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	Figure 1. Immunoselected native SMN complexes associate in vitro with 7S RNA. (A) Human 7S RNA sequence. The secondary structure was established by previous studies (75,76). Stems are numbered 2 to 8 according to the criteria of previous study (76). (B) Native SMN complexes (SMN complex) were purified by immunoprecipitation with anti-Flag antibodies using total extracts prepared from HeLa TET-off cells that stably expressed Flag-tagged Gemin2. The protein composition of the complexes was analyzed by SDS–PAGE followed by silver staining. The non-specific proteins immunoselected from HeLa TET-off cells that do not express Flag-Gemin2 are shown as a negative control (Control). Proteins were identified according to their molecular weight as referred to molecular weight markers (MW) and to (39) and (68). The identity of the proteins was confirmed by western blotting and mass spectrometry (data not shown). (C) The purified native SMN complex (SMN complex) shown in (B) or the non-specific proteins purified from parental cells (Control) were incubated with in vitro transcribed [32P]UTP-labeled 7S RNA, U1 snRNA and U1ΔSL1 in the constant presence of U6 snRNA as an internal negative control. Subsequently, bound RNAs were isolated after washing and analyzed by electrophoresis on denaturing polyacrylamide gels and autoradiography. Lanes labeled ‘Total’ represent 10% of input.
	Figure 2. U1 and U2 snRNAs compete with 7S RNA for binding to the SMN complex. Purified native SMN complexes (SMN complex) were pre-incubated with an excess of unlabeled U1, U1ΔSL1, U2, U4, U5 and 7S. A pre-incubation was also done without the presence of any RNA (/) as a control. Non-specific proteins purified from parental cells (Control) were also pre-incubated. Then, in vitro transcribed [32P]UTP-labeled 7S RNA was added to the pre-incubated mixtures and further incubated. Bound RNAs were isolated after washing and analyzed by electrophoresis on denaturing polyacrylamide gels and autoradiography. Lanes labeled ‘Total’ represent 10% of input. The intensity of signal in each condition was quantified using the Typhoon 9410 (Amersham).
	Figure 3. The overall 7S RNA molecule is required for efficient interaction with the purified SMN complex in vitro. (A) Schematic representation of the 7S RNA domains used in the study. (B) The purified native SMN complex (SMN complex) or the non-specific proteins purified from parental cells (Control) were incubated with in vitro transcribed [32P]UTP-labeled 7S RNA WT or the individual 7S RNA domains, in the constant presence of U6 snRNA as an internal negative control. Subsequently, bound RNAs were isolated after washing and analyzed by electrophoresis on denaturing polyacrylamide gels and autoradiography. Lanes labeled ‘Total’ represent 10% of input.
	Figure 4. The SMN complex protects three regions of the 7S RNA. (A) In vitro transcribed 7S RNA was incubated in the absence (Transcript) or in the presence (SMN complex) of the purified SMN complex. The RNA was then digested by RNases T1, T2 and V1 as described in the ‘Materials and Methods’ section. The cleavage products were analyzed by electrophoresis on denaturing polyacrylamide gels. An alkaline hydrolysis is shown (H). Nucleotides are numbered on the left and were designed by the use of RNase T1 in denaturing conditions and by a longer fractionation on polyacrylamide gel to identify residues in the 5′-end (data not shown). (B) The protected regions within the 7S RNA are indicated in red, and the G residues that were more sensitive to T1 RNase cleavages are indicated by blue arrows.
	Figure 5. Gemin5 binds to 7S RNA. (A) Immunoprecipitation experiments using anti-Flag antibody were carried out on total extracts prepared from HeLa cells transiently expressing Flag-tagged Gemin5 in the absence (−) or in the presence (+) of 1% Empigen-BB detergent. The immunoprecipitated proteins were analyzed by SDS–PAGE and western blotting using antibodies directed against the indicated proteins. In total, 5% of the extracts used in the experiments is shown. (B) After immunoprecipitations as in (A) in the presence of 1% Empigen-BB detergent, purified Flag-Gemin5 was incubated with [32P]UTP-labeled 7S RNA, U4 snRNA or U1 snRNA, in the constant presence of U6 snRNA used as an internal negative control. (C) Recombinant GST-Gemin5, GST-SMN or GST were expressed in E. coli and bound to glