Sequence-specific interaction of U1 snRNA with the SMN complex
2002; Springer Nature; Volume: 21; Issue: 5 Linguagem: Inglês
10.1093/emboj/21.5.1188
ISSN1460-2075
Autores Tópico(s)Cancer-related gene regulation
ResumoArticle1 March 2002free access Sequence-specific interaction of U1 snRNA with the SMN complex Jeongsik Yong Jeongsik Yong Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104-6148 USA Search for more papers by this author Livio Pellizzoni Livio Pellizzoni Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104-6148 USA Search for more papers by this author Gideon Dreyfuss Corresponding Author Gideon Dreyfuss Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104-6148 USA Search for more papers by this author Jeongsik Yong Jeongsik Yong Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104-6148 USA Search for more papers by this author Livio Pellizzoni Livio Pellizzoni Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104-6148 USA Search for more papers by this author Gideon Dreyfuss Corresponding Author Gideon Dreyfuss Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104-6148 USA Search for more papers by this author Author Information Jeongsik Yong1, Livio Pellizzoni1 and Gideon Dreyfuss 1 1Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104-6148 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1188-1196https://doi.org/10.1093/emboj/21.5.1188 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The survival of motor neurons (SMN) protein complex functions in the biogenesis of spliceosomal small nuclear ribonucleoprotein particles (snRNPs) and prob ably other RNPs. All spliceosomal snRNPs have a common core of seven Sm proteins. To mediate the assembly of snRNPs, the SMN complex must be able to bring together Sm proteins with U snRNAs. We showed previously that SMN and other components of the SMN complex interact directly with several Sm proteins. Here, we show that the SMN complex also interacts specifically with U1 snRNA. The stem–loop 1 domain of U1 (SL1) is necessary and sufficient for SMN complex binding in vivo and in vitro. Substitution of three nucleotides in the SL1 loop (SL1A3) abolishes SMN interaction, and the corresponding U1 snRNA (U1A3) is impaired in U1 snRNP biogenesis. Microinjection of excess SL1 but not SL1A3 into Xenopus oocytes inhibits SMN complex binding to U1 snRNA and U1 snRNP assembly. These findings indicate that SMN complex interaction with SL1 is sequence-specific and critical for U1 snRNP biogenesis, further supporting the direct role of the SMN complex in RNP biogenesis. Introduction Pre-mRNA splicing in the nucleus of eukaryotic cells is mediated by the spliceosome. The small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U5 and U4/U6 are major components of the spliceosome. Each snRNP consists of one snRNA (U1, U2, U5 or U4/U6), an Sm protein core and a set of proteins that are specific to individual snRNAs (Will and Lührmann, 2001). The Sm proteins B/B′, D1, D2, D3, E, F and G are common to all spliceosomal snRNPs and are arranged into a seven-membered ring (Kambach et al., 1999; Stark et al., 2001) that assembles for each snRNA on a consensus sequence motif (PuAU4–6GPu) called the Sm site (Branlant et al., 1982; Nagai et al., 2001). This assembly process takes place in the cytoplasm shortly after the nuclear export of nascent snRNAs. After the formation of the Sm core, the 7-methyl guanosine (m7G) cap of these snRNAs is hypermethylated to become a 2,2,7-trimethyl guanosine (m3G or TMG) (Mattaj, 1986; Plessel et al., 1994). Properly assembled Sm core, cap hypermethylation and 3′ end processing of the snRNAs are required for the subsequent nuclear import of the snRNPs, where they function in splicing (Mattaj and De Robertis, 1985; Fischer and Lührmann, 1990; Hamm et al., 1990b; Fischer et al., 1993; Mattaj et al., 1993; Jarmolowski et al., 1994; Will and Lührmann, 2001). Important and unexpected insights into the process of snRNP assembly came from studies on the function of the survival of motor neurons (SMN) protein (Liu and Dreyfuss, 1996; Fischer et al., 1997; Liu et al., 1997; Mattaj, 1998). SMN is the protein product