FRET analyses of the U2AF complex localize the U2AF35/U2AF65 interaction in vivo and reveal a novel self-interaction of U2AF35

Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
RNA (Impact Factor: 4.94). 09/2005; 11(8):1201-14. DOI: 10.1261/rna.7277705
Source: PubMed


We have analyzed the interaction between the U2AF subunits U2AF35 and U2AF65 in vivo using fluorescence resonance energy transfer (FRET) microscopy. U2 snRNP Auxiliary Factor (U2AF) is an essential pre-mRNA splicing factor complex, comprising 35-kDa (U2AF35) and 65-kDa (U2AF65) subunits. U2AF65 interacts directly with the polypyrimidine tract and promotes binding of U2 snRNP to the pre-mRNA branchpoint, while U2AF35 associates with the conserved AG dinucleotide at the 3' end of the intron and has multiple functions in the splicing process. Using two different approaches for measuring FRET, we have identified and spatially localized sites of direct interaction between U2AF35 and U2AF65 in vivo in live cell nuclei. While U2AF is thought to function as a heterodimeric complex, the FRET data have also revealed a novel U2AF35 self-interaction in vivo, which is confirmed in vitro using biochemical assays. These results suggest that the stoichiometry of the U2AF complex may, at least in part, differ in vivo from the expected heterodimeric complex. The data show that FRET studies offer a valuable approach for probing interactions between pre-mRNA splicing factors in vivo.

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Available from: Janet Karpinski
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    • "U2AF extensively interacts with splicing factors, but where and when these interactions occur within cells was not known. FRET studies revealed that the subunits of U2AF directly interact with each other even in the absence of transcription, suggesting that the U2AF heterodimer is preassembled before binding to pre-mRNA and stored in distinct foci called nuclear speckles [47]. U2AF65 also associates with splicing factor 1 (SF1) enhancing its binding to the pre-mRNA branch point sequence. "
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    ABSTRACT: Förster resonance energy transfer (FRET) microscopy is a powerful technique routinely used to monitor interactions between biomolecules. Here, we focus on the techniques that are used for investigating the structure and interactions of nucleic acids (NAs). We present a brief overview of the most commonly used FRET microscopy techniques, their advantages and drawbacks. We list experimental approaches recently used for either in vitro or in vivo studies. Next, we summarize how FRET contributed to the understanding of pre-mRNA splicing and spliceosome assembly.
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    • "EGFP-SRSF2 construct was generated by subcloning SRSF2 fragment from mCherry-SRSF2 as described previously [48]. EGFP-U2AF65 construct was generated by EcoR1/BamH1 digestion and gel extraction from EYFP-U2AF65 [62] and ligation into pEGFP-C1. pSV2-mMALAT1 [25] construct was a kind gift from Dr. KV. Prasanth, University of Illinois at Urbana-Champaign, USA. "
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    ABSTRACT: Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3' splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ∼20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA.
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    • "Compared to untreated controls, HMFG1- and B27.29-positive nuclear speckles in DRB-treated cells were larger and rounder with fewer interconnections (Fig. 6B). Similar results were found for U2AF65 and spliceosomal proteins, consistent with previous reports for the effects of transcriptional inhibition on splicing factor expression [52], [60], [62]. "
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