Nuclear splicing speckles are thought to be storage sites for pre-mRNA splicing factors. From here, splicing factors can be recruited to RNA polymerase II (pol II) transcription sites and nascent transcripts in the nucleoplasm. It is currently thought that varying the concentration of antagonistic splicing factors in the nucleoplasm may control alternative pre-mRNA splicing. Thus, speckles may function as reservoirs of splicing factors. In addition, speckles have been identified in a wide variety of metazoan species, underscoring the functional significance of this compartment.

Nuclear Speckle Structure

Electron microscopy analysis suggests that speckles may correspond to two distinct structural elements, interchromatin granule clusters (IGCs) and perichromatin fibrils (PFs). The IGCs have an apparent diameter of 0.8-1.8 um and are composed of smaller, 20-25 nm particles (Monneron and Bernhard, 1969). IGCs, unlike

PFs do not appear to incorporate [3H]-UTP or Br-UTP and do not colocalize with DNA, suggesting that IGCs cannot be correlated with transcription sites or nascent transcripts (Fakan and Bernhard, 1971; Wansink et al., 1993). PFs are structures found at the periphery of IGCs and have a diameter of 5-8 nm. PFs are thought to contain nascent transcripts, as they are sensitive to RNase treatment and are labeled rapidly with Br-UTP and [3H]-UTP (Cmarko et al., 1999). One possible relationship between IGCs and PFs is that splicing factors may be recruited from their depots in IGCs to nascent transcripts present in PFs. IGCs contain poly adenyated (poly A) RNA, however it is unlikely that IGCs are sites of pre-mRNA splicing. Recent experiments employing laser scanning confocal microscopy and very high dilutions of primary anti-bodies against known speckle components has revealed that speckles may be much more heterogeneous and perhaps dynamic then previously thought. Mintz and Spector (2001) demonstrated
Figure 1. Recruitment of SR proteins to transcription sites

Click figures to enlarge
that speckles are composed of numerous, smaller subdomains (subspeckles) ranging in size from 0.2-0.5 um in diameter and are often arranged in loops. The loops of subspeckles are dependent on pol II transcription and thus may be involved in targeting splicing factors to sites of transcription or RNA processing.

Protein components of Nuclear Speckles

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).

Nuclear Speckles are dynamic structures

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.


Click figures to enlarge
<-- Figure 3. Phosphoproteins involved in pre-mRNA splicing

Figure 3. Nuclear ratios of antagonistic splicing factors may control splicing patterns-->


Click figures to enlarge
At the biochemical level, the higher phosphorylation state of liberated SR protein favours specific interactions with consensus sequences within the pre-mRNA as well as protein-protein interactions with the U1 snRNP (i.e. U1 70K ) and perhaps U2AF35 (see figure 2 and Xiao and Manley, 1997; Xiao and Manley, 1998). Once the spliceosome has assembled, dephosphorylation of SR proteins is necessary for catalysis and re-targeting of SR proteins to speckles (for review see Misteli, 1999). Thus, speckles can be thought of as storage sites for pre-mRNA splicing factors. Although SR proteins accumulate in the nucleus, a subset appear to shuttle continuously and rapidly between the nucleus and the cytoplasm, possibly escorting the spliced mRNA to its' final destination in the cytoplasm (Cáceres et al., 1998; Huang and Steitz, 2001). The nucleocytoplasmic shuttling activity of SR proteins may be regulated by their state of phosphorylation and could prove to be a key regulatory target for the cell. Splice site selection in vivo is sensitive to the nuclear concentration of SR proteins and their antagonists, therefore modulation of SR protein localization may be an effective way for the cell to control patterns of pre-mRNA splicing (figure 3, Cáceres et al., 1994; van der Houven van Oordt et al., 2000).