of the spinal muscular atrophy (SMA) disease gene (Lefebvre et al., 1995). SMA is a severe neuromuscular disease characterized by degeneration of motor neurons in the spinal cord (Melki, 1997). Over 98% of SMA patients have deletions or mutations of the telomeric copy of the gene (SMN1) and produce markedly reduced levels of the SMN protein (Lefebvre et al., 1995; Burghes, 1997). SMN is part of a large multiprotein complex that also contains Gemin2 (Liu et al., 1997), the DEAD box RNA helicase Gemin3 (Charroux et al., 1999), Gemin4 (Charroux et al., 2000) and several additional as yet uncharacterized proteins. The SMN complex is present in both the nucleus and the cytoplasm of all metazoan cells, suggesting that it may have multiple functions in cells (Liu and Dreyfuss, 1996). Most lines of evidence indicate that the SMN complex functions in the assembly and metabolism of various RNPs, including snRNPs, snoRNPs, and the machineries that carry out transcription and pre-mRNA splicing (Fischer et al., 1997; Liu et al., 1997; Pellizzoni et al., 1998, 1999, 2001a, b; Buhler et al., 1999; Friesen and Dreyfuss, 2000; Meister et al., 2000, 2001; Jones et al., 2001; Mourelatos et al., 2001). The process of snRNP assembly can be most readily studied in Xenopus oocytes, where specific reagents and intermediates can be microinjected into either the nucleus or the cytoplasm and where dissection of nuclear and cytoplasmic fractions can be readily performed. Such experiments have revealed that the SMN complex associates with spliceosomal U1, U4 and U5 snRNAs in the cytoplasm (Fischer et al., 1997; Buhler et al., 1999; Charroux et al., 2000). Antibodies against components of the SMN complex microinjected into Xenopus oocytes also inhibit the assembly of snRNPs, indicating that the SMN complex plays a crucial role in the biogenesis of snRNPs (Fischer et al., 1997; Buhler et al., 1999). In addition, overexpression of a dominant-negative SMN mutant blocks snRNP assembly in the cytoplasm of somatic cells, suggesting a general function for the SMN complex in the cytoplasmic phase of U snRNAs biogenesis (Pellizzoni et al., 1998). Recent studies have further demonstrated that the SMN complex is necessary for assembly of U1 snRNP in Xenopus egg extracts (Meister et al., 2001). The capacity of the SMN complex to associate with and mediate the assembly of snRNPs is probably due, at least in part, to interactions between the SMN complex and snRNP proteins. Several of the components of the SMN complex interact directly with Sm proteins (Liu et al., 1997; Buhler et al., 1999; Charroux et al., 1999, 2000; Pellizzoni et al., 1999; Friesen and Dreyfuss, 2000). In particular, SMN binds avidly to RG-rich C-terminal domains that are found in the Sm proteins B, D1 and D3, whereas several SMN mutants found in SMA patients are defective in Sm protein binding (Buhler et al., 1999; Pellizzoni et al., 1999; Friesen and Dreyfuss, 2000). Importantly, SMN binds preferentially to the RG domains of D1 and D3 after arginines in specific positions are converted to symmetric dimethylarginines (sDMAs) (Friesen et al., 2001a). Thus, arginine dimethylation has a key role in the protein substrate recognition by the SMN complex, and RNP assembly is likely to be regulated by arginine methylation (Friesen et al., 2001a). To serve in snRNP assembly, the SMN–Sm protein complex must also recruit the snRNAs. Here, we investigated the binding of U1 snRNA, an abundant and high-avidity substrate, to the SMN complex. We show that the binding is sequence-specific and is mediated by the loop of stem–loop 1 domain (SL1) of U1 snRNA. SL1 is both necessary and sufficient for the interaction with the SMN complex in vivo and in vitro. We further demonstrate that this interaction is required for U1 snRNP assembly and thus mediates the function of the SMN complex in U1 snRNP biogenesis. Results The association of the SMN complex with U1 snRNA is independent of Sm core assembly Immunoprecipitation experiments of microinjected RNAs demonstrated an association of the SMN complex with U1 and U5, and to a lesser extent U4, snRNAs (Fischer et al., 1997; Buhler et al., 