circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA

Trine Line Hauge Okholm; Andreas Bjerregaard Kamstrup; Morten Muhlig Nielsen; Anne Kruse Hollensen; Mette Laugesen Graversgaard; Matilde Helbo Sørensen; Lasse Sommer Kristensen; Søren Vang; Samuel S. Park; Eugene Yeo; Lars Dyrskjøt; Jørgen Kjems; Jakob Skou Pedersen; Christian Kroun Damgaard

doi:10.7554/eLife.91783

circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA

Trine Line Hauge Okholm^*, Andreas Bjerregaard Kamstrup, Morten Muhlig Nielsen, Anne Kruse Hollensen, Mette Laugesen Graversgaard, Matilde Helbo Sørensen, Lasse Sommer Kristensen, Søren Vang, Samuel S. Park, Eugene Yeo, Lars Dyrskjøt, Jørgen Kjems, Jakob Skou Pedersen^*, Christian Kroun Damgaard^*

^*Corresponding author for this work

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

Abstract

Circular RNAs represent a class of endogenous RNAs that regulate gene expression and influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Using time-course depletion of circHIPK3 and specific candidate RNA-binding proteins, we identify several perturbed genes by RNA sequencing analyses. Expression-coupled motif analyses identify an 11-mer motif within circHIPK3, which also becomes enriched in genes that are downregulated upon circHIPK3 depletion. By mining eCLIP datasets and combined with RNA immunoprecipitation assays, we demonstrate that the 11-mer motif constitutes a strong binding site for IGF2BP2 in bladder cancer cell lines. Our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and, thereby, STAT3 mRNA levels. Surprisingly, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Our results support a model where a few cellular circHIPK3 molecules can induce IGF2BP2 condensation, thereby regulating key factors for cell proliferation.

Original language	English
Article number	RP91783
Journal	eLife
Volume	13
ISSN	2050-084X
DOIs	https://doi.org/10.7554/eLife.91783
Publication status	Published - 2024

Access to Document

10.7554/eLife.91783

Cite this

@article{b5c5ee5fb5c94cd1bab1d5a33ee3d9ea,

title = "circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA",

abstract = "Circular RNAs represent a class of endogenous RNAs that regulate gene expression and influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Using time-course depletion of circHIPK3 and specific candidate RNA-binding proteins, we identify several perturbed genes by RNA sequencing analyses. Expression-coupled motif analyses identify an 11-mer motif within circHIPK3, which also becomes enriched in genes that are downregulated upon circHIPK3 depletion. By mining eCLIP datasets and combined with RNA immunoprecipitation assays, we demonstrate that the 11-mer motif constitutes a strong binding site for IGF2BP2 in bladder cancer cell lines. Our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and, thereby, STAT3 mRNA levels. Surprisingly, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Our results support a model where a few cellular circHIPK3 molecules can induce IGF2BP2 condensation, thereby regulating key factors for cell proliferation.",

author = "Okholm, {Trine Line Hauge} and Kamstrup, {Andreas Bjerregaard} and Nielsen, {Morten Muhlig} and Hollensen, {Anne Kruse} and Graversgaard, {Mette Laugesen} and S{\o}rensen, {Matilde Helbo} and Kristensen, {Lasse Sommer} and S{\o}ren Vang and Park, {Samuel S.} and Eugene Yeo and Lars Dyrskj{\o}t and J{\o}rgen Kjems and Pedersen, {Jakob Skou} and Damgaard, {Christian Kroun}",

note = "Publisher Copyright: {\textcopyright} Okholm et al.",

year = "2024",

doi = "10.7554/eLife.91783",

language = "English",

volume = "13",

journal = "eLife",

issn = "2050-084X",

publisher = "eLife Sciences Publications Ltd",

}

TY - JOUR

T1 - circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA

AU - Okholm, Trine Line Hauge

AU - Kamstrup, Andreas Bjerregaard

AU - Nielsen, Morten Muhlig

AU - Hollensen, Anne Kruse

AU - Graversgaard, Mette Laugesen

AU - Sørensen, Matilde Helbo

AU - Kristensen, Lasse Sommer

AU - Vang, Søren

AU - Park, Samuel S.

AU - Yeo, Eugene

AU - Dyrskjøt, Lars

AU - Kjems, Jørgen

AU - Pedersen, Jakob Skou

AU - Damgaard, Christian Kroun

PY - 2024

Y1 - 2024

N2 - Circular RNAs represent a class of endogenous RNAs that regulate gene expression and influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Using time-course depletion of circHIPK3 and specific candidate RNA-binding proteins, we identify several perturbed genes by RNA sequencing analyses. Expression-coupled motif analyses identify an 11-mer motif within circHIPK3, which also becomes enriched in genes that are downregulated upon circHIPK3 depletion. By mining eCLIP datasets and combined with RNA immunoprecipitation assays, we demonstrate that the 11-mer motif constitutes a strong binding site for IGF2BP2 in bladder cancer cell lines. Our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and, thereby, STAT3 mRNA levels. Surprisingly, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Our results support a model where a few cellular circHIPK3 molecules can induce IGF2BP2 condensation, thereby regulating key factors for cell proliferation.

AB - Circular RNAs represent a class of endogenous RNAs that regulate gene expression and influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Using time-course depletion of circHIPK3 and specific candidate RNA-binding proteins, we identify several perturbed genes by RNA sequencing analyses. Expression-coupled motif analyses identify an 11-mer motif within circHIPK3, which also becomes enriched in genes that are downregulated upon circHIPK3 depletion. By mining eCLIP datasets and combined with RNA immunoprecipitation assays, we demonstrate that the 11-mer motif constitutes a strong binding site for IGF2BP2 in bladder cancer cell lines. Our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and, thereby, STAT3 mRNA levels. Surprisingly, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Our results support a model where a few cellular circHIPK3 molecules can induce IGF2BP2 condensation, thereby regulating key factors for cell proliferation.

UR - http://www.scopus.com/inward/record.url?scp=85200982798&partnerID=8YFLogxK

U2 - 10.7554/eLife.91783

DO - 10.7554/eLife.91783

M3 - Journal article

C2 - 39041323

SN - 2050-084X

VL - 13

JO - eLife

JF - eLife

M1 - RP91783

ER -

circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA

Abstract

Access to Document

Other files and links

Fingerprint

Cite this