  • Lamond Lab Image Gallery --Contains images and movies of splicing speckles

  • Splicing Proteins in Drosophila --from the Interactive Fly

  • Spector Lab Home Page -- contains images and movies of splicing speckles

  • Caceres Lab Pages
    1- Nuclear functions of SR and hnRNP proteins
    2- Nucleo-cytoplasmic shuttling of SR and hnRNP proteins
    3- Identification of novel splicing regulators based on subcellular localization

  • Ben Blencowe Lab-- mechanisms underlying the regulation of pre-mRNA processing (srm160/300)

  • A Red-Green Stereo 3-D timelapse series on a human cell expressing the alternate splicing factor (ASF)-GFP chimeric protein--from www.cellnucleus.com

  • 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

    Cáceres, J. F., Misteli, T., Screaton, G. R., Spector, D. L., and Krainer, A. R. (1997). Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity, JCB 138, 225-38.

    Cáceres, J. F., Screaton, G. R., and Krainer, A. R. (1998). A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm, Genes Dev 12, 55-66.

    Cáceres, J. F., Stamm, S., Helfman, D. M., and Krainer, A. R. (1994). Regulation of alternative splicing in vivo by overexpression of antagonistic pre-mRNA splicing factors, Science 265, 1706-1709.

    Cmarko, D., Verschure, P. J., Martin, T. E., Dahmus, M. E., Krause, S., Fu, X. D., van Driel, R., and Fakan, S. (1999). Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection, Mol Biol Cell 10, 211-23.

    Colwill, K., Pawson, T., Andrews, B., Prasad, J., Manley, J. L., Bell, J. C., and Duncan, P. I. (1996). The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution, EMBO J 15, 265-275.

    Fakan, S., and Bernhard, W. (1971). Localization of rapidly and slowly labelled nuclear RNA as visualized by high resolution autoradiography, Exp Cell Res 67, 129-41.

    Fu, X.-D. (1995). The superfamily of arginine/serine-rich splicing factors, RNA 1, 663-680.

    Graveley, B. R. (2000). Sorting out the complexity of SR protein functions, RNA 6, 1197-1211.

    Gui, J.-F., Lane, W. S., and Fu, X.-D. (1994). A serine kinase regulates intracellular localization of splicing factors in the cell cycle, Nature 369, 678-682.

    Huang, Y., and Steitz, J. A. (2001). Splicing factors SRp20 and 9G8 promote the nucleocytoplasmic export of mRNA, Mol Cell 7, 899-905.

    Mintz, P. J., Patterson, S. D., Neuwald, A. F., Spahr, C. S., and Spector, D. L. (1999). Purification and biochemical characterization of interchromatin granule clusters, Embo J 18, 4308-20.

    Misteli, T. (1999). RNA splicing: What has phosphorylation got to do with it?, Curr Biol 9, R198-200.

    Misteli, T., Cáceres, J. F., Clement, J. Q., Krainer, A. R., Wilkinson, M. F., and Spector, D. L. (1998). Serine phosphorylation of SR proteins is required for their recruitment to sites of transcription in vivo, JCB 143, 297-307.

    Misteli, T., Caceres, J. F., and Spector, D. L. (1997). The dynamics of a pre-mRNA splicing factor in living cells, Nature 387, 523-527.

    Misteli, T., and Spector, D. L. (1999). RNA polymerase II targets pre-mRNA splicing factors to transcription sites in vivo, Mol Cell 3, 697-705.

    Monneron, A., and Bernhard, W. (1969). Fine structural organization of the interphase nucleus in some mammalian cells, J Ultrastruct Res 27, 266-88.

    van der Houven van Oordt, W., Diaz-Meco, M. T., Lozano, J., Krainer, A. R., Moscat, J., and Caceres, J. F. (2000). The MKK(3/6)-p38-signaling cascade alters the subcellular distribution of hnRNP A1 and modulates alternative splicing regulation, JCB 149, 307-16.

    Wansink, D. G., Schul, W., van der Kraan, I., van Steensel, B., van Driel, R., and de Jong, L. (1993). Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus, J Cell Biol 122, 283-93.

    Xiao, S.-H., and Manley, J. L. (1997). Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing, Genes Dev 11, 334-344.

    Xiao, S. H., and Manley, J. L. (1998). Phosphorylation-dephosphorylation differentially affects activities of splicing factor ASF/SF2, EMBO J 17, 6359-6367.

    Zahler, A. M., Neugebauer, K. M., Lane, W. S., and Roth, M. B. (1993). Distinct functions of SR proteins in alternative pre-mRNA splicing, Science 260, 219-222.