1999; Charroux et al., 2000). In light of earlier studies demonstrating protein–protein interactions between components of the SMN complex and Sm proteins (Liu et al., 1997; Buhler et al., 1999; Pellizzoni et al., 1999; Charroux et al., 1999, 2000; Friesen and Dreyfuss, 2000) and the suggestion that SMN can bind RNA (Liu et al., 1996; Lorson and Androphy, 1998), we asked whether the interaction of the SMN complex with the U snRNAs is direct or occurs through the Sm proteins, which bind to the Sm site. To address this, we studied U1 snRNA interaction with the SMN complex. U1 snRNA was chosen for these experiments because it has high affinity for the SMN complex and is the most abundant and extensively characterized snRNA (Fischer et al., 1997; Charroux et al., 2000; Will and Lührmann, 2001). Vertebrate U1 snRNA contains four distinct stem–loop structures and the Sm site (Figure 1) (Branlant et al., 1981; Mount and Steitz, 1981). The U1ΔSm RNA, the Sm site sequence UAAUUU of which is substituted by the hexanucleotide sequence CUCGAG, does not bind Sm proteins (Hamm et al., 1987). Several RNAs including U1ΔSm and the spliceosomal U1 and U6 snRNAs were transcribed in vitro in the presence of [32P]UTP. Various mixtures of these RNAs were injected into the cytoplasm of oocytes and, after 15 h of incubation, oocytes were dissected manually into nuclear and cytoplasmic fractions, and immunoprecipitation was carried out with anti-SMN monoclonal antibody (2B1) and anti-Sm monoclonal antibody (Y12) (Figure 2A). Figure 2B and C shows that both the wild-type (wt) U1 snRNA and U1ΔSm, but not U6 snRNA, associate with the SMN complex with similar affinity. As expected, U1ΔSm was not immunoprecipitated using anti-Sm antibodies, indicating that this mutant is not assembled with the Sm proteins, and was not imported into the nucleus (Figure 2C). These data demonstrate that the interaction between the SMN complex and U1 snRNA is independent of the Sm site and therefore does not require Sm core assembly. Figure 1.The secondary structure of Xenopus laevis U1 snRNA and the deletion or substitution mutant U1 snRNAs used in this study. The positions of the structural elements deleted or altered in U1 snRNA are listed in the table. ΔSL1 has the entire SL1 structure deleted. ΔSL2/3 has the entire SL2 and SL3 structures deleted. The nucleotides mutated in the loop domain of SL1 of U1A3 RNA are boxed. The secondary structure is according to Branlant et al. (1981) and Mount and Steitz (1981). Download figure Download PowerPoint Figure 2.Stem–loop 1 of U1 snRNA is necessary for efficient interaction of the SMN complex with U1 snRNA. (A) Experimental strategy used in this experiment. A mixture of 32P-labeled U1 and U6 snRNAs was injected into the cytoplasm of oocytes. After incubation for 15 h, the oocytes were dissected manually into nuclear (N) and cytoplasmic (C) fractions. Immunoprecipitation was then carried out from both fractions with either the anti-Sm (Y12), anti-SMN (2B1) or control non-immune (SP2/0) antibodies. (B) Microinjection and immunoprecipitation experiments of wt U1 and U6 32P-labeled snRNAs. 'Total' lanes correspond to 10% of the fractions used for each immunoprecipitation. Immunoprecipitation with Y12 was carried out in RSB-500 buffer (500 mM NaCl). 2B1 immunoprecipitation was performed in RSB-150 buffer. After immunoprecipitation, RNAs were purified and analyzed by electrophoresis on a 7 M urea/8% acrylamide gel and autoradiography. (C) The SMN complex binding to U1 snRNA is independent of Sm core assembly. A mixture of 32P-labeled U1ΔSm and U6 snRNAs was microinjected and analyzed for the SMN complex interaction by immunoprecipitation as described in (B). (D) SL1 domain of U1 snRNA is necessary for U1 snRNA binding of the SMN complex. A mixture of 32P-labeled U1, U6 and U1ΔSL1 snRNAs was microinjected and analyzed for the SMN complex interaction by immunoprecipitation as described in (B). (E) SL2 and SL3 domains of U1 snRNA are not necessary for the SMN complex–U1 snRNA interaction. A mixture of 32P-labeled U1, U6 and U1ΔSL2/3 snRNAs was microinjected and analyzed for the SMN complex interaction by