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circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA

Circular RNAs (circRNAs) represent a class of widespread endogenous RNAs that regulate gene expression and thereby 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 larg...

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Autores principales: Okholm, Trine Line Hauge, Kamstrup, Andreas Bjerregaard, Nielsen, Morten Muhlig, Hollensen, Anne Kruse, Graversgaard, Mette Laugesen, Kristensen, Lasse Sommer, Vang, Søren, Park, Samuel S., Yeo, Gene W., Dyrskjøt, Lars, Kjems, Jørgen, Pedersen, Jakob Skou, Damgaard, Christian Kroun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10515936/
https://www.ncbi.nlm.nih.gov/pubmed/37745562
http://dx.doi.org/10.1101/2023.09.14.557527
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author Okholm, Trine Line Hauge
Kamstrup, Andreas Bjerregaard
Nielsen, Morten Muhlig
Hollensen, Anne Kruse
Graversgaard, Mette Laugesen
Kristensen, Lasse Sommer
Vang, Søren
Park, Samuel S.
Yeo, Gene W.
Dyrskjøt, Lars
Kjems, Jørgen
Pedersen, Jakob Skou
Damgaard, Christian Kroun
author_facet Okholm, Trine Line Hauge
Kamstrup, Andreas Bjerregaard
Nielsen, Morten Muhlig
Hollensen, Anne Kruse
Graversgaard, Mette Laugesen
Kristensen, Lasse Sommer
Vang, Søren
Park, Samuel S.
Yeo, Gene W.
Dyrskjøt, Lars
Kjems, Jørgen
Pedersen, Jakob Skou
Damgaard, Christian Kroun
author_sort Okholm, Trine Line Hauge
collection PubMed
description Circular RNAs (circRNAs) represent a class of widespread endogenous RNAs that regulate gene expression and thereby 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. Specifically, we use temporal depletion of circHIPK3 or specific RNA binding proteins (RBPs) and identify several perturbed genes by RNA sequencing analyses. Using expression-coupled motif analyses of mRNA expression data from various knockdown experiments, we identify an 11-mer motif within circHIPK3, which is also enriched in genes that become downregulated upon circHIPK3 depletion. By mining eCLIP datasets, we find that the 11-mer motif constitutes a strong binding site for IGF2BP2 and validate this circHIPK3-IGF2BP2 interaction experimentally using RNA-immunoprecipitation and competition assays in bladder cancer cell lines. Our results suggest that circHIPK3 and IGF2BP2 mRNA targets compete for binding. Since the identified 11-mer motif found in circHIPK3 is enriched in upregulated genes following IGF2BP2 knockdown, and since IGF2BP2 depletion conversely globally antagonizes the effect of circHIPK3 knockdown on target genes, 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. However, 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. Finally, we show that circHIPK3 expression correlates with overall survival of patients with bladder cancer. Our results are consistent with a model where relatively few cellular circHIPK3 molecules function as inducers of IGF2BP2 condensation thereby regulating STAT3 and other key factors for cell proliferation and potentially cancer progression.
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spelling pubmed-105159362023-09-23 circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA Okholm, Trine Line Hauge Kamstrup, Andreas Bjerregaard Nielsen, Morten Muhlig Hollensen, Anne Kruse Graversgaard, Mette Laugesen Kristensen, Lasse Sommer Vang, Søren Park, Samuel S. Yeo, Gene W. Dyrskjøt, Lars Kjems, Jørgen Pedersen, Jakob Skou Damgaard, Christian Kroun bioRxiv Article Circular RNAs (circRNAs) represent a class of widespread endogenous RNAs that regulate gene expression and thereby 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. Specifically, we use temporal depletion of circHIPK3 or specific RNA binding proteins (RBPs) and identify several perturbed genes by RNA sequencing analyses. Using expression-coupled motif analyses of mRNA expression data from various knockdown experiments, we identify an 11-mer motif within circHIPK3, which is also enriched in genes that become downregulated upon circHIPK3 depletion. By mining eCLIP datasets, we find that the 11-mer motif constitutes a strong binding site for IGF2BP2 and validate this circHIPK3-IGF2BP2 interaction experimentally using RNA-immunoprecipitation and competition assays in bladder cancer cell lines. Our results suggest that circHIPK3 and IGF2BP2 mRNA targets compete for binding. Since the identified 11-mer motif found in circHIPK3 is enriched in upregulated genes following IGF2BP2 knockdown, and since IGF2BP2 depletion conversely globally antagonizes the effect of circHIPK3 knockdown on target genes, 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. However, 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. Finally, we show that circHIPK3 expression correlates with overall survival of patients with bladder cancer. Our results are consistent with a model where relatively few cellular circHIPK3 molecules function as inducers of IGF2BP2 condensation thereby regulating STAT3 and other key factors for cell proliferation and potentially cancer progression. Cold Spring Harbor Laboratory 2023-09-14 /pmc/articles/PMC10515936/ /pubmed/37745562 http://dx.doi.org/10.1101/2023.09.14.557527 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
spellingShingle Article
Okholm, Trine Line Hauge
Kamstrup, Andreas Bjerregaard
Nielsen, Morten Muhlig
Hollensen, Anne Kruse
Graversgaard, Mette Laugesen
Kristensen, Lasse Sommer
Vang, Søren
Park, Samuel S.
Yeo, Gene W.
Dyrskjøt, Lars
Kjems, Jørgen
Pedersen, Jakob Skou
Damgaard, Christian Kroun
circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA
title circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA
title_full circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA
title_fullStr circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA
title_full_unstemmed circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA
title_short circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA
title_sort circhipk3 nucleates igf2bp2 and functions as a competing endogenous rna
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10515936/
https://www.ncbi.nlm.nih.gov/pubmed/37745562
http://dx.doi.org/10.1101/2023.09.14.557527
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