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Splicing Enhancers at Intron–Exon Borders Participate in Acceptor Splice Sites Recognition

Acceptor splice site recognition (3′ splice site: 3′ss) is a fundamental step in precursor messenger RNA (pre-mRNA) splicing. Generally, the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF) heterodimer recognizes the 3′ss, of which U2AF35 has a dual function: (i) It binds to the in...

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Autores principales: Kováčová, Tatiana, Souček, Přemysl, Hujová, Pavla, Freiberger, Tomáš, Grodecká, Lucie
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554774/
https://www.ncbi.nlm.nih.gov/pubmed/32911621
http://dx.doi.org/10.3390/ijms21186553
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author Kováčová, Tatiana
Souček, Přemysl
Hujová, Pavla
Freiberger, Tomáš
Grodecká, Lucie
author_facet Kováčová, Tatiana
Souček, Přemysl
Hujová, Pavla
Freiberger, Tomáš
Grodecká, Lucie
author_sort Kováčová, Tatiana
collection PubMed
description Acceptor splice site recognition (3′ splice site: 3′ss) is a fundamental step in precursor messenger RNA (pre-mRNA) splicing. Generally, the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF) heterodimer recognizes the 3′ss, of which U2AF35 has a dual function: (i) It binds to the intron–exon border of some 3′ss and (ii) mediates enhancer-binding splicing activators’ interactions with the spliceosome. Alternative mechanisms for 3′ss recognition have been suggested, yet they are still not thoroughly understood. Here, we analyzed 3′ss recognition where the intron–exon border is bound by a ubiquitous splicing regulator SRSF1. Using the minigene analysis of two model exons and their mutants, BRCA2 exon 12 and VARS2 exon 17, we showed that the exon inclusion correlated much better with the predicted SRSF1 affinity than 3′ss quality, which were assessed using the Catalog of Inferred Sequence Binding Preferences of RNA binding proteins (CISBP-RNA) database and maximum entropy algorithm (MaxEnt) predictor and the U2AF35 consensus matrix, respectively. RNA affinity purification proved SRSF1 binding to the model 3′ss. On the other hand, knockdown experiments revealed that U2AF35 also plays a role in these exons’ inclusion. Most probably, both factors stochastically bind the 3′ss, supporting exon recognition, more apparently in VARS2 exon 17. Identifying splicing activators as 3′ss recognition factors is crucial for both a basic understanding of splicing regulation and human genetic diagnostics when assessing variants’ effects on splicing.
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spelling pubmed-75547742020-10-14 Splicing Enhancers at Intron–Exon Borders Participate in Acceptor Splice Sites Recognition Kováčová, Tatiana Souček, Přemysl Hujová, Pavla Freiberger, Tomáš Grodecká, Lucie Int J Mol Sci Article Acceptor splice site recognition (3′ splice site: 3′ss) is a fundamental step in precursor messenger RNA (pre-mRNA) splicing. Generally, the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF) heterodimer recognizes the 3′ss, of which U2AF35 has a dual function: (i) It binds to the intron–exon border of some 3′ss and (ii) mediates enhancer-binding splicing activators’ interactions with the spliceosome. Alternative mechanisms for 3′ss recognition have been suggested, yet they are still not thoroughly understood. Here, we analyzed 3′ss recognition where the intron–exon border is bound by a ubiquitous splicing regulator SRSF1. Using the minigene analysis of two model exons and their mutants, BRCA2 exon 12 and VARS2 exon 17, we showed that the exon inclusion correlated much better with the predicted SRSF1 affinity than 3′ss quality, which were assessed using the Catalog of Inferred Sequence Binding Preferences of RNA binding proteins (CISBP-RNA) database and maximum entropy algorithm (MaxEnt) predictor and the U2AF35 consensus matrix, respectively. RNA affinity purification proved SRSF1 binding to the model 3′ss. On the other hand, knockdown experiments revealed that U2AF35 also plays a role in these exons’ inclusion. Most probably, both factors stochastically bind the 3′ss, supporting exon recognition, more apparently in VARS2 exon 17. Identifying splicing activators as 3′ss recognition factors is crucial for both a basic understanding of splicing regulation and human genetic diagnostics when assessing variants’ effects on splicing. MDPI 2020-09-08 /pmc/articles/PMC7554774/ /pubmed/32911621 http://dx.doi.org/10.3390/ijms21186553 Text en © 2020 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Kováčová, Tatiana
Souček, Přemysl
Hujová, Pavla
Freiberger, Tomáš
Grodecká, Lucie
Splicing Enhancers at Intron–Exon Borders Participate in Acceptor Splice Sites Recognition
title Splicing Enhancers at Intron–Exon Borders Participate in Acceptor Splice Sites Recognition
title_full Splicing Enhancers at Intron–Exon Borders Participate in Acceptor Splice Sites Recognition
title_fullStr Splicing Enhancers at Intron–Exon Borders Participate in Acceptor Splice Sites Recognition
title_full_unstemmed Splicing Enhancers at Intron–Exon Borders Participate in Acceptor Splice Sites Recognition
title_short Splicing Enhancers at Intron–Exon Borders Participate in Acceptor Splice Sites Recognition
title_sort splicing enhancers at intron–exon borders participate in acceptor splice sites recognition
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554774/
https://www.ncbi.nlm.nih.gov/pubmed/32911621
http://dx.doi.org/10.3390/ijms21186553
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