immunoprecipitation as described in (B). Download figure Download PowerPoint SL1 of U1 snRNA is necessary for SMN complex–U1 snRNA interaction The efficient interaction of U1ΔSm with the SMN complex suggested that other sequences of U1 snRNA mediate this interaction. To identify this sequence(s), we analyzed the binding of several deletion mutants of U1 snRNA shown in Figure 1 using the experimental procedures described above. Figure 2D shows that deletion of SL1 (U1ΔSL1) strongly impaired the association of U1 snRNA with the SMN complex. Immunoprecipitation with the Y12 antibody shows that both U1 snRNA and U1ΔSL1 were associated with the Sm proteins in the cytoplasm. However, Y12 immunoprecipitation of U1ΔSL1 from the nucleus was greatly diminished (∼4-fold compared with that of wt U1 snRNA), suggesting that deletion of SL1 strongly impairs the biogenesis of U1 snRNP. Deletion of SL2 and SL3 (U1ΔSL2/3) did not reduce the association of U1 snRNA with the SMN complex (Figure 2E). In addition, immunoprecipitation with Y12 from the nuclear and cytoplasmic fractions showed that U1ΔSL2/3 is assembled with the Sm proteins in the cytoplasm and is imported efficiently into the nucleus. Thus, unlike deletion of SL1, deletion of SL2 and SL3 does not impair the biogenesis of U1 snRNP. Similar results were obtained with individual deletions of either SL2 or SL3 (data not shown). We conclude that SL1 is necessary for efficient interaction of U1 snRNA with the SMN complex and is important for U1 snRNP biogenesis, as reflected by U1ΔSL1 accumulation in the cytoplasm. SL1 of U1 snRNA is sufficient for SMN complex–U1 snRNA interaction Because the experiments described above indicated that SL1 of U1 snRNA is necessary for the efficient interaction with the SMN complex, we wished to determine next whether it is also sufficient for this interaction. To do so, radiolabeled SL1 was injected into the cytoplasm of oocytes. Immunoprecipitation with 2B1 from oocyte extracts carried out after incubation for 3 h (SL1 is less stable in the oocyte than constructs containing 3′ stem loops) showed that SL1 alone interacts with the SMN complex as efficiently as wt U1 snRNA (Figure 3A and B). This indicates that SL1 is sufficient for interaction with the SMN complex. Figure 3.Sequence-specific interaction of the SMN complex with SL1 of U1 snRNA. (A) Experimental strategy used in this experiment. A mixture of 32P-labeled RNAs was injected into the cytoplasm of oocytes. Following a 3 h incubation, the oocytes were homogenized, and immunoprecipitated with 2B1 or SP2/0 antibodies. Immunoprecipitated RNAs were analyzed as described in Figure 2B. (B) SL1 is sufficient for U1 snRNA interaction with the SMN complex. A mixture of 32P-labeled U1, U6 snRNAs and SL1 was microinjected and analyzed as described in (A). (C) The SMN complex binding to the SL1 of U1 snRNA is sequence specific. A mixture of 32P-labeled U1, U6 snRNAs and SL1A3 was microinjected and analyzed as described in (A). Download figure Download PowerPoint To further address the specificity of the interaction of SL1 with the SMN complex we examined the effect of mutations in SL1 on this interaction. It has been reported that mutations in SL1 can affect the binding to the U1-specific proteins U170K, U1A or U1C (Hamm et al., 1988, 1990b; Surowy et al., 1989). Several point mutants of SL1 were tested by microinjection and immunoprecipitation experiments. One previously described mutant of particular relevance is SL1A3, which contains three nucleotide changes in the loop sequence of SL1 (U27G, A29C and U30C; boxed in Figure 1) and impairs U170K binding to U1 snRNA (Hamm et al., 1990b). wt U1 and wt U6 snRNAs were injected along with SL1A3 and analyzed by immunoprecipitation with 2B1. As shown in Figure 3C, SL1A3 does not associate with the SMN complex. Although U170K interaction is dependent on Sm core assembly and is thought to take place in the nucleus (Hamm et al., 1987; Nelissen et al., 1994; Will and Lührmann, 2001), U170K binds to SL1 but not to SL1A3 and could therefore mediate the interaction with the SMN complex. To test this possibility, the point mutant A29G, which