Quantifying full-length circular RNAs in cancer

Circular RNAs (circRNAs) are abundantly expressed in cancer. Their resistance to exonucleases enables them to have potentially stable interactions with different types of biomolecules. Alternative splicing can create different circRNA isoforms that have different sequences and unequal interaction potentials. The study of circRNA function thus requires knowledge of complete circRNA sequences. Here we describe psirc, a method that can identify full-length circRNA isoforms and quantify their expression levels from RNA sequencing data. We confirm the effectiveness and computational efficiency of psirc using both simulated and actual experimental data. Applying psirc on transcriptome profiles from nasopharyngeal carcinoma and normal nasopharynx samples, we discover and validate circRNA isoforms differentially expressed between the two groups. Compared to the assumed circular isoforms derived from linear transcript annotations, some of the alternatively spliced circular isoforms have 100 times higher expression and contain substantially fewer microRNA response elements, demonstrating the importance of quantifying full-length circRNA isoforms.

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Quantifying full-length circular RNAs in cancer

10.1101/2021.02.04.429722 ◽

2021 ◽

Author(s):

Ken Hung-On Yu ◽

Christina Huan Shi ◽

Bo Wang ◽

Savio Ho-Chit Chow ◽

Grace Tin-Yun Chung ◽

...

Keyword(s):

Experimental Data ◽

Alternative Splicing ◽

Nasopharyngeal Carcinoma ◽

Computational Efficiency ◽

Full Length ◽

Circular Rnas ◽

Sequencing Data ◽

Response Elements ◽

Different Types ◽

Alternatively Spliced

AbstractCircular RNAs (circRNAs) are abundantly expressed in cancer. Their resistance to exonucleases enables them to have potentially stable interactions with different types of biomolecules. Alternative splicing can create different circRNA isoforms that have different sequences and unequal interaction potentials. The study of circRNA function thus requires knowledge of complete circRNA sequences. Here we describe psirc, a method that can identify full-length circRNA isoforms and quantify their expression levels from RNA sequencing data. We confirm the effectiveness and computational efficiency of psirc using both simulated and actual experimental data. Applying psirc on transcriptome profiles from nasopharyngeal carcinoma and normal nasopharynx samples, we discover and validate circRNA isoforms differentially expressed between the two groups. Compared to the assumed circular isoforms derived from linear transcript annotations, some of the alternatively spliced circular isoforms have 100 times higher expression and contain substantially fewer microRNA response elements, demonstrating the importance of quantifying full-length circRNA isoforms.

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The interplay between microRNA and alternative splicing of linear and circular RNAs in eleven plant species

Bioinformatics ◽

10.1093/bioinformatics/btz038 ◽

2019 ◽

Vol 35 (17) ◽

pp. 3119-3126 ◽

Cited By ~ 5

Author(s):

Huiyuan Wang ◽

Huihui Wang ◽

Hangxiao Zhang ◽

Sheng Liu ◽

Yongsheng Wang ◽

...

Keyword(s):

Alternative Splicing ◽

Plant Species ◽

Single Molecule ◽

Mirna Target ◽

Supplementary Information ◽

Circular Rnas ◽

Sequencing Data ◽

Genomic Locus ◽

Target Sites ◽

Alternatively Spliced

Abstract Motivation MicroRNA (miRNA) and alternative splicing (AS)-mediated post-transcriptional regulation has been extensively studied in most eukaryotes. However, the interplay between AS and miRNAs has not been explored in plants. To our knowledge, the overall profile of miRNA target sites in circular RNAs (circRNA) generated by alternative back splicing has never been reported previously. To address the challenge, we identified miRNA target sites located in alternatively spliced regions of the linear and circular splice isoforms using the up-to-date single-molecule real-time (SMRT) isoform sequencing (Iso-Seq) and Illumina sequencing data in eleven plant species. Results In total, we identified 399 401 and 114 574 AS events from linear and circular RNAs, respectively. Among them, there were 64 781 and 41 146 miRNA target sites located in linear and circular AS region, respectively. In addition, we found 38 913 circRNAs to be overlapping with 45 648 AS events of its own parent isoforms, suggesting circRNA regulation of AS of linear RNAs by forming R-loop with the genomic locus. Here, we present a comprehensive database of miRNA targets in alternatively spliced linear and circRNAs (ASmiR) and a web server for deposition and identification of miRNA target sites located in the alternatively spliced region of linear and circular RNAs. This database is accompanied by an easy-to-use web query interface for meaningful downstream analysis. Plant research community can submit user-defined datasets to the web service to search AS regions harboring small RNA target sites. In conclusion, this study provides an unprecedented resource to understand regulatory relationships between miRNAs and AS in both gymnosperms and angiosperms. Availability and implementation The readily accessible database and web-based tools are available at http://forestry.fafu.edu.cn/bioinfor/db/ASmiR. Supplementary information Supplementary data are available at Bioinformatics online.

