Spinal muscular atrophy (SMA) is a genetic disorder caused by mutations in the human survival of motor neuron 1 gene, SMN1. SMN protein is part of a large complex that is required for biogenesis of various small nuclear ribonucleoproteins (snRNPs). Here, we report that SMN interacts directly with the Cajal body signature protein, coilin, and that this interaction mediates recruitment of the SMN complex to Cajal bodies. Mutation or deletion of specific RG dipeptide residues within coilin inhibits the interaction both in vivo and in vitro. Interestingly, GST-pulldown experiments show that coilin also binds directly to SmB. Competition studies show that coilin competes with SmB for binding sites on SMN. Ectopic expression of SMN and coilin constructs in mouse embryonic fibroblasts lacking endogenous coilin confirms that recruitment of SMN and splicing snRNPs to Cajal bodies depends on the coilin C-terminal RG motif. A cardinal feature of SMA patient cells is a defect in the targeting of SMN to nuclear foci; our results uncover a role for coilin in this process.

We have found that coilin, the marker protein for Cajal bodies (coiled bodies, CBs), is a self-interacting protein, and we have mapped the domain responsible for this activity to the amino-terminus. Together with a nuclear localization signal, the self-interaction domain is necessary and sufficient for localization to CBs. Overexpression of various wild-type and mutant coilin constructs in HeLa cells results in disruption of both CBs and survival motor neurons (SMN) gems. Additionally, we have identified a cryptic nucleolar localization signal (NoLS), within the coilin protein, which may be exposed in specific coilin phospho-isoforms. The implications of these findings are discussed in light of the fact that other proteins known to localize within nuclear bodies (e. g., PML, SMN and Sam68) can also self-associate. Thus protein self-interaction appears to be a general feature of nuclear body marker proteins.

Cajal bodies (CBs) are subnuclear domains implicated in small nuclear ribonucleoprotein (snRNP) biogenesis. In most cell types, CBs coincide with nuclear gems, which contain the survival of motor neurons (SMN) complex, an essential snRNP assembly factor. Here, we analyze the exchange kinetics of multiple components of CBs and gems in living cells using photobleaching microscopy. We demonstrate differences in dissociation kinetics of CB constituents and relate them to their functions. Coilin and SMN complex members exhibit relatively long CB residence times, whereas components of snRNPs, small nucleolar RNPs, and factors shared with the nucleolus have significantly shorter residence times. Comparison of the dissociation kinetics of these shared proteins from either the nucleolus or the CB suggests the existence of compartment-specific retention mechanisms. The dynamic properties of several CB components do not depend on their interaction with coilin because their dissociation kinetics are unaltered in residual nuclear bodies of coilin knockout cells. Photobleaching and fluorescence resonance energy transfer experiments demonstrate that coilin and SMN can interact within CBs, but their interaction is not the major determinant of their residence times. These results suggest that CBs and gems are kinetically independent structures.

