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Cooperative Binding of SRSF3 to Structured 3’ss-α Exon RNA during α Exon Inclusion in the ZO-1 mRNA

ZO-1α+ and ZO-1α− proteins are expressed in hermetic and leaky tight junctions, respectively. Two cis-acting distant exonic elements partly activate the 240 nucleotide-long α exon producing the ZO-1α+ isoform. However, the elements within and around the α exon and their respective factors involved i...

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Autores principales: Ruiz-Luis, Tea Anastasia, Ortuño-Pineda, Carlos, Galindo-Rosales, José Manuel, Saucedo-Cárdenas, Odila, Valdés, Jesús
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9857539/
https://www.ncbi.nlm.nih.gov/pubmed/36661525
http://dx.doi.org/10.3390/cimb45010039
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author Ruiz-Luis, Tea Anastasia
Ortuño-Pineda, Carlos
Galindo-Rosales, José Manuel
Saucedo-Cárdenas, Odila
Valdés, Jesús
author_facet Ruiz-Luis, Tea Anastasia
Ortuño-Pineda, Carlos
Galindo-Rosales, José Manuel
Saucedo-Cárdenas, Odila
Valdés, Jesús
author_sort Ruiz-Luis, Tea Anastasia
collection PubMed
description ZO-1α+ and ZO-1α− proteins are expressed in hermetic and leaky tight junctions, respectively. Two cis-acting distant exonic elements partly activate the 240 nucleotide-long α exon producing the ZO-1α+ isoform. However, the elements within and around the α exon and their respective factors involved in its splicing are unknown. To study the dynamic interaction between SRSF3 and its bioinformatically predicted target sites around the 3’ss upstream of the α exon during its activation, we performed EMSA, crosslinking, and in vivo splicing assays by ZO-1 minigene expression and siRNA-mediated silencing in transfected cells. Using V1 RNase, we probed the possible formation of a hairpin RNA structure between the intronic and proximal exonic SRSF3 binding sites. The hairpin sufficed for complex formations in the EMSA. The interaction of SRSF3 with the intronic site promoted the cooperative binding of SRSF3 to the exonic site. Finally, SRSF3 restored α exon activation in SRSF3 knockdown transfectants. Altogether, our results show that SRSF3–hairpin RNA interaction is crucial in the early recognition of 3’ss for α exon activation. It remains to be explored whether SRSF3 recruits or stabilizes the binding of other factors or brings separate splice sites into proximity.
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spelling pubmed-98575392023-01-21 Cooperative Binding of SRSF3 to Structured 3’ss-α Exon RNA during α Exon Inclusion in the ZO-1 mRNA Ruiz-Luis, Tea Anastasia Ortuño-Pineda, Carlos Galindo-Rosales, José Manuel Saucedo-Cárdenas, Odila Valdés, Jesús Curr Issues Mol Biol Article ZO-1α+ and ZO-1α− proteins are expressed in hermetic and leaky tight junctions, respectively. Two cis-acting distant exonic elements partly activate the 240 nucleotide-long α exon producing the ZO-1α+ isoform. However, the elements within and around the α exon and their respective factors involved in its splicing are unknown. To study the dynamic interaction between SRSF3 and its bioinformatically predicted target sites around the 3’ss upstream of the α exon during its activation, we performed EMSA, crosslinking, and in vivo splicing assays by ZO-1 minigene expression and siRNA-mediated silencing in transfected cells. Using V1 RNase, we probed the possible formation of a hairpin RNA structure between the intronic and proximal exonic SRSF3 binding sites. The hairpin sufficed for complex formations in the EMSA. The interaction of SRSF3 with the intronic site promoted the cooperative binding of SRSF3 to the exonic site. Finally, SRSF3 restored α exon activation in SRSF3 knockdown transfectants. Altogether, our results show that SRSF3–hairpin RNA interaction is crucial in the early recognition of 3’ss for α exon activation. It remains to be explored whether SRSF3 recruits or stabilizes the binding of other factors or brings separate splice sites into proximity. MDPI 2023-01-09 /pmc/articles/PMC9857539/ /pubmed/36661525 http://dx.doi.org/10.3390/cimb45010039 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ruiz-Luis, Tea Anastasia
Ortuño-Pineda, Carlos
Galindo-Rosales, José Manuel
Saucedo-Cárdenas, Odila
Valdés, Jesús
Cooperative Binding of SRSF3 to Structured 3’ss-α Exon RNA during α Exon Inclusion in the ZO-1 mRNA
title Cooperative Binding of SRSF3 to Structured 3’ss-α Exon RNA during α Exon Inclusion in the ZO-1 mRNA
title_full Cooperative Binding of SRSF3 to Structured 3’ss-α Exon RNA during α Exon Inclusion in the ZO-1 mRNA
title_fullStr Cooperative Binding of SRSF3 to Structured 3’ss-α Exon RNA during α Exon Inclusion in the ZO-1 mRNA
title_full_unstemmed Cooperative Binding of SRSF3 to Structured 3’ss-α Exon RNA during α Exon Inclusion in the ZO-1 mRNA
title_short Cooperative Binding of SRSF3 to Structured 3’ss-α Exon RNA during α Exon Inclusion in the ZO-1 mRNA
title_sort cooperative binding of srsf3 to structured 3’ss-α exon rna during α exon inclusion in the zo-1 mrna
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9857539/
https://www.ncbi.nlm.nih.gov/pubmed/36661525
http://dx.doi.org/10.3390/cimb45010039
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