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EPCO-14. MULTIFACETED TRANSCRIPTOMIC AND PROTEOMIC ANALYSES IDENTIFIED PUTATIVE ALTERNATIVE SPLICING-DERIVED CELL SURFACE ANTIGENS IN GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noab196.013 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi4-vi4

Author(s):

Takahide Nejo ◽

Darwin Kwok ◽

Kevin Leung ◽

Lin Wang ◽

Albert Wang ◽

...

Keyword(s):

Alternative Splicing ◽

Cell Surface ◽

Surface Antigens ◽

Amino Acid Sequences ◽

Full Length ◽

Validation Dataset ◽

Sequencing Data ◽

Cell Surface Antigens ◽

Proteomic Analyses ◽

Full Length Transcript

Abstract BACKGROUND To develop effective immunotherapy for gliomas, it is crucial to expand the repertoire of targetable antigens. Recent studies have suggested that alternative splicing (AS), or its deriving tumor-specific junctions (“neojunctions”), could generate cryptic amino acid sequences that can be a source of neoantigens. In this study, we investigated neojunctions based on multifaceted transcriptomic and proteomic analyses, seeking the potential cell surface antigens that may be targeted by CAR. METHODS For screening, we analyzed bulk RNA-sequencing data of TCGA-GBM/LGG with high tumor purity (n = 429) and GTEx normal tissues (n = 9,166). Cohorts of spatially mapped intratumoral samples and longitudinally collected tumors were used to determine clonality and stability of the candidate neojunctions. Nanopore long-read amplicon sequencing was deployed to confirm the full-length transcript sequence. Their protein-level expression was explored by analyzing the Clinical Proteomic Tumor Analysis Consortium (CPTAC)-GBM proteomics dataset. RESULTS In the screening analysis comparing TCGA and GTEx datasets, we identified 218 neojunctions with adequate expression, prevalence, and tumor-specificity. Of these, 12 were predicted to be cell-surface antigens. Eight of the 12, such as BCAN, DLL3, and PTPRZ1, were also observed in multiple cases of another validation dataset. In the analysis of tumors with spatially mapped intratumoral samples, 7 of the 12 were recurrently detected in no less than 50% of the samples in multiple cases. In addition, 5 of the 12 were found to be conserved in primary and recurrent pairs of tumors in multiple cases. Full-length transcript sequencing corroborated our predictions based on short reads, and also demonstrated more complex AS patterns. Finally, CPTAC-GBM proteomics analysis identified one cryptic peptide that substantiated the corresponding transcriptome-based prediction. CONCLUSION: We identified neojunctions with the potential to generate cell-surface antigens. These multifaceted transcriptomic and proteomic analyses provide the rationale to pursue the development of immunotherapy targeting neojunction-derived antigens.

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CircAST: Full-length Assembly and Quantification of Alternatively Spliced Isoforms in Circular RNAs

Genomics Proteomics & Bioinformatics ◽

10.1016/j.gpb.2019.03.004 ◽

2019 ◽

Vol 17 (5) ◽

pp. 522-534 ◽

Cited By ~ 6

Author(s):

Jing Wu ◽

Yan Li ◽

Cheng Wang ◽

Yiqiang Cui ◽

Tianyi Xu ◽

...

Keyword(s):

Full Length ◽

Circular Rnas ◽

Alternatively Spliced

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Global Survey of the Full-Length Cabbage Transcriptome (Brassica oleracea Var. capitata L.) Reveals Key Alternative Splicing Events Involved in Growth and Disease Response

International Journal of Molecular Sciences ◽

10.3390/ijms221910443 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10443

Author(s):

Yong Wang ◽

Jialei Ji ◽

Long Tong ◽

Zhiyuan Fang ◽

Limei Yang ◽

...

Keyword(s):

