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

Trine Line Hauge Okholm; Shashank Sathe; Samuel S.  Park; Andreas Bjerregaard Kamstrup; Asta Mannstaedt Rasmussen; Archana Shankar; Zong Ming Chua; Niels Fristrup; Morten Muhlig Nielsen; Søren Vang; Lars Dyrskjøt; Stefan Aigner; Christian Kroun Damgaard; Gene W. Yeo; Jakob Skou Pedersen

doi:10.1186/s13073-020-00812-8

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

Trine Line Hauge Okholm, Shashank Sathe, Samuel S. Park, Andreas Bjerregaard Kamstrup, Asta Mannstaedt Rasmussen, Archana Shankar, Zong Ming Chua, Niels Fristrup, Morten Muhlig Nielsen, Søren Vang, Lars Dyrskjøt, Stefan Aigner, Christian Kroun Damgaard, Gene W. Yeo, Jakob Skou Pedersen

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Abstract

Background: 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 distribution of these interactions in different biological contexts. Methods: We conduct an extensive screen of circRNA-RBP interactions in the ENCODE cell lines HepG2 and K562. We profile circRNAs in deep-sequenced total RNA samples and analyze circRNA-RBP interactions using a large set of eCLIP data with binding sites of 150 RBPs. We validate interactions for select circRNAs and RBPs by performing RNA immunoprecipitation and functionally characterize our most interesting candidates by conducting knockdown studies followed by RNA-Seq. Results: We generate a comprehensive catalog of circRNA-RBP interactions in HepG2 and K562 cells. We show that KHSRP binding sites are enriched in flanking introns of circRNAs and that KHSRP depletion affects circRNA biogenesis. We identify circRNAs that are highly covered by RBP binding sites and experimentally validate individual circRNA-RBP interactions. We show that circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, is almost completely covered with GRWD1 binding sites in HepG2 cells, and that circCDYL depletion counteracts the effect of GRWD1 depletion. 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. Finally, we show that elevated levels of circCDYL are associated with overall survival of bladder cancer patients. Conclusions: Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis.

Original language	English
Article number	112
Journal	Genome Medicine
Volume	12
Issue	112
ISSN	1756-994X
DOIs	https://doi.org/10.1186/s13073-020-00812-8
Publication status	Published - Dec 2020

Keywords

Bladder cancer
Circular RNAs
RBP sponges
RNA-binding proteins
Tumorigenesis
circCDYL

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Okholm, T. L. H., Sathe, S., Park, S. S., Kamstrup, A. B., Rasmussen, A. M., Shankar, A., Chua, Z. M., Fristrup, N., Nielsen, M. M., Vang, S., Dyrskjøt, L., Aigner, S., Damgaard, C. K., Yeo, G. W., & Pedersen, J. S. (2020). Transcriptome-wide profiles of circular RNA and RNA-binding protein interactions reveal effects on circular RNA biogenesis and cancer pathway expression. Genome Medicine, 12(112), Article 112. https://doi.org/10.1186/s13073-020-00812-8

@article{386d256c44b5490882dc7ec8fc8bf6a3,

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

abstract = "Background: 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 distribution of these interactions in different biological contexts. Methods: We conduct an extensive screen of circRNA-RBP interactions in the ENCODE cell lines HepG2 and K562. We profile circRNAs in deep-sequenced total RNA samples and analyze circRNA-RBP interactions using a large set of eCLIP data with binding sites of 150 RBPs. We validate interactions for select circRNAs and RBPs by performing RNA immunoprecipitation and functionally characterize our most interesting candidates by conducting knockdown studies followed by RNA-Seq. Results: We generate a comprehensive catalog of circRNA-RBP interactions in HepG2 and K562 cells. We show that KHSRP binding sites are enriched in flanking introns of circRNAs and that KHSRP depletion affects circRNA biogenesis. We identify circRNAs that are highly covered by RBP binding sites and experimentally validate individual circRNA-RBP interactions. We show that circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, is almost completely covered with GRWD1 binding sites in HepG2 cells, and that circCDYL depletion counteracts the effect of GRWD1 depletion. 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. Finally, we show that elevated levels of circCDYL are associated with overall survival of bladder cancer patients. Conclusions: Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis.",

keywords = "Bladder cancer, Circular RNAs, RBP sponges, RNA-binding proteins, Tumorigenesis, circCDYL",

author = "Okholm, {Trine Line Hauge} and Shashank Sathe and Park, {Samuel S.} and Kamstrup, {Andreas Bjerregaard} and Rasmussen, {Asta Mannstaedt} and Archana Shankar and Chua, {Zong Ming} and Niels Fristrup and Nielsen, {Morten Muhlig} and S{\o}ren Vang and Lars Dyrskj{\o}t and Stefan Aigner and Damgaard, {Christian Kroun} and Yeo, {Gene W.} and Pedersen, {Jakob Skou}",