was previously shown not to interact with U170K (Surowy et al., 1989), was also studied. Immunoprecipitation with 2B1 showed that the A29G mutant associates efficiently with the SMN complex (data not shown), suggesting that the interaction between the SMN complex and SL1 is not likely to be mediated by U170K. Taken together, these results demonstrate that the interaction of the SMN complex with SL1 is sequence specific. Purified SMN complexes interact directly with SL1 of U1 snRNA in vitro To examine the specific interaction of the SMN complex with the SL1 in a more purified and biochemically defined in vitro system, we carried out binding experiments with purified SMN complexes. Recently, we have described the purification of SMN complexes from HeLa cells under stringent conditions (500 mM NaCl) (Pellizzoni et al., 2002). These complexes are purified from cells expressing tagged SMN or Gemin2 and contain all the integral components of the complex at the native stoichiometry. Native SMN complexes were isolated by affinity chromatography and peptide elution from extracts of a stable cell line that expresses sub-stoichiometric levels of Flag-tagged Gemin2 under the control of a tetracycline-inducible promoter (see Materials and methods). Total cell extracts from the Flag-Gemin2 cells (SMN) or the parental HeLa Tet-ON cells (control) were immunoprecipitated with anti-Flag antibodies, and bound proteins were analyzed by PAGE and silver staining. These complexes isolated via Flag-Gemin2 contain all of the known components of the SMN complex, including SMN, Gemin2, Gemin3, Gemin4, Gemin5 (Gubitz et al., 2002) and Gemin6 (Pellizzoni et al., 2002), as well as smaller amounts of Sm proteins, U1A and a few as yet unidentified proteins (Figure 4A and B). Western blotting with specific antibodies showed that U170K is not present in these purified complexes (Figure 4B). Figure 4.The purified native SMN complex interacts directly and specifically with the SL1 of U1 snRNA. (A) Native SMN complex purified from HeLa cells. The SMN complex (SMN) was purified as described in Materials and methods and visualized on SDS–PAGE by silver staining. Non-specific proteins purified from HeLa cells that do not express Flag-Gemin2 (control) are also shown. The known components of the SMN complex are identified. Molecular weight markers (MW) are indicated. (B) Purified native SMN complex shown in (A) was analyzed by western blotting using indicated antibodies. 'Total' lanes contain 5% of the input. (C) The purified SMN complex (SMN) or non-specific proteins (control) as in (A) were mixed with several 32P-labeled RNAs. The RNA–protein complexes formed were analyzed by immunoprecipitation with anti-SMN (2B1) antibody in RSB-100 containing 0.02% NP-40. Each RNA (25 000 c.p.m.) was mixed as indicated and used for the binding experiments. 'Total' lanes contain 25% of the input. (D) The purified SMN complex (SMN complex) or non-specific proteins (control) as in (A) were mixed with SL1 or SL1A3. The specific interaction between SL1 and the SMN complex was analyzed by in vitro gel mobility-shift assay in native PAGE. Download figure Download PowerPoint To demonstrate that the interaction of the SMN complex with SL1 is direct, we carried out in vitro binding experiments using purified SMN complexes and in vitro transcribed snRNAs. SMN complexes purified from Flag-Gemin2 cells (SMN) or the non-specific proteins purified from HeLa Tet-ON cells (control) were incubated for 1 h with in vitro transcribed [32P]UTP-labeled U1, U1A3, SL1 or SL1A3 RNAs in the presence of U6 snRNA as an internal control. Immunoprecipitation experiments from these mixtures were carried out with anti-SMN antibodies (2B1), and bound RNAs were analyzed by denaturing PAGE and autoradiography. As shown in Figure 4C, purified SMN complexes interact efficiently with U1 snRNA in vitro. This interaction is specific because U6 snRNA is not immunoprecipitated and the interaction of the SMN complex with the U1A3 mutant is severely impaired. Furthermore, the SMN complex interacts efficiently with SL1, but not with SL1A3 mutant. The residual interaction with U1A3, but