Alternative Splicing ◽

Brassica Oleracea ◽

Single Molecule ◽

Alternative Polyadenylation ◽

Full Length ◽

Genomic Research ◽

Accurate Information ◽

Sequencing Data ◽

Specific Expression ◽

Global Survey

Cabbage (Brassica oleracea L. var. capitata L.) is an important vegetable crop cultivated around the world. Previous studies of cabbage gene transcripts were primarily based on next-generation sequencing (NGS) technology which cannot provide accurate information concerning transcript assembly and structure analysis. To overcome these issues and analyze the whole cabbage transcriptome at the isoform level, PacBio RS II Single-Molecule Real-Time (SMRT) sequencing technology was used for a global survey of the full-length transcriptomes of five cabbage tissue types (root, stem, leaf, flower, and silique). A total of 77,048 isoforms, capturing 18,183 annotated genes, were discovered from the sequencing data generated through SMRT. The patterns of both alternative splicing (AS) and alternative polyadenylation (APA) were comprehensively analyzed. In total, we detected 13,468 genes which had isoforms containing APA sites and 8978 genes which underwent AS events. Moreover, 5272 long non-coding RNAs (lncRNAs) were discovered, and most exhibited tissue-specific expression. In total, 3147 transcription factors (TFs) were detected and 10 significant gene co-expression network modules were identified. In addition, we found that Fusarium wilt, black rot and clubroot infection significantly influenced AS in resistant cabbage. In summary, this study provides abundant cabbage isoform transcriptome data, which promotes reannotation of the cabbage genome, deepens our understanding of their post-transcriptional regulation mechanisms, and can be used for future functional genomic research.

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SRPK1/2 and PP1α exert opposite functions by modulating SRSF1-guided MKNK2 alternative splicing in colon adenocarcinoma

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01877-y ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Hongda Liu ◽

Zheng Gong ◽

Kangshuai Li ◽

Qun Zhang ◽

Zekuan Xu ◽

...

Keyword(s):

Alternative Splicing ◽

Colon Adenocarcinoma ◽

Mapk Signaling ◽

Splicing Factor ◽

Tumor Proliferation ◽

Sequencing Data ◽

Alternatively Spliced ◽

Xenograft Models ◽

Splicing Isoforms ◽

Splicing Factor 1

Abstract Background The Mnk2 kinase, encoded by MKNK2 gene, plays critical roles in MAPK signaling and was involved in oncogenesis. Human MKNK2 pre-mRNA can be alternatively spliced into two splicing isoforms, the MKNK2a and MKNK2b, thus yielding Mnk2a and Mnk2b proteins with different domains. The involvement of Mnk2 alternative splicing in colon cancer has been implicated based on RNA-sequencing data from TCGA database. This study aimed at investigating the upstream modulators and clinical relevance of Mnk2 alternative splicing in colon adenocarcinoma (CAC). Methods PCR, western blotting and immunohistochemistry (IHC) were performed to assess the expression of Mnk2 and upstream proteins in CAC. The function of Mnk2 and its regulators were demonstrated in different CAC cell lines as well as in xenograft models. Two independent cohorts of CAC patients were used to reveal the clinical significance of MKNK2 alternative splicing. Results Comparing with adjacent nontumorous tissue, CAC specimen showed a decreased MKNK2a level and an increased MKNK2b level, which were correlated with KRAS mutation and tumor size. The SRSF1 (serine/arginine-rich splicing factor 1) was further confirmed to be the major splicing factor targeting MKNK2 in CAC cells. Higher expression of SRPK1/2 or decreased activity of PP1α were responsible for enhancing SRSF1 phosphorylation and nucleus translocation, subsequently resulted in a switch of MKNK2 alternative splicing. Conclusions Our data showed that phosphorylation and subcellular localization of SRSF1 were balanced by SRPK1/2 and PP1α in CAC cells. High nucleus SRSF1 promoted MKNK2 splicing into MKNK2b instead of MNK2a, consequently enhanced tumor proliferation.

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Alternative splicing of follicle-stimulating hormone receptor pre-mRNA: cloning and characterization of two alternatively spliced mRNA transcripts

Journal of Endocrinology ◽

10.1677/joe.0.1580127 ◽

1998 ◽

Vol 158 (1) ◽

pp. 127-136 ◽

Cited By ~ 32

Author(s):

R Kraaij ◽

M Verhoef-Post ◽

JA Grootegoed ◽

AP Themmen

Keyword(s):

Signal Transduction ◽

Alternative Splicing ◽

Follicle Stimulating Hormone ◽

Extracellular Domain ◽

Full Length ◽

Fsh Receptor ◽

Receptor Mrna ◽

Alternatively Spliced ◽

Stimulating Hormone

Glycoprotein hormone receptors contain a large extracellular domain that is encoded by multiple exons, facilitating the possibility of expressing alternatively spliced transcripts. We have cloned two new splice variants of the rat follicle-stimulating hormone (FSH) receptor gene: FSH-R1 and FSH-R2. The splice variant FSH-R1 differs from the full-length FSH receptor mRNA by the inclusion of a small extra exon between exons 9 and 10. FSH-R2 lacks the first three base pairs of exon 4, contains an extra exon between exons 4 and 5, and has an extended 3'-untranslated region. According to the predicted open reading frames, both mRNAs encode truncated FSH receptor proteins, consisting of the entire extracellular domain (FSH-R1) or the amino-terminal half of the extracellular domain (FSH-R2), and are expressed at a low level in testes and ovaries. The levels of expression of the FSH-R1 and FSH-R2 mRNAs in the gonads show a constant ratio to the expression level of the full-length FSH receptor mRNA. Furthermore, in vitro co-expression of either one of the truncated proteins with the full-length FSH receptor in COS1 cells did not affect signal transduction through the full-length FSH receptor. The absence of a function of the truncated FSH receptors in FSH signal transduction in vitro, and the lack of differential regulation of the alternative transcripts, indicate that there is no clear function for alternative splicing of the FSH receptor pre-mRNA in the postnatal testis and the cycling adult ovary.

