Recent biochemical purification and characterisation of IGCs has determined that at least 75 proteins are enriched in speckles, with the majority being RNA processing factors (Mintz et al., 1999)
. Interestingly, this approach also identified several novel gene products that may help to elucidate connections between RNA processing and other regulatory or metabolic pathways. The most extensively characterised denizens of speckles are the serine-arginine-rich protein (SR protein) family of essential pre-mRNA splicing factors (for review see Fu, 1995)
. SR proteins are a highly related, evolutionarily conserved family of nuclear phosphoproteins involved in both constitutive and alternative splicing. The primary structure of SR proteins consists of one or two, amino-terminal RNA recognition motifs (RRMs) and a carboxyl terminal domain that is enriched in serine-arginine dipeptides (RS domain). Sequence specific RNA binding is conveyed by the RRMs while the RS domain facilitates protein-protein interactions and contributes to their proper subcellular localization (Cáceres et al., 1997)
. The modular domain structure of SR proteins reflects their roles as adapter molecules, mediating interactions between the pre-mRNA and the assembling spliceosome (for review see, Graveley, 2000)
.
Speckles are dynamic structures that respond to the levels of pol II transcription in the nucleus. Fusion proteins between the green fluorescent fusion protein (GFP) and the SR protein SF2/ASF has been a powerful tool in elucidating the functional relationship between speckles and pre-mRNA splicing. A variety of studies suggest that SR proteins are released from their storage sites in speckles and targeted to transcription sites in the nucleoplasm. Recruitment of SR proteins to transcription sites requires both the RS domain of SR proteins as well as the carboxyl terminal domain (CTD) of pol II (Misteli et al., 1997; Misteli and Spector, 1999)
. The extent of serine phosphorylation within the RS domain is critical for regulating SR protein activities in vitro and in vivo. Accordingly, several kinases have been described that can induce the dissociation of SR proteins from speckles, thereby increasing their nucleoplasmic concentration (Colwill et al., 1996; Gui et al., 1994)
. The emerging picture of pre-mRNA splicing in vivo involves release of SR proteins from speckles by one or more kinase activities (see figure 1 and Misteli et al., 1998; Misteli et al., 1997)
. Once free, SR proteins are targeted to nascent transcripts via interactions with the CTD of pol II, where spliceosome assembly begins.
Published Movies on Nuclear Speckle Dynamics
Spector
Lab Movies Movies from Misteli, T., J.F. Cáceres and D.L.
Spector. 1997. The dynamics of a pre-mRNA splicing factor in living
cells. Nature 387, 523-527.
MBC
11:413 Link to "Quantitative Imaging of Pre-mRNA Splicing Factors
in Living Cells". R. Eils et al. (2000). Contains several movies
quantitating speckle dynamics.
JCB
150:41 Link to "Reduced Mobility of the Alternate Splicing
Factor (ASF) through the Nucleoplasm and Steady State Speckle Compartments."
Contains a 4-D movie a 2-D timelapse movie of speckle dynamics.
REFERENCES
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