not with SL1A3, suggests the presence of additional weaker interactions of the SMN complex with regions of U1 snRNA other than SL1 (see also Figure 5A). Further analysis of the specific interaction between the SMN complex and SL1 was carried out using in vitro gel mobility-shift assay. Purified SMN complexes were mixed with [32P]UTP-labeled SL1 or SL1A3 and incubated for 1 h at 30°C. As shown in Figure 4D, purified SMN complexes specifically bind SL1 and form a stable RNA–protein complex in a native PAGE. Consistent with the microinjection experiments shown above, these experiments demonstrate that purified SMN complexes interact directly and efficiently with the stem–loop 1 domain of U1 snRNA in vitro. Figure 5.U1A3 snRNA is defective in 5′ cap hypermethylation and interaction with the SMN complex. (A) A mixture of [32P]UTP-labeled U1A3, U6 and U1ΔSL2/3 snRNAs was injected into the cytoplasm of oocytes and incubated for 15 h. Nuclear and cytoplasmic fractions were used for immunoprecipitation experiments with the indicated antibodies. R1131 is a polyclonal antibody specific for trimethyl guanosine cap structure and normal rabbit serum (NRS) was used as its negative control. (B) A mixture of [32P]UTP-labeled wt U1 snRNA (U1 wt) and U1ΔSL2/3 was injected into the cytoplasm of oocytes and incubated as indicated. Nuclear and cytoplasmic fractions were used for immunoprecipitation experiments with the Y12 antibody. (C) The same experiments as described in (B) were carried out with a mixture of [32P]UTP-labeled U1A3 and U1ΔSL2/3. Download figure Download PowerPoint U1A3 snRNA is defective in binding to the SMN complex and in cap hypermethylation in vivo Next, we investigated the effect of mutations in SL1 on the interaction of U1 snRNA with the SMN complex and on U1 snRNP biogenesis. [32P]UTP-labeled U1A3 was injected, along with U1ΔSL2/3 and U6 RNAs, and immunoprecipitation was carried out. Consistent with the results described above, the association of U1A3 mutant with the SMN complex is almost completely abolished in the cytoplasm (Figure 5A, 2B1). Immunoprecipitation with Y12 showed that U1A3 associates with the Sm proteins in the cytoplasm of oocytes, but its nuclear import is markedly reduced (∼2-fold) compared with U1ΔSL2/3 (Figure 5A). This effect was similar to that of U1ΔSL1 (see Figure 2D). To further investigate the defect in U1A3 RNA snRNP biogenesis, we analyzed the hypermethylation of its cap structure. Proper snRNP assembly is required for TMG formation (Fischer and Lührmann, 1990; Hamm et al., 1990a). Strikingly, U1A3 from the cytoplasm as well as from the nucleus could not be efficiently immunoprecipitated with the anti-TMG-specific antibody (R1131), indicating a defect in TMG formation (Figure 5A). In contrast, hypermethylation of U1ΔSL2/3 was efficient and similar to that of U1 snRNA (Figure 5A; data not shown). Thus, both the hypermethylation of the cap structure and the association with the SMN complex are impaired in the U1A3 mutant RNA. Because the hypermethylation of the cap structure requires an assembled Sm core, it is likely that the Sm core is not properly assembled in the U1A3 mutant. To further investigate whether the impaired import of U1A3 is a reflection of a kinetic delay in the assembly of the Sm proteins, the time course of Sm protein assembly on U1A3 was monitored. [32P]UTP-labeled wt U1 or U1A3 was injected along with U1ΔSL2/3 and immunoprecipitation with Y12 was carried out at the indicated time points. As shown in Figure 5B and C, the assembly kinetics of the Sm core of U1A3 are delayed compared with those of wt U1. These data suggest that binding of the SMN complex to the SL1 domain of U1 snRNA is important for the efficient biogenesis of U1 snRNP. An excess of SL1 inhibits U1 snRNP assembly In order to investigate further the functional relevance of the interaction between the SMN complex and SL1 of U1 snRNA, we tested the effect of an excess of SL1 on U1 snRNP assembly. Unlabeled in vitro transcribed SL1, SL1A3 or wt U1 snRNA was microinjected into the cytoplasm of oocytes at the concentration of 300 fmol/oocyte. This represents 100-fold excess over a