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Noncoding RNAs regulate alternative splicing in Cancer

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-020-01798-2 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Yunze Liu ◽

Xin Liu ◽

Changwei Lin ◽

Xianhong Jia ◽

Hongmei Zhu ◽

...

Keyword(s):

Alternative Splicing ◽

Cancer Progression ◽

Cancer Biology ◽

Noncoding Rnas ◽

Circular Rnas ◽

Mrna Transcription ◽

Apoptosis Resistance ◽

Cis Acting ◽

Alternatively Spliced ◽

Multiple Levels

AbstractAS (alternative splicing) is a fundamental process by which a gene can generate multiple distinct mRNA transcripts to increase protein diversity. Defects in AS influence the occurrence and development of many diseases, including cancers, and are frequently found to participate in various aspects of cancer biology, such as promoting invasion, metastasis, apoptosis resistance and drug resistance. NcRNAs (noncoding RNAs) are an abundant class of RNAs that do not encode proteins. NcRNAs include miRNAs (microRNAs), lncRNAs (long noncoding RNAs), circRNAs (circular RNAs) and snRNAs (small nuclear RNAs) and have been proven to act as regulatory molecules that mediate cancer processes through AS. NcRNAs can directly or indirectly influence a plethora of molecular targets to regulate cis-acting elements, trans-acting factors, or pre-mRNA transcription at multiple levels, affecting the AS process and generating alternatively spliced isoforms. Consequently, ncRNA-mediated AS outcomes affect multiple cellular signaling pathways that promote or suppress cancer progression. In this review, we summarize the current mechanisms by which ncRNAs regulate AS in cancers and discuss their potential clinical applications as biomarkers and therapeutic targets.

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The mechanism and detection of alternative splicing events in circular RNAs

PeerJ ◽

10.7717/peerj.10032 ◽

2020 ◽

Vol 8 ◽

pp. e10032

Author(s):

Xiaohan Li ◽

Bing Zhang ◽

Fuyu Li ◽

Kequan Yu ◽

Yunfei Bai

Keyword(s):

Alternative Splicing ◽

Large Scale ◽

Single Gene ◽

Expression Patterns ◽

Functional Characterization ◽

Full Length ◽

Circular Rnas ◽

Specific Expression ◽

Enrichment Protocol

Circular RNAs (circRNAs) are considered as functional biomolecules with tissue/development-specific expression patterns. Generally, a single gene may generate multiple circRNA variants by alternative splicing, which contain different combinations of exons and/or introns. Due to the low abundance of circRNAs as well as overlapped with their linear counterparts, circRNA enrichment protocol is needed prior to sequencing. Compared with numerous algorithms, which use back-splicing reads for detection and functional characterization of circRNAs, original bioinformatic analyzing tools have been developed to large-scale determination of full-length circRNAs and accurate quantification. This review provides insights into the complexity of circRNA biogenesis and surveys the recent progresses in the experimental and bioinformatic methodologies that focus on accurately full-length circRNAs identification.

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Integration of Bioinformatic Predictions and Experimental Data to Identify circRNA-miRNA Associations

Genes ◽

10.3390/genes10090642 ◽

2019 ◽

Vol 10 (9) ◽

pp. 642 ◽

Cited By ~ 8

Author(s):

Martina Dori ◽

Silvio Bicciato

Keyword(s):

Experimental Data ◽

Single Case ◽

Circular Rnas ◽

General Function ◽

Response Elements ◽

Disease Biomarker ◽

Mirna Sponges ◽

Covalently Linked

Circular RNAs (circRNAs) have recently emerged as a novel class of transcripts, characterized by covalently linked 3′–5′ ends that result in the so-called backsplice junction. During the last few years, thousands of circRNAs have been identified in different organisms. Yet, despite their role as disease biomarker started to emerge, depicting their function remains challenging. Different studies have shown that certain circRNAs act as miRNA sponges, but any attempt to generalize from the single case to the “circ-ome” has failed so far. In this review, we explore the potential to define miRNA “sponging” as a more general function of circRNAs and describe the different approaches to predict miRNA response elements (MREs) in known or novel circRNA sequences. Moreover, we discuss how experiments based on Ago2-IP and experimentally validated miRNA:target duplexes can be used to either prioritize or validate putative miRNA-circRNA associations.

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