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Spatial mapping of splicing factor complexes involved in exon and intron definition

We have analyzed the interaction between serine/arginine-rich (SR) proteins and splicing components that recognize either the 5′ or 3′ splice site. Previously, these interactions have been extensively characterized biochemically and are critical for both intron and exon definition. We use fluorescen...

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Autores principales: Ellis, Jonathan D., Llères, David, Denegri, Marco, Lamond, Angus I., Cáceres, Javier F.
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2426932/
https://www.ncbi.nlm.nih.gov/pubmed/18559666
http://dx.doi.org/10.1083/jcb.200710051
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author Ellis, Jonathan D.
Llères, David
Denegri, Marco
Lamond, Angus I.
Cáceres, Javier F.
author_facet Ellis, Jonathan D.
Llères, David
Denegri, Marco
Lamond, Angus I.
Cáceres, Javier F.
author_sort Ellis, Jonathan D.
collection PubMed
description We have analyzed the interaction between serine/arginine-rich (SR) proteins and splicing components that recognize either the 5′ or 3′ splice site. Previously, these interactions have been extensively characterized biochemically and are critical for both intron and exon definition. We use fluorescence resonance energy transfer (FRET) microscopy to identify interactions of individual SR proteins with the U1 small nuclear ribonucleoprotein (snRNP)–associated 70-kD protein (U1 70K) and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) in live-cell nuclei. We find that these interactions occur in the presence of RNA polymerase II inhibitors, demonstrating that they are not exclusively cotranscriptional. Using FRET imaging by means of fluorescence lifetime imaging microscopy (FLIM), we map these interactions to specific sites in the nucleus. The FLIM data also reveal a previously unknown interaction between HCC1, a factor related to U2AF65, with both subunits of U2AF. Spatial mapping using FLIM-FRET reveals differences in splicing factors interactions within complexes located in separate subnuclear domains.
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spelling pubmed-24269322008-12-16 Spatial mapping of splicing factor complexes involved in exon and intron definition Ellis, Jonathan D. Llères, David Denegri, Marco Lamond, Angus I. Cáceres, Javier F. J Cell Biol Research Articles We have analyzed the interaction between serine/arginine-rich (SR) proteins and splicing components that recognize either the 5′ or 3′ splice site. Previously, these interactions have been extensively characterized biochemically and are critical for both intron and exon definition. We use fluorescence resonance energy transfer (FRET) microscopy to identify interactions of individual SR proteins with the U1 small nuclear ribonucleoprotein (snRNP)–associated 70-kD protein (U1 70K) and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) in live-cell nuclei. We find that these interactions occur in the presence of RNA polymerase II inhibitors, demonstrating that they are not exclusively cotranscriptional. Using FRET imaging by means of fluorescence lifetime imaging microscopy (FLIM), we map these interactions to specific sites in the nucleus. The FLIM data also reveal a previously unknown interaction between HCC1, a factor related to U2AF65, with both subunits of U2AF. Spatial mapping using FLIM-FRET reveals differences in splicing factors interactions within complexes located in separate subnuclear domains. The Rockefeller University Press 2008-06-16 /pmc/articles/PMC2426932/ /pubmed/18559666 http://dx.doi.org/10.1083/jcb.200710051 Text en © 2008 Ellis et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
spellingShingle Research Articles
Ellis, Jonathan D.
Llères, David
Denegri, Marco
Lamond, Angus I.
Cáceres, Javier F.
Spatial mapping of splicing factor complexes involved in exon and intron definition
title Spatial mapping of splicing factor complexes involved in exon and intron definition
title_full Spatial mapping of splicing factor complexes involved in exon and intron definition
title_fullStr Spatial mapping of splicing factor complexes involved in exon and intron definition
title_full_unstemmed Spatial mapping of splicing factor complexes involved in exon and intron definition
title_short Spatial mapping of splicing factor complexes involved in exon and intron definition
title_sort spatial mapping of splicing factor complexes involved in exon and intron definition
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2426932/
https://www.ncbi.nlm.nih.gov/pubmed/18559666
http://dx.doi.org/10.1083/jcb.200710051
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