-related ceRNA network and lncRNA-related ceRNA network might regulate the formation of the WAT-like phenotype of BAT via MAPK and Ras signaling pathways, respectively.ConclusionsOur work systematically revealed ceRNA networks during BAT whitening in rabbits and might provide new insight into BAT-based obesity treatments.

Circular RNAs dysregulation in ischemic heart failure

Abstract Introduction Circular RNAs (circRNAs) are an emerging class of non-coding RNAs originating from the splicing and circularization of pre-mRNAs and long non-coding RNAs. CircRNAs can regulate transcription and splicing, sequester microRNAs acting as “sponge” and inducing the respective targets, and bind to RNA binding proteins. CircRNAs have been found deregulated in several cardiovascular diseases, including heart failure (HF). However, incomplete and sometimes contradictory results have been reported on their regulation and function in HF, possibly for the heterogeneity of patients analyzed, indicating that our understanding of the regulation and role of cardiac circRNAs is still very limited. Purpose We aim to identify novel circRNA candidates deregulated in ischemic HF and to functionally characterize them in HF. Methods To define the circRNA expression pattern in ischemic HF, a high-depth RNA-seq was performed on the remote zone of left ventricle (LV) samples of 20 non end-stage ischemic HF patients with reduced LV ejection fraction and 20 matched controls. Differentially expressed circRNAs identified using CIRIquant tool were validated by qRT-PCR. Samples from the border zone of non end-stage HF patients and LV biopsies from end-stage HF patients were also used to measure circRNAs expression. Results By RNA-seq, we identified 17 circRNAs as differentially expressed with FDR<0.1. qRT-PCR results confirmed the differential expression of 4 circRNAs, cSLC6A6, cNAA16, cMLIP and cHDCA9, which were upregulated in HF samples (t-test, p-value <0.001). To evaluate circRNAs deregulated independently of their linear counterparts, we also estimated the ratio between circular and linear isoforms. cSLC6A6, cMLIP and cHDCA9 showed also a statistically significant (t-test, p-value <0.002) increase of the circular-to-linear ratio between HF and control patients. To complement the unbiased strategy of RNA-seq, we evaluated the modulation of 18 candidate circRNAs identified by a literature analysis as dysregulated in ischemic or not-ischemic cardiomyopathies or in biological mechanisms relevant for ischemic HF. cPVT1, cANKRD17, cBPTF, displaying a concordant deregulation in different stages of the disease (non end-stage and end-stage HF) and in different regions of LV myocardium (remote and border zone), were chosen for further analysis. Using siRNAs targeting the backsplice junction, cPVT1 was specifically knockdown in a human cardiomyocyte cell line. RNA-seq analysis of cPVT1 knockdown samples identified 3577 significantly differentially expressed genes (FDR<0.01). Gene ontology and pathway analysis showed that these genes were mainly involved in the formation of extracellular matrix, fibrosis and in cellular senescence, suggesting the involvement of cPVT1 in these HF-related disease mechanisms. Conclusion We identified new deregulated circRNAs in ischemic HF patients that might play a pathogenic role in HF. Funding Acknowledgement Type of funding sources: Private hospital(s). Main funding source(s): Italian Ministry of Health, Ricerca Corrente 2021-2023 “CircularRNAs in heart failure: regulation, function and interaction with the transcriptome”

Screening circular RNAs with functional potential using the RfxCas13d/BSJ-gRNA system.

Circular RNAs (circRNAs) are covalently enclosed, single-stranded RNAs produced by back-splicing of pre-mRNA exons that have recently emerged as an important class of molecules in gene expression regulation. circRNAs share overlapping sequences with their cognate linear mRNAs except the back-splicing junction (BSJ) sites. This feature makes it difficult to discriminate between the functions of circRNAs and their cognate mRNAs. We previously reported that the programmable RNA-guided, RNA-targeting CRISPR-Cas13 (RfxCas13d) system effectively and specifically discriminates circRNAs from mRNAs by using guide RNAs (gRNAs) targeting sequences across BSJ sites. Here, we describe a detailed protocol based on this RfxCas13d/BSJ-gRNA system for large-scale functional circRNA screening in human cell lines. The protocol includes gRNA library design, construction and transduction, analysis of screening results and validation of functional circRNA candidates. In total, it takes ~3-4 months of collaborative work between a well-trained molecular biologist and a bioinformatic expert. This protocol can be applied both in cells and in vivo to identify highly expressed circRNAs affecting cell growth, either in unperturbed conditions or under environmental stimulation, without disturbing their cognate linear mRNAs.