functional homolog, yeast Rmt1/Hmt1, encoding the bulk of the activity (Tang et al., 2000; McBride et al., Center for Human Genetics and Program in Cell Biology 2000). The only type II PRMT defined to date is the Janus kinase binding protein, JBP1/PRMT5 (Branscombe et Case Western Reserve University and University Hospitals of Cleveland al., 2001). There are only five known targets of PRMT5: myelin Cleveland, Ohio 44106 basic protein (Baldwin and Carnegie, 1971) and the spliceosomal proteins SmB/BЈ, SmD1, SmD3, and Lsm4 (Brahms et al., 2000, 2001). Importantly, symmetrical Summary dimethylation of the Sm and Sm-like proteins is important for binding to the survival of motor neurons (SMN) Cajal bodies (CBs) are nuclear suborganelles involved in biogenesis of small RNAs. Twin structures, called protein in vivo (Friesen et al., 2001a; Brahms et al., 2001). The SMN1 gene is mutated in Ͼ95% of patients with gems, contain high concentrations of the survival motor neurons (SMN) protein complex. CBs and gems spinal muscular atrophy (SMA), a recessive neurodegenerative disease, which is the leading genetic cause of often colocalize, and communication between these subdomains is mediated by coilin, the CB marker. Coi-childhood mortality (Pearn, 1980). SMN is part of a large protein complex that is required for assembly of Sm lin contains symmetrical dimethylarginines that modulate its affinity for SMN, and, thus, localization of SMN proteins onto spliceosomal U snRNAs (Liu et al., 1997; Fischer et al., 1997; Meister et al., 2000, 2001a). The complexes to CBs. Inhibition of methylation or mutation of the coilin RG box dramatically decreases bind-protein is localized diffusely throughout the cytoplasm as well as to nuclear Cajal bodies (Liu and Dreyfuss, ing of coilin to SMN, resulting in gem formation. Coilin is hypomethylated in cells that display gems, but not 1996; Matera and Frey, 1998; Carvalho et al., 1999). We have recently shown that coilin, the Cajal body (formerly in those that primarily contain CBs. Likewise, extracts prepared from cells that display gems are less efficient coiled body) marker protein, interacts with SMN both genetically (Tucker et al., 2001) and physically (Hebert in methylating coilin and Sm constructs in vitro. These results demonstrate that alterations in protein methyl-et al., 2001). The interaction between coilin and SMN is direct, and recruitment of the SMN complex to Cajal ation status can affect nuclear organization. bodies (CBs) depends upon the presence of an arginineand glycine-rich domain (the RG box motif) within coilin

Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a Cterminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB′ binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB′ than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB.

The survival of motor neuron (SMN) protein is mutated in patients with spinal muscular atrophy (SMA). SMN is part of a multiprotein complex required for biogenesis of the Sm class of small nuclear ribonucleoproteins (snRNPs). Following assembly of the Sm core domain, snRNPs are transported to the nucleus via importin beta. Sm snRNPs contain a nuclear localization signal (NLS) consisting of a 2,2,7-trimethylguanosine (TMG) cap and the Sm core. Snurportin1 (SPN) is the adaptor protein that recognizes both the TMG cap and importin beta. Here, we report that a mutant SPN construct lacking the importin beta binding domain (IBB), but containing an intact TMG cap-binding domain, localizes primarily to the nucleus, whereas full-length SPN localizes to the cytoplasm. The nuclear localization of the mutant SPN was not a result of passive diffusion through the nuclear pores. Importantly, we found that SPN interacts with SMN, Gemin3, Sm snRNPs and importin beta. In the presence of ribonucleases, the interactions with SMN and Sm proteins were abolished, indicating that snRNAs mediate this interplay. Cell fractionation studies showed that SPN binds preferentially to cytoplasmic SMN complexes. Notably, we found that SMN directly interacts with importin beta in a GST-pulldown assay, suggesting that the SMN complex might represent the Sm core NLS receptor predicted by previous studies. Therefore, we conclude that, following Sm protein assembly, the SMN complex persists until the final stages of cytoplasmic snRNP maturation and may provide somatic cell RNPs with an alternative NLS.

Spliceosomal small nuclear ribonucleoproteins (snRNPs) are enriched in the Cajal body (CB). Guide RNAs, known as small Cajal body-specific RNAs (scaRNAs), direct modification of the small nuclear RNA (snRNA) component of the snRNP. The protein WRAP53 binds a sequence motif (the CAB box) found in many scaRNAs and the RNA component of telomerase (hTR) and targets these RNAs to the CB. We have previously reported that coilin, the CB marker protein, associates with certain non-coding RNAs. For a more comprehensive examination of the RNAs associated with coilin, we have sequenced the RNA isolated from coilin immunocomplexes. A striking preferential association of coilin with the box C/D scaRNAs 2 and 9, which lack a CAB box, was observed. This association varied by treatment condition and WRAP53 knockdown. In contrast, reduction of WRAP53 did not alter the level of coilin association with hTR. Additional studies showed that coilin degrades/processes scaRNA 2 and 9, associates with active telomerase and can influence telomerase activity. These findings suggest that coilin plays a novel role in the biogenesis of box C/D scaRNPs and telomerase.

Transcription-dependent colocalization of the U1, U2, U4/U6 and U5 snRNPs in coiled bodies.