Splicing factors are also reduced in NLA-injected cells (Spann et al., 2002); on the other hand, other treatments such as overexpression of Clk/STY protein kinase result in the redistribution of splicing factors from speckles to a diffuse pattern (Colwill et al., 1996; Sacco-Bubulya and Spector, 2002). Our results suggest a unique role for lamin speckles in the spatial organization of RNA splicing factors and pol II transcription in the nucleus. lamin B1 (Ellis et al., 1997) leads to defects in lamina assembly, disruption of the lamina, and inhibition of DNA replication. Mutations in human lamin A cause debilitating diseases such as Emery-Dreifuss muscular dystrophy, cardiomyopathy, partial lipodystrophy and axonal neuropathy (Bonne et al., 1999; Fatkin et al., 1999; Cao and Hegele, 2000; Shackleton et al., 2000; De Sandre-Giovannoli et al., 2002). A few of these lamin mutant proteins cause gross defects in the peripheral lamina and also assemble aberrantly, but other mutants do not show an obvious phenotype (?stlund et al., 2001; Raharjo et al., 2001; Vigouroux et al., 2001). The presence of morphologically distinct nuclear compartments that are enriched for specific proteins is now well established (for reviews see Spector, 1993; Lamond and Earnshaw, 1998). RNA splicing factors are present in high concentrations in compartments or speckles called splicing factor compartments (SFCs)* that correspond at the electron microscopic level to interchromatin granule clusters (IGCs) and are also dispersed in the nucleoplasm on perichromatin fibrils (PFs), which contain nascent transcripts (for reviews see Fakan and Puvion, 1980; Spector, 1993; Fakan, 1994). The splicing of pre-mRNAs occurs concomitantly with transcription Thiamet G on PFs (Beyer et al., 1988) and away from, or at the periphery of, SFCs for most transcripts (Jackson et al., 1993; Wansink et al., 1993; Cmarko et al., 1999). Transcription by RNA polymerase II (pol II) has been visualized on hundreds of small foci throughout the nucleoplasm (Jackson et al., 1993; Wansink et al., 1993; Bregman et al., 1995). The SFCs are dynamic compartments involved in the storage/recruitment of splicing factors (Misteli et al., 1997). Their size can change depending on RNA splicing or transcription levels in the cell; for example, they become considerably enlarged due to reduced dissociation of splicing factors in the presence of transcriptional inhibitors (Carmo-Fonseca et al., 1992; Spector, 1993), in pathological conditions (Fakan and Puvion, 1980), or upon inhibition of splicing (O’Keefe et al., 1994). The gene-specific positioning of transcription sites with respect to SFCs (Smith et al., 1999) and recruitment of splicing factors from SFCs upon gene activation CDKN2A (Misteli et al., 1997) point to significant spatial coordination of transcription and pre-mRNA splicing. A key issue that has not yet been resolved is the importance of nuclear architecture in the spatial organization of transcription and pre-mRNA splicing. It has been proposed that SFCs are generated by interactions with the nucleoskeletal framework (Kruhlak et al., 2000), or, alternatively, that self-organization of splicing factors leads to the assembly of SFCs (Misteli, 2001). The association of transcription sites or active pol II with an insoluble nuclear framework or matrix has been well documented (Jackson et al., 1993; Wansink et al., 1993; Kimura et al., 1999; Wei et al., 1999), several transcription factors have been localized to the nuclear matrix (for review see Stein et al., 2000), and SFCs have also been observed to be attached to a detergent-insoluble nuclear structure (Spector, 1993). Importantly, Hendzel et al. (1999) have demonstrated the presence of an underlying protein architecture in IGCs that physically connects the relatively dispersed granules within the cluster, by Thiamet G using energy transmission electron microscopy in intact cells and thus avoiding the problems associated with typical nuclear matrix isolation protocols (Pederson, 2000; Nickerson, 2001). The main candidate protein constituents of the nuclear framework are the lamins, previously identified at the nuclear periphery but now also observed in the nuclear interior, and actin, which can bind to snRNPs (Nakayasu and Ueda, 1984). A role for lamins in controlling gene expression has been proposed earlier (Wilson, 2000), and in vitro binding of lamin A to the retinoblastoma protein, an important transcriptional regulator, has been reported (Ozaki et al., 1994). More recently, an NH2-terminal deletion lamin A mutant, Thiamet G NLA, that disrupts the lamina has been observed to inhibit transcription (Spann et al., 2002). However, the involvement of internal lamins in the spatial organization of transcription or splicing has not been demonstrated. The aim of this study is to understand the functional role of internal lamin A/C speckles that have been observed to colocalize with SFCs in a variety of cell types using a monoclonal antibody to rat lamin A that has certain unique properties (Jagatheesan et al., 1999). This antibody, mAb LA-2H10, exclusively stains intranuclear speckles in interphase cells without labeling the peripheral.