Profile of TREM2-Derived circRNA and mRNA Variants in the Entorhinal Cortex of Alzheimer's Disease Patients.

Genetic variants in TREM2, a microglia-related gene, are well-known risk factors for Alzheimer’s disease (AD). Here, we report that TREM2 originates from circular RNAs (circRNAs), a novel class of non-coding RNAs characterized by a covalent and stable closed-loop structure. First, divergent primers were designed to amplify circRNAs by RT-PCR, which were further assessed by Sanger sequencing. Then, additional primer sets were used to confirm back-splicing junctions. In addition, HMC3 cells were used to assess the microglial expression of circTREM2s. Three candidate circTREM2s were identified in control and AD human entorhinal samples. One of the circRNAs, circTREM2_1, was consistently amplified by all divergent primer sets in control and AD entorhinal cortex samples as well as in HMC3 cells. In AD cases, a moderate negative correlation (r = −0.434) was found between the global average area of Aβ deposits in the entorhinal cortex and circTREM2_1 expression level. In addition, by bioinformatics tools, a total of 16 miRNAs were predicted to join with circTREM2s. Finally, TREM2 mRNA corresponding to four isoforms was profiled by RT-qPCR. TREM2 mRNA levels were found elevated in entorhinal samples of AD patients with low or intermediate ABC scores compared to controls. To sum up, a novel circRNA derived from the TREM2 gene, circTREM2_1, has been identified in the human entorhinal cortex and TREM2 mRNA expression has been detected to increase in AD compared to controls. Unraveling the molecular genetics of the TREM2 gene may help to better know the innate immune response in AD.

Identification of Novel Circular RNAs of the Human Protein Arginine Methyltransferase 1 (PRMT1) Gene, Expressed in Breast Cancer Cells.

Circular RNAs (circRNAs) constitute a type of RNA formed through back-splicing. In breast cancer, circRNAs are implicated in tumor onset and progression. Although histone methylation by PRMT1 is largely involved in breast cancer development and metastasis, the effect of circular transcripts deriving from this gene has not been examined. In this study, total RNA was extracted from four breast cancer cell lines and reversely transcribed using random hexamer primers. Next, first- and second-round PCRs were performed using gene-specific divergent primers. Sanger sequencing followed for the determination of the sequence of each novel PRMT1 circRNA. Lastly, bioinformatics analysis was conducted to predict the functions of the novel circRNAs. In total, nine novel circRNAs were identified, comprising both complete and truncated exons of the PRMT1 gene. Interestingly, we demonstrated that the back-splice junctions consist of novel splice sites of the PRMT1 exons. Moreover, the circRNA expression pattern differed among these four breast cancer cell lines. All the novel circRNAs are predicted to act as miRNA and/or protein sponges, while five circRNAs also possess an open reading frame. In summary, we described the complete sequence of nine novel circRNAs of the PRMT1 gene, comprising distinct back-splice junctions and probably having different molecular properties.

Target-Initiated Cascade Signal Amplification Lights up a G-Quadruplex for a Label-Free Detection of Circular Ribonucleic Acids.

Circular ribonucleic acids (circRNAs) are a type of RNA that originates through back-splicing events from linear primary transcripts. CircRNAs display high structural resistance and tissue specificity. Accurate quantification of the circRNA expression level is of vital importance to disease diagnosis. Herein, we construct a label-free fluorescent biosensor for ultrasensitive analysis of circRNAs based on the integration of target-initiated cascade signal amplification strategy with a light-up G-quadruplex. This assay involves only one assistant probe that targets the circRNA-specific back-splice junction. When circRNA is present, it hybridizes with the assistant probe to initiate the duplex-specific nuclease (DSN)-catalyzed cyclic cleavage reaction, producing abundant triggers with 3'OH termini. Then, terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of dGTP and dATP at the 3'-OH termini of the resultant triggers to obtain abundant long G-rich DNA sequences that can form efficient G-quadruplex products. The addition of Thioflavin T (ThT) can light up G-quadruplex, generating an enhanced fluorescence. This assay may be performed isothermally without the involvement of any nucleic acid templates, exogenous primers, and specific labeled probes. Importantly, this biosensor can discriminate target circRNA from one-base mismatched circRNA and exhibits good performance in human serum. Moreover, it can accurately detect circRNA in cancer cells at a single-cell level and even differentiate the circRNA levels in the tissues of healthy persons and nonsmall cell lung cancer (NSCLC) patients, with promising applications in circRNA-related cancer diagnosis and therapeutics.