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

Jonathan D. Ellis, David Lleres, Marco Denegri, Angus I. Lamond, Javier F. Caceres

    Research output: Contribution to journalArticlepeer-review

    39 Citations (Scopus)
    381 Downloads (Pure)

    Abstract

    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.

    © 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/).

    Original languageEnglish
    Pages (from-to)921-934
    Number of pages14
    JournalJournal of Cell Biology
    Volume181
    Issue number6
    DOIs
    Publication statusPublished - 16 Jun 2008

    Keywords

    • PRE-MESSENGER-RNA
    • PROTEIN-PROTEIN INTERACTIONS
    • GREEN FLUORESCENT PROTEIN
    • SR PROTEINS
    • IN-VIVO
    • FACTOR U2AF(35)
    • NUCLEAR SPECKLES
    • SITE SELECTION
    • SNRNP PROTEIN
    • CELL-NUCLEUS

    Cite this