year = "2020",

month = dec,

doi = "10.1186/s13073-020-00812-8",

language = "English",

volume = "12",

journal = "Genome Medicine",

issn = "1756-994X",

publisher = "BioMed Central Ltd.",

number = "112",

}

Okholm, TLH, Sathe, S, Park, SS, Kamstrup, AB, Rasmussen, AM, Shankar, A, Chua, ZM, Fristrup, N , Nielsen, MM , Vang, S , Dyrskjøt, L, Aigner, S, Damgaard, CK, Yeo, GW & Pedersen, JS 2020, 'Transcriptome-wide profiles of circular RNA and RNA-binding protein interactions reveal effects on circular RNA biogenesis and cancer pathway expression', Genome Medicine, vol. 12, no. 112, 112. https://doi.org/10.1186/s13073-020-00812-8

Transcriptome-wide profiles of circular RNA and RNA-binding protein interactions reveal effects on circular RNA biogenesis and cancer pathway expression. / Okholm, Trine Line Hauge; Sathe, Shashank; Park, Samuel S. et al.
In: Genome Medicine, Vol. 12, No. 112, 112, 12.2020.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - Transcriptome-wide profiles of circular RNA and RNA-binding protein interactions reveal effects on circular RNA biogenesis and cancer pathway expression

AU - Okholm, Trine Line Hauge

AU - Sathe, Shashank

AU - Park, Samuel S.

AU - Kamstrup, Andreas Bjerregaard

AU - Rasmussen, Asta Mannstaedt

AU - Shankar, Archana

AU - Chua, Zong Ming

AU - Fristrup, Niels

AU - Nielsen, Morten Muhlig

AU - Vang, Søren

AU - Dyrskjøt, Lars

AU - Aigner, Stefan

AU - Damgaard, Christian Kroun

AU - Yeo, Gene W.

AU - Pedersen, Jakob Skou

PY - 2020/12

Y1 - 2020/12

N2 - Background: 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 distribution of these interactions in different biological contexts. Methods: We conduct an extensive screen of circRNA-RBP interactions in the ENCODE cell lines HepG2 and K562. We profile circRNAs in deep-sequenced total RNA samples and analyze circRNA-RBP interactions using a large set of eCLIP data with binding sites of 150 RBPs. We validate interactions for select circRNAs and RBPs by performing RNA immunoprecipitation and functionally characterize our most interesting candidates by conducting knockdown studies followed by RNA-Seq. Results: We generate a comprehensive catalog of circRNA-RBP interactions in HepG2 and K562 cells. We show that KHSRP binding sites are enriched in flanking introns of circRNAs and that KHSRP depletion affects circRNA biogenesis. We identify circRNAs that are highly covered by RBP binding sites and experimentally validate individual circRNA-RBP interactions. We show that circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, is almost completely covered with GRWD1 binding sites in HepG2 cells, and that circCDYL depletion counteracts the effect of GRWD1 depletion. 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. Finally, we show that elevated levels of circCDYL are associated with overall survival of bladder cancer patients. Conclusions: Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis.

AB - Background: 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 distribution of these interactions in different biological contexts. Methods: We conduct an extensive screen of circRNA-RBP interactions in the ENCODE cell lines HepG2 and K562. We profile circRNAs in deep-sequenced total RNA samples and analyze circRNA-RBP interactions using a large set of eCLIP data with binding sites of 150 RBPs. We validate interactions for select circRNAs and RBPs by performing RNA immunoprecipitation and functionally characterize our most interesting candidates by conducting knockdown studies followed by RNA-Seq. Results: We generate a comprehensive catalog of circRNA-RBP interactions in HepG2 and K562 cells. We show that KHSRP binding sites are enriched in flanking introns of circRNAs and that KHSRP depletion affects circRNA biogenesis. We identify circRNAs that are highly covered by RBP binding sites and experimentally validate individual circRNA-RBP interactions. We show that circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, is almost completely covered with GRWD1 binding sites in HepG2 cells, and that circCDYL depletion counteracts the effect of GRWD1 depletion. 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. Finally, we show that elevated levels of circCDYL are associated with overall survival of bladder cancer patients. Conclusions: Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis.

KW - Bladder cancer

KW - Circular RNAs

KW - RBP sponges

KW - RNA-binding proteins

KW - Tumorigenesis

KW - circCDYL

UR - http://www.scopus.com/inward/record.url?scp=85097237432&partnerID=8YFLogxK

U2 - 10.1186/s13073-020-00812-8

DO - 10.1186/s13073-020-00812-8

M3 - Journal article

C2 - 33287884

SN - 1756-994X

VL - 12

JO - Genome Medicine

JF - Genome Medicine

IS - 112

M1 - 112

ER -

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

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