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Analyses of in vivo interaction and mobility of two spliceosomal proteins using FRAP and BiFC.

ABSTRACT: U1-70K, a U1 snRNP-specific protein, and serine/arginine-rich (SR) proteins are components of the spliceosome and play critical roles in both constitutive and alternative pre-mRNA splicing. However, the mobility properties of U1-70K, its in vivo interaction with SR proteins, and the mobility of the U1-70K-SR protein complex have not been studied in any system. Here, we studied the in vivo interaction of U1-70K with an SR protein (SR45) and the mobility of the U1-70K/SR protein complex using bimolecular fluorescence complementation (BiFC) and fluorescence recovery after photobleaching (FRAP). Our results show that U1-70K exchanges between speckles and the nucleoplasmic pool very rapidly and that this exchange is sensitive to ongoing transcription and phosphorylation. BiFC analyses showed that U1-70K and SR45 interacted primarily in speckles and that this interaction is mediated by the RS1 or RS2 domain of SR45. FRAP analyses showed considerably slower recovery of the SR45/U1-70K complex than either protein alone indicating that SR45/U1-70K complexes remain in the speckles for a longer duration. Furthermore, FRAP analyses with SR45/U1-70K complex in the presence of inhibitors of phosphorylation did not reveal any significant change compared to control cells, suggesting that the mobility of the complex is not affected by the status of protein phosphorylation. These results indicate that U1-70K, like SR splicing factors, moves rapidly in the nucleus ensuring its availability at various sites of splicing. Furthermore, although it appears that U1-70K moves by diffusion its mobility is regulated by phosphorylation and transcription.

SUBMITTER: Ali GS

PROVIDER: S-EPMC2278372 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Analyses of in vivo interaction and mobility of two spliceosomal proteins using FRAP and BiFC.

Ali Gul Shad GS Prasad K V S K KV Hanumappa M M Reddy A S N AS

PloS one 20080416 4

U1-70K, a U1 snRNP-specific protein, and serine/arginine-rich (SR) proteins are components of the spliceosome and play critical roles in both constitutive and alternative pre-mRNA splicing. However, the mobility properties of U1-70K, its in vivo interaction with SR proteins, and the mobility of the U1-70K-SR protein complex have not been studied in any system. Here, we studied the in vivo interaction of U1-70K with an SR protein (SR45) and the mobility of the U1-70K/SR protein complex using bimo ...[more]

PMID: 18414657

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Project description:Bimolecular Fluorescence Complementation (BiFC) assay is a method used to directly visualize protein-protein interaction in vivo using live-cell imaging or fixed cells. This protocol described here is based on our recent paper describing the functional association of human chromatin adaptor and transcription cofactor Brd4 with p53 tumor suppressor protein (Wu et al., 2013). BiFC was first described by Hu et al. (2002) using two non-fluorescent protein fragments of enhanced yellow fluorescent protein (EYFP), which is an Aequorea victoria GFP variant protein, fused respectively to a Rel family protein and a bZIP family transcription factor to investigate interactions between these two family members in living cells. The YFP was later improved by introducing mutations to reduce its sensitivity to pH and chloride ions, thus generating a super-enhanced YFP, named Venus fluorescent protein, without showing diminished fluorescence at 37 °C as typically observed with EYFP (Nagai et al., 2006). The fluorescence signal is regenerated by complementation of two non-fluorescent fragments (e.g., the Venus N-terminal 1-158 amino acid residues, called Venus-N, and its C-terminal 159-239 amino acid residues, named Venus-C; see Figure 1A and Gully et al., 2012; Ding et al., 2006; Kerppola, 2006) that are brought together by interaction between their respective fusion partners (e.g., Venus-N to p53, and Venus-C to the PDID domain of human Brd4; see Figure 1B and 1C). The intensity and cellular location of the regenerated fluorescence signals can be detected by fluorescence microscope. The advantages of the proximity-based BiFC assay are: first, it allows a direct visualization of spatial and temporal interaction between two partner proteins in vivo; second, the fluorescence signal provides a sensitive readout for detecting protein-protein interaction even at a low expression level comparable to that of the endogenous proteins; third, the intensity of the fluorescence signal is proportional to the strength of protein-protein interaction (Morell et al., 2008); and fourth, the BiFC signals are derived from intrinsic protein-protein interaction, rather than from extrinsic fluorophores that may not reflect true protein-protein interaction due to their nonspecific association with cellular macromolecules or subcellular compartments. However, some limitations of BiFC include slow maturation (T1/2 ~ 1 h) of an eventually stable BiFC complex (Hu et al., 2002), making it unsuitable for real-time observation of transient interaction that disappears prior to BiFC detection, and enhanced BiFC background at high expression levels due to fusion-independent association between two non-fluorescent fragments association. BiFC signals generated by in vivo protein-protein interaction can be validated by amino acid mutation introduced at the protein-protein contact surfaces. This imaging technique has been widely used in different cell types and organisms (Kerppola, 2006).

Project description:The homodimeric transmembrane receptor endoglin (CD105) plays an important role in angiogenesis. This is highlighted by mutations in its gene, causing the vascular disorder HHT1. The main role of endoglin function has been assigned to the modulation of transforming growth factor β and bone morphogenetic protein signalling in endothelial cells. Nevertheless, other functions of endoglin have been revealed to be involved in different cellular functions and in other cell types than endothelial cells. Compared to the exploration of its natural function, little experimental data have been gathered about the mode of action of endoglin HHT mutations at the cellular level, especially missense mutations, and to what degree these might interfere with normal endoglin function. In this paper, we have used fluorescence-based microscopic techniques, such as bimolecular fluorescence complementation (BiFC), immunofluorescence staining with the endoglin specific monoclonal antibody SN6, and protein interaction studies by Förster Resonance Energy Transfer (FRET) to investigate the formation and cellular localisation of possible homo- and heterodimers composed of endoglin wild-type and endoglin missense mutant proteins. The results show that all of the investigated missense mutants dimerise with themselves, as well as with wild-type endoglin, and localise, depending on the position of the affected amino acid, either in the rough endoplasmic reticulum (rER) or in the plasma membrane of the cells. We show that the rER retained mutants reduce the amount of endogenous wild-type endoglin on the plasma membrane through interception in the rER when transiently or stably expressed in HMEC-1 endothelial cells. As a result of this, endoglin modulated TGF-β1 signal transduction is also abrogated, which is not due to TGF-β receptor ER trafficking interference. Protein interaction analyses by FRET show that rER located endoglin missense mutants do not perturb protein processing of other membrane receptors, such as TβRII, ALK5 or ALK1.

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Analyses of in vivo interaction and mobility of two spliceosomal proteins using FRAP and BiFC.

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Analyses of in vivo interaction and mobility of two spliceosomal proteins using FRAP and BiFC.

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