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Discovery of novel circular RNAs through integration of short- and long-read RNA sequencing

ABSTRACT: Circular RNAs (circRNAs) are associated with crucial hallmarks of tumorigenesis. Select circRNAs contain circular open reading frames (cORFs) and impact tumorigenesis through encoded small peptides. However, current circRNA detection approaches bias towards using short-read RNA-seq for detecting circRNA backsplice junctions (BSJs) without reliably reconstructing complete circRNA sequences, inhibiting accurate cORF prediction. To address these challenges, we performed long-read sequencing to enrich for full-length circRNAs that could serve as a guide for short-read alignment. This approach “rescued” circRNAs eluding existing tools focused on circRNA detection from short reads and enabled the development of an open-source bioinformatics workflow that characterizes and rescues circRNAs by integrating short- and long-read RNA-seq: CHRIS (CHaracterizing CircRNAs by Integrative Sequencing). Application of the approach to colorectal cancer (CRC) cell lines and patient samples revealed 6,445 non-canonical isoforms of known circRNAs of which 69 were altered during cancer metastasis. Validation experiments in CRC cell lines confirmed endogenous expression of 11 high-confidence circRNAs rescued by CHRIS. Next, proteogenomic analysis using 67,326 circRNAs detected by CHRIS and mass spectrometry data from 261 CRC patients from Clinical Proteomic Tumor Analysis Consortium (CPTAC) identified 6,848 peptides encoded by circRNAs, including 994 only detectable with long-read integration and 914 potential neoantigens. Overall, this research develops an approach that can facilitate circRNA detection and provide valuable resources for future circRNA tumor biology research.

ORGANISM(S): Homo sapiens

PROVIDER: GSE250483 | GEO | 2025/11/12

REPOSITORIES: GEO

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Project description:In platelets, splicing and translation occur in the absence of a nucleus. However, the integrity and stability of mRNAs derived from megakaryocyte progenitor cells remain poorly quantified on a transcriptome-wide level. As circular RNAs (circRNAs) are resistant to degradation by exonucleases, their abundance relative to linear RNAs can be used as a surrogate marker for mRNA stability in the absence of transcription. Here we show that circRNAs are enriched in human platelets 17-188 fold relative to nucleated tissues, and 14-26 fold relative to samples digested with RNAseR to selectively remove linear RNA. We compare RNAseq read depths inside and outside circRNAs to provide in silico evidence of transcript circularity, show that exons within circRNAs are enriched ~13X in platelets relative to nucleated tissues, and identify 3162 genes significantly enriched for circRNAs including some where all RNAs appear to be derived from circular molecules. We also confirm that this is a feature of other anucleate cells through transcriptome sequencing of mature erythrocytes, demonstrate that circRNAs are not enriched in megakaryocytes, and that linear RNAs decay more rapidly than circRNAs in platelet preparations. Collectively, these results suggest that circulating platelets have lost on aveage over 90% of their progenitor mRNAs, and that translation in platelets occurrs against the backdrop of a highly degraded transcriptome. Finally, we find that transcripts classified as products of reverse transcriptase template switching are both enriched in platelets and resistant to decay, countering the recent suggestion that up to 50% of rearranged RNAs are artefacts. A single rRNA depleted total RNA sample was sequenced. This together with 25 publicly available rRNA depleted total RNA samples (including 3 from platelets) were analysed using PTESFinder v 1 (http://sourceforge.net/projects/ptesfinder-v1/) to identify back-splice junctions, characteristic of circRNA transcripts. The contribution of circRNA producing exons was analysed on a gene by gene basis as follows: All circRNA transcripts identified in any sample were first pooled to define exons which can contribute to circRNA generation using custom scripts (available on request). For each sample, expression estimates (RPKMI) across all circRNA producing exons were computed for each locus using the total size of exons (in bp) and the read counts mapping to them. Similarly, total size and exonic read counts for exons for which no circRNA were detected in any sample were used to compute expression estimates (RPKME) for non-circRNA producing exons for each locus. Abundance ratios (RPKMI/RPKME and RPKMI/RPKMI+RPKME) were calculated and compared between Platelets and human tissues using Wilcoxon signed-rank test. Please note that the '25sample_info_accn_no.txt' contains the accession numbers and tissue/cell type information for 25 samples analyzed together.

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Discovery of novel circular RNAs through integration of short- and long-read RNA sequencing

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