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A better way to see splicing partners

Splicing factor interactions have been well-characterized in vitro, but less so in vivo. Ellis et al. have now used live cell imaging techniques to study these interactions in vivo and to discover where in the nucleus they occur.

Coimmunoprecipitation can't capture the dynamic nature of protein interactions, and the resolution of conventional fluorescence microscopy is too poor to distinguish truly interacting proteins from ones that are merely close to each other. In contrast, a positive FRET (fluorescence resonance energy transfer) signal implies two proteins are close enough to interact, and photobleaching or FLIM (fluorescence lifetime imaging microscopy) can detect changes in an interacting pair over time.

Using these techniques, the authors confirmed that the splice site protein, SF2/ASF, and the 5' splice site factor, U1 70K, interact in a type of nuclear body called a "speckle" and, to a lesser extent, in the nucleoplasm. When transcription was blocked, nucleoplasmic, but not speckle-localized, interactions were diminished. This finding bolsters the case for speckles as storage or assembly sites for splicing factors rather than sites of transcription-associated splicing.

The same localization and response to transcription inhibition was seen for interactions between SF2/ASF and the 3' splice site binder, U2AF35. But apparently not all splicing factors accumulate in speckles: interactions between HCC1 and either U2AF35 or U2AF65 were most prominent in the nucleoplasm, even when transcription was blocked.

There is clearly still much to uncover about splicing factor interactions in vivo, but by using FLIM-FRET, the authors have a powerful tool to dig deeper.

Ellis, J.D., et al. 2008. J. Cell Biol. doi:10.1083/jcb.200710051.[Abstract/Free Full Text]

Richard Robinson

rrobinson{at}nasw.org

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This Article Full Text (PDF, 1149K) Alert me when this article is cited Services Email this article Similar articles in this journal Alert me to new content in the JCB Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Robinson, R. Search for Related Content PubMed Articles by Robinson, R. Social Bookmarking                            What's this?