mixture of 32P-labeled U1, U2 and U6 snRNAs that was injected into the cytoplasm of the same oocytes 2 h later. The oocytes were then incubated for an additional 2 h, and immunoprecipitation with anti-SMN (2B1), anti-Sm (Y12) or SP2/0 antibodies was carried out. In the presence of excess amounts of unlabeled wt U1 in the cytoplasm of oocytes, the interaction of the SMN complex with radiolabeled U1 snRNA was abolished, indicating that the SMN complex was saturated by the unlabeled RNAs (compare 2B1 immunoprecipitation lanes in Figure 6A). Similarly, immunoprecipitation with Y12 showed that excess amounts of unlabeled wt U1 snRNA completed the Sm core assembly on radiolabeled U snRNAs (Figure 6A). Excess amount of unlabeled SL1 RNA in the cytoplasm of oocytes strongly reduced the binding of the SMN complex to U1 snRNA (Figure 6B). The inhibition is not complete compared with wt U1 snRNA, possibly because SL1 is less stable in vivo (data not shown). Interestingly, immunoprecipitation with Y12 showed that the assembly of U1 snRNP was inhibited by an excess of SL1, while U2 snRNP assembly was not affected severely (Figure 6B). In contrast, an excess of unlabeled SL1A3 RNA, which does not bind to the SMN complex in vivo or in vitro, did not prevent, and possibly increased, the interaction between the SMN complex and U1 snRNA. SL1A3 also did not inhibit the assembly of Sm proteins with U1 and U2 snRNAs (Figure 6B). These results are consistent with the possibility that reduction in the interaction of the SMN complex with U1 snRNA results in reduced U1 snRNP assembly. These data indicate that the interaction of the SMN complex with SL1 of U1 snRNA is important for U1 snRNP biogenesis. Figure 6.An excess of SL1 inhibits SMN complex interaction with U1 snRNA and U1 snRNP assembly. Non-radioactive (cold) U1 snRNA, SL1 and SL1A3 RNAs were transcribed separately in vitro. Each RNA was injected separately into the cytoplasm of oocytes at a concentration of 300 fmol/oocyte and incubated for 2 h. The oocytes were then reinjected into the cytoplasm with a mixture of 32P-labeled U1, U2 and U6 snRNAs and incubated for an additional 2 h. The oocytes were homogenized and RNAs were immunoprecipitated with 2B1, Y12 or SP2/0 antibodies. (A) Immunoprecipitation of U snRNAs following pre-injection of water (Control) or cold U1 snRNA (+ U1 wt). (B) Immunoprecipitation of U snRNAs following pre-injection of SL1 (+SL1) or SL1A3 (+SL1A3) RNAs. Download figure Download PowerPoint Discussion The SMN complex plays an important role in the biogenesis of spliceosomal U snRNPs and probably of other RNPs such as snoRNPs (Fischer et al., 1997; Pellizzoni et al., 1998, 2001a, b; Buhler et al., 1999; Meister et al., 2000, 2001; Jones et al., 2001; Mourelatos et al., 2001). Immunoprecipitation experiments of microinjected RNAs into Xenopus oocytes showed that the SMN complex interacts particularly avidly and stably with U1 snRNA (Fischer et al., 1997; Buhler et al., 1999; Charroux et al., 2000) and we therefore chose to study the interaction of SMN complex with this snRNA. We show here that this interaction is sequence-specific and is mediated by the loop sequence of SL1. We conclude that to fulfill its function in the assembly of U1 snRNA, and perhaps more generally with the myriad RNPs that the SMN complex assembles or restructures, it interacts with both the protein and the RNA components in a sequence-specific manner. The interaction with the protein components is mediated by RG domains, specifically with sDMA-modified RG domains in the case of Sm proteins (Friesen and Dreyfuss, 2000; Friesen et al., 2001a). RG domains are present in most, if not all, of the substrates of the SMN complex, including SmB, SmD1, SmD3, Lsm4, fibrillarin, GAR1, RNA helicase A, hnRNP U, hnRNP Q and coilin (Liu and Dreyfuss, 1996; Friesen and Dreyfuss, 2000; Friesen et al., 2001a; Hebert et al., 2001; Jones et al., 2001; Mourelatos et al., 2001; Pellizzoni et al., 2001a, b). For the Sm proteins SmD1 and SmD3, this requires the activity of the protein arginine methyltransferase JBP1 (PRMT5) in the conte
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