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Knockdown of Splicing Complex Protein PCBP2 Reduces Extravillous Trophoblast Differentiation Through Transcript Switching

Mutations in the LINC-HELLP non-coding RNA (HELLPAR) have been associated with familial forms of the pregnancy-specific HELLP syndrome. These mutations negatively affect extravillous trophoblast (EVT) differentiation from a proliferative to an invasive state and disturb the binding of RNA splicing c...

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Autores principales: Georgiadou, Danai, Boussata, Souad, Keijser, Remco, Janssen, Dianta A. M., Afink, Gijs B., van Dijk, Marie
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8172583/
https://www.ncbi.nlm.nih.gov/pubmed/34095140
http://dx.doi.org/10.3389/fcell.2021.671806
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author Georgiadou, Danai
Boussata, Souad
Keijser, Remco
Janssen, Dianta A. M.
Afink, Gijs B.
van Dijk, Marie
author_facet Georgiadou, Danai
Boussata, Souad
Keijser, Remco
Janssen, Dianta A. M.
Afink, Gijs B.
van Dijk, Marie
author_sort Georgiadou, Danai
collection PubMed
description Mutations in the LINC-HELLP non-coding RNA (HELLPAR) have been associated with familial forms of the pregnancy-specific HELLP syndrome. These mutations negatively affect extravillous trophoblast (EVT) differentiation from a proliferative to an invasive state and disturb the binding of RNA splicing complex proteins PCBP1, PCBP2, and YBX1 to LINC-HELLP. In this study, by using both in vitro and ex vivo experiments, we investigate if these proteins are involved in the regulation of EVT invasion during placentation. Additionally, we study if this regulation is due to alternative mRNA splicing. HTR-8/SVneo extravillous trophoblasts and human first trimester placental explants were used to investigate the effect of siRNA-mediated downregulation of PCBP1, PCBP2, and YBX1 genes on the differentiation of EVTs. Transwell invasion assays and proliferation assays indicated that upon knockdown of PCBP2 and, to a lesser extent, YBX1 and PCBP1, EVTs fail to differentiate toward an invasive phenotype. The same pattern was observed in placental explants where PCBP2 knockdown led to approximately 80% reduction in the number of explants showing any EVT outgrowth. Of the ones that still did show EVT outgrowth, the percentage of proliferating EVTs was significantly higher compared to explants transfected with non-targeting control siRNAs. To further investigate this effect of PCBP2 silencing on EVTs, we performed whole transcriptome sequencing (RNA-seq) on HTR-8/SVneo cells after PCBP2 knockdown. PCBP2 knockdown was found to have minimal effect on mRNA expression levels. In contrast, PCBP2 silencing led to a switch in splicing for a large number of genes with predominant functions in cellular assembly and organization, cellular function and maintenance, and cellular growth and proliferation and the cell cycle. EVTs, upon differentiation, alter their function to be able to invade the decidua of the mother by changing their cellular assembly and their proliferative activity by exiting the cell cycle. PCBP2 appears to be a paramount regulator of these differentiation mechanisms, where its disturbed binding to LINC-HELLP could contribute to dysfunctional placental development as seen in the HELLP syndrome.
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spelling pubmed-81725832021-06-04 Knockdown of Splicing Complex Protein PCBP2 Reduces Extravillous Trophoblast Differentiation Through Transcript Switching Georgiadou, Danai Boussata, Souad Keijser, Remco Janssen, Dianta A. M. Afink, Gijs B. van Dijk, Marie Front Cell Dev Biol Cell and Developmental Biology Mutations in the LINC-HELLP non-coding RNA (HELLPAR) have been associated with familial forms of the pregnancy-specific HELLP syndrome. These mutations negatively affect extravillous trophoblast (EVT) differentiation from a proliferative to an invasive state and disturb the binding of RNA splicing complex proteins PCBP1, PCBP2, and YBX1 to LINC-HELLP. In this study, by using both in vitro and ex vivo experiments, we investigate if these proteins are involved in the regulation of EVT invasion during placentation. Additionally, we study if this regulation is due to alternative mRNA splicing. HTR-8/SVneo extravillous trophoblasts and human first trimester placental explants were used to investigate the effect of siRNA-mediated downregulation of PCBP1, PCBP2, and YBX1 genes on the differentiation of EVTs. Transwell invasion assays and proliferation assays indicated that upon knockdown of PCBP2 and, to a lesser extent, YBX1 and PCBP1, EVTs fail to differentiate toward an invasive phenotype. The same pattern was observed in placental explants where PCBP2 knockdown led to approximately 80% reduction in the number of explants showing any EVT outgrowth. Of the ones that still did show EVT outgrowth, the percentage of proliferating EVTs was significantly higher compared to explants transfected with non-targeting control siRNAs. To further investigate this effect of PCBP2 silencing on EVTs, we performed whole transcriptome sequencing (RNA-seq) on HTR-8/SVneo cells after PCBP2 knockdown. PCBP2 knockdown was found to have minimal effect on mRNA expression levels. In contrast, PCBP2 silencing led to a switch in splicing for a large number of genes with predominant functions in cellular assembly and organization, cellular function and maintenance, and cellular growth and proliferation and the cell cycle. EVTs, upon differentiation, alter their function to be able to invade the decidua of the mother by changing their cellular assembly and their proliferative activity by exiting the cell cycle. PCBP2 appears to be a paramount regulator of these differentiation mechanisms, where its disturbed binding to LINC-HELLP could contribute to dysfunctional placental development as seen in the HELLP syndrome. Frontiers Media S.A. 2021-05-20 /pmc/articles/PMC8172583/ /pubmed/34095140 http://dx.doi.org/10.3389/fcell.2021.671806 Text en Copyright © 2021 Georgiadou, Boussata, Keijser, Janssen, Afink and van Dijk. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cell and Developmental Biology
Georgiadou, Danai
Boussata, Souad
Keijser, Remco
Janssen, Dianta A. M.
Afink, Gijs B.
van Dijk, Marie
Knockdown of Splicing Complex Protein PCBP2 Reduces Extravillous Trophoblast Differentiation Through Transcript Switching
title Knockdown of Splicing Complex Protein PCBP2 Reduces Extravillous Trophoblast Differentiation Through Transcript Switching
title_full Knockdown of Splicing Complex Protein PCBP2 Reduces Extravillous Trophoblast Differentiation Through Transcript Switching
title_fullStr Knockdown of Splicing Complex Protein PCBP2 Reduces Extravillous Trophoblast Differentiation Through Transcript Switching
title_full_unstemmed Knockdown of Splicing Complex Protein PCBP2 Reduces Extravillous Trophoblast Differentiation Through Transcript Switching
title_short Knockdown of Splicing Complex Protein PCBP2 Reduces Extravillous Trophoblast Differentiation Through Transcript Switching
title_sort knockdown of splicing complex protein pcbp2 reduces extravillous trophoblast differentiation through transcript switching
topic Cell and Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8172583/
https://www.ncbi.nlm.nih.gov/pubmed/34095140
http://dx.doi.org/10.3389/fcell.2021.671806
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