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In both skin fibroblasts and Ntera-2 cells, gems/CBs were rarely, if ever, stained by anti-gemin5 mAbs (data not shown)

In both skin fibroblasts and Ntera-2 cells, gems/CBs were rarely, if ever, stained by anti-gemin5 mAbs (data not shown). Open in a separate window Figure 7 Distribution of gemins and other proteins between cytoplasmic and nuclear fractions of HeLa and Ntera2 cells. abundant in the cytoplasm. In a cytoplasmic extract obtained by moderate disruption Dimebon 2HCl of HeLa cells, nearly all the SMN and gemins 2C4 were in large complexes, but most of the gemin5 sedimented separately with a lower S value. Most of the unrip sedimented with gemins 6 and 7 near the top of the sucrose density gradients, individual from both SMN and gemin5. Anti-SMN mAbs pulled down gemin5 from cytoplasmic extracts, but not from nuclear extracts, and gemin5 did not co-sediment with large SMN complexes in nuclear extracts. These data suggest that gemin5 is usually very easily detached from SMN-gemin complexes in the nucleus. By immuno-histochemistry, gemin5 was rarely detectable in nuclear gems/Cajal body, although it was accessible to antibody and very easily detectable when present. This suggests that gemin5 is normally absent from SMN complexes in these nuclear storage sites. Conclusion We conclude that SMN complexes usually exist without gemin5 in nuclear gems/Cajal body. Gemin5 is usually believed to be involved in capturing snRNA into SMN complexes in the cytoplasm for transport into the nucleus. We hypothesize that gemin5, though present in the nucleus, is usually no longer needed for SMN complex function during the time these complexes are stored in gems/Cajal body. Background The SMN protein forms a stable complex with a group of proteins named gemins [examined in [1,2]]. The gemins colocalize with SMN in gems/Cajal body (CBs) and are also present throughout the cytoplasm and in the Dimebon 2HCl nucleoplasm [1], although gemin4 has also been reported in the nucleolus [3]. An early view of the structure of the SMN complex was that gemins 2, 3, 5 and 7 bind directly to SMN, while gemins 4 and 6 associated by conversation with gemins 3 and 7, respectively [4]. It was later shown that a novel protein, gemin8, Dimebon 2HCl mediates the binding to SMN of the subcomplex of gemins 6 and 7 and a protein called unrip [5]. A recent study suggests that SMN interacts directly only with gemins 2, 3 and 8, while the subcomplex of gemin7 with gemin6 binds through gemin8, unrip binds through gemin7, gemin5 binds through gemin2, and gemin4 binds to both gemins 3 and 8 [2]. SMN complexes clearly have an important and well-documented role in both assembly of cytoplasmic snRNPs and their transport to the nucleus [5-8]. However, a significant amount of SMN is also found in the cytoplasm of motor neuron axons, suggesting that SMN may have motor neuron-specific functions impartial of snRNP assembly [9-17]. Immunopurification of a 300-kDa SMN-gemin2 complex showed that it also contained spliceosomal snRNP core proteins B/B’, D1, D2, D3, E, F and G [6]. There is some controversy in the literature on whether there is an SMN conversation site for Sm core proteins near the C-terminus (residues 240C267; [6]) or at residues 120C160 in the exon3-encoded tudor domain [18]. Charroux et al [3] explained an 800 kDa complex that included SMN, gemin2 and gemins 3 and 4. Gemins 3 and 4 were also found without SMN in a separate 15S complex that contains eukaryotic initiation factor 2C and numerous microRNAs [19]. Meister et al [20] isolated two unique SMN complexes from HeLa nuclei, NSC1 and NSC2, that migrated in sucrose gradients at 20S and 18S respectively. NSC1 was U snRNA-free, but contained at least 10 proteins, including SMN, gemin2, gemins 3 and 4 and Sm proteins D1, D2 and F. They later explained a complex in both nucleus and Dimebon 2HCl cytoplasm that contains all gemins and Sm core proteins, plus unrip and hsc70 [21]. Unrip is an interacting partner of unr, a cytoplasmic RNA binding protein involved in the translation of viral RNAs [22]. The functions of individual proteins in SMN complexes and how they ALCAM contribute to the overall function of the complex remains unclear. A role for gemin2 in the oligomerization of the SMN complex was recently.