Widespread and Functional RNA Circularization in Localized Prostate Cancer

Chen, Sujun; Huang, Vincent; Xu, Xin; Livingstone, Julie; Soares, Fraser; Jeon, Jouhyun; Zeng, Yong; Hua, Junjie; Petricca, Jessica; Guo, Haiyang; Wang, Miranda; Yousif, Fouad; Zhang, Yuzhe; Donmez, Nilgun; Ahmed, Musaddeque; Volik, Stas; Lapuk, Anna; Chua, Melvin L.K.; Heisler, Lawrence E.; Foucal, Adrien; Fox, Natalie S.; Fraser, Michael; Bhandari, Vinayak; Shiah, Yu Jia; Guan, Jiansheng; Li, Jixi; Orain, Michèle; Picard, Valérie; Hovington, Hélène; Bergeron, Alain; Lacombe, Louis; Fradet, Yves; Têtu, Bernard; Liu, Stanley K.; Feng, Felix Y.; Wu, Xue; Shao, Yang; Komor, Malgorzata A.; Sahinalp, Cenk; Collins, Colin C.; Hoogstrate, Youri; Jong, Mark de; Fijneman, Remond J.A.; Teng, Fei; Jenster, Guido; Kwast, Theodorus van der; Bristow, Robert G.; Boutros, Paul C.; He, Housheng Hansen

doi:10.1016/j.cell.2019.01.025

Cited by 369 publications

(325 citation statements)

References 82 publications

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“…Their functions largely remain elusive, partly owing to the lack of effective tools to interfere with circular but not linear RNA levels in cells 1 . Short hairpin RNAs (shRNAs) or specific small interfering RNAs (siRNAs) targeting BSJ sites of circRNAs have been used to knock down circRNAs [3][4][5] . However, even partial complementarity of a half-RNAi (~10 nt) to linear mRNAs may affect parental gene expression (Extended Data Fig.…”

Section: Introductionmentioning

confidence: 99%

Screening for functional circular RNAs using the CRISPR-Cas13 system

Xue³

et al. 2020

Preprint

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Circular RNAs (circRNAs) produced from back-spliced exons are widely expressed, but individual circRNA functions remain poorly understood due to inadequate methods, such as RNAi and genome engineering, in distinguishing overlapped exons in circRNAs from those in linear cognate mRNAs 1,2 . Here we report that the programable RNA-guided, RNA-targeting CRISPR-Cas13, RfxCas13d, effectively and specifically discriminates circRNAs from mRNAs, using guide (g)RNAs targeting sequences spanning the back-splicing junction (BSJ) sites featured in RNA circles. Using a lentiviral library that targets sequences across BSJ sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type specific manner and that a common oncogenic circRNA, circFAM120A, promotes cell proliferation in vitro and in vivo by preventing FAM120A mRNA from binding the translation inhibitor IGF2BP2 for efficient translation. Application of RfxCas13d/BSJ-gRNA screening has also uncovered circMan1a2 with regulatory potential in mouse embryo preimplantation development. Together, these results establish CRISPR-RfxCas13d as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels.

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Section: Introductionmentioning

confidence: 99%

Screening for functional circular RNAs using the CRISPR-Cas13 system

Xue³

et al. 2020

Preprint

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“…Twenty years later, the advent of high-throughput sequencing of non-polyadenylated transcriptomes and bioinformatic analyses have made it possible to detect thousands of circRNAs, many of which are highly abundant and conserved across species (3)(4)(5) . Accumulating evidence links circRNAs to development and progression of different diseases (reviewed in (6) ) and several recent studies have shown that circRNAs are involved in tumorigenesis (7,8) . Due to their structural stability (4) , tissue specificity (3) , and relatively high expression levels in exosomes (9) , blood (10) , and plasma (11) , circRNAs have been suggested as a new class of biomarkers and potential therapeutic targets.…”

Section: Introductionmentioning

confidence: 99%

Transcriptome-wide profiles of circular RNA and RNA binding protein interactions reveal effects on circular RNA biogenesis and cancer pathway expression

Okholm

Sathe

Park

et al. 2020

Preprint

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Circular RNAs (circRNAs) are stable, often highly expressed RNA transcripts with potential to modulate other regulatory RNAs. A few circRNAs have been shown to bind RNA binding proteins (RBPs), however, little is known about the prevalence and strength of these interactions in different biological contexts. Here, we comprehensively evaluate the interplay between circRNAs and RBPs in the ENCODE cell lines, HepG2 and K562, by profiling the expression of circRNAs in fractionated total RNA-sequencing samples and analyzing binding sites of 150 RBPs in large eCLIP data sets. We show that KHSRP binding sites are enriched in flanking introns of circRNAs in both HepG2 and K562 cells, and that KHSRP depletion affects circRNA biogenesis. Additionally, we show that exons forming circRNAs are generally enriched with RBP binding sites compared to non-circularizing exons. To detect individual circRNAs with regulatory potency, we computationally identify circRNAs that are highly covered by RBP binding sites and experimentally validate circRNA-RBP interactions by RNA immunoprecipitations. We characterize circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, which is covered with GRWD1 binding sites. We confirm that circCDYL binds GRWD1 in vivo and functionally characterizes the effect of circCDYL-GRWD1 interactions on target genes in HepG2. Furthermore, we confirm interactions between circCDYL and RBPs in bladder cancer cells and demonstrate that circCDYL depletion affects hallmarks of cancer and perturbs the expression of key cancer genes, e.g. TP53 and MYC . Finally, we show that elevated levels of highly RBP-covered circRNAs, including circCDYL, are associated with overall survival of bladder cancer patients. Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis.

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“…Recently, shRNA-based functional screen has been employed to understand circRNA essentiality 1 . However, in our study, we observed frequent discrepancies between shRNAmediated circRNA knockdown efficiency and the corresponding biological effect on cell proliferation (see below), raising concerns about the robustness of using RNAi to study the function of circRNAs.…”

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confidence: 99%

Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying essential circRNAs

Zhang

Nguyen

Zhang

et al. 2020

Preprint

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Circular RNAs (circRNAs) are widely expressed, but their functions remain largely unknown.To study circRNAs in a high-throughput manner, short hairpin RNA (shRNA) screens 1 have recently been used to deplete circRNAs by targeting their unique back-splicing junction (BSJ) sites. Here, we report frequent discrepancies between shRNA-mediated circRNA knockdown efficiency and the corresponding biological effect, raising pressing concerns about the robustness of shRNA screening for functional circRNAs. To address this issue, we leveraged the CRISPR/Cas13d system 2 for circRNAs functional screenings. We optimized a strategy for designing single guide RNAs to deplete circRNAs. We then performed shRNA and CRISPR/Cas13d parallel screenings and demonstrated that shRNA-mediated circRNAs screening yielded a high rate of false positives phenotypes, while optimized CRISPR/Cas13d led to the identification of bona-fide functional circRNAs. Collectively, we developed a specific and reliable approach to functionalize circRNAs in a high-throughput manner. MainCircular RNAs (circRNAs) are covalently closed, single stranded transcripts, which are produced by back-splicing of precursor mRNAs (pre-mRNAs). Thousands of circRNAs have been discovered across species with cell-type-and tissue-specific expression patterns 3-6 . However, the functional repertoire of circRNAs remains mostly uncharacterized to date, which is mainly due to the unique properties of circRNAs and limitations of current approaches in circRNA studies 7,8 . The rapid development of CRISPR-Cas9 based genomics screens has dramatically enhanced speed and precision of functional characterization of both coding genes and linear non-coning RNAs 9 . Nevertheless, the majority of circRNAs are generated from protein-coding genes 10 , and hence the sequences of circRNAs are completely overlapping with their cognate linear RNAs

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Widespread and Functional RNA Circularization in Localized Prostate Cancer

Cited by 369 publications

References 82 publications

Screening for functional circular RNAs using the CRISPR-Cas13 system

Screening for functional circular RNAs using the CRISPR-Cas13 system

Transcriptome-wide profiles of circular RNA and RNA binding protein interactions reveal effects on circular RNA biogenesis and cancer pathway expression

Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying essential circRNAs

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