We have produced mAbs to Gemin3 and show by immunofluorescence microscopy that it colocalizes with SMN in gems. with them (Liu and Dreyfuss 1996; Liu et al. Pyronaridine Tetraphosphate 1997). In addition to SMN and SIP1, the large cytoplasmic complex of which they are part of also contains additional proteins, including some of the Sm proteins that are common components of spliceosomal small nuclear ribonucleoproteins (snRNPs; Liu et al. 1997). The presence of the Sm proteins in the SMNCSIP1 complex is a result of a direct interaction between SMN and several of the Sm proteins (Liu et al. 1997; Pellizzoni et al. 1999). The role of the cytoplasmic pool of Pyronaridine Tetraphosphate the SMNCSIP1 complex has been studied by antibody microinjection experiments in oocytes. These experiments revealed that SIP1 has a critical role in the assembly of snRNPs, a process which takes place in the cytoplasm where the Sm proteins combine with snRNAs that were exported from the nucleus (Mattaj and De Robertis 1985; Mattaj 1988; Luhrmann et al. 1990; Fischer et al. 1997). Once properly assembled and modified, the snRNPs recruit the necessary nuclear import receptors and translocate into the nucleus where they function in pre-mRNA splicing (Mattaj 1986, Mattaj 1988; Luhrmann et al. 1990; Neuman de Vegvar and Dahlberg 1990; Zieve and Sauterer 1990). In contrast to the inhibitory effects of anti-SIP1 antibodies, the anti-SMN antibodies show some stimulation of snRNP assembly (Liu et al. 1997). By transfection of a dominant negative form of SMN (SMNN27) in HeLa cells, we found that SMN also plays a critical role in the cytoplasmic assembly of snRNPs (Pellizzoni et al. 1998). In the nucleus, the SMNN27 protein causes a striking rearrangement of the snRNPs, colocalizing them with the mutant SMNN27 in enlarged gems (Pellizzoni et al. 1998). Using in vitro experiments, we have shown that SMN is required for pre-mRNA splicing, likely for the regeneration or recycling of snRNPs (Pellizzoni et al. 1998). SMN mutants found in SMA patients lack this activity because they are Rabbit polyclonal to C-EBP-beta.The protein encoded by this intronless gene is a bZIP transcription factor which can bind as a homodimer to certain DNA regulatory regions. defective in their interaction with the Sm proteins (Pellizzoni et al. 1999). Unlike the several recycling factors described so far that are essential for splicing, SMN and SIP1 do not contain DEAD/ DEAH motifs (reviewed in Staley and Guthrie 1998). Here, we report the molecular cloning and characterization of a protein designated Gemin3 (for protein component of gems number 3 3) that associates with SMN in vitro and in vivo. Gemin3 is a novel DEAD box protein and is, therefore, a putative RNA helicase. We have produced mAbs to Pyronaridine Tetraphosphate Gemin3 and show Pyronaridine Tetraphosphate by immunofluorescence microscopy that it colocalizes with Pyronaridine Tetraphosphate SMN in gems. Like SMN and SIP1, Gemin3 can be isolated in a complex with several spliceosomal snRNP proteins. We further found that Gemin3 interacts directly with SMN and with several of the spliceosomal snRNP core Sm proteins, including the B and D2-3 proteins. The unique COOH-terminal domain of Gemin3 mediates its interaction with SMN and its localization to gems. The discovery of a DEAD box protein, a likely RNA helicase, in the SMN complex is of particular interest as the functions revealed so far suggest that this complex has crucial activities in the biogenesis of RNPs. To perform such functions, including assembly of the snRNPs and the regeneration of active components of the spliceosome, it would be expected that the SMN complex can affect structural changes in its RNP targets. Of the known components of the SMN complex, the DEAD box protein Gemin3 is the most likely protein to have the capacity to perform such a function. Importantly, SMN proteins with mutations found in SMA individuals display a significantly reduced connection with Gemin3, suggesting the SMN complexes in these individuals will become deficient with this protein. Materials and Methods Recognition of p105 Protein by Mass Spectrometry The p105 protein was coimmunoprecipitated with anti-SMN mAb 2B1 and the band was excised from a single one-dimensional Coomassie stained polyacrylamide gel and in-gel digested with trypsin (unmodified, sequencing grade; Boehringer Mannheim Corp.) mainly because explained in Shevchenko et al. 1996. Tryptic peptides were recovered from gel items by extraction with 5% formic acid and acetonitrile. The combined components were pooled collectively, dried inside a rate vac, redissolved in 5% formic acid, and analyzed by nanoelectrospray tandem mass spectrometry (nano-ES MS/MS) as explained in Wilm et al. 1996. Nano-ES MS/MS was performed on a API III triple quadrupole instrument (PE Sciex) equipped with a nano-ES ion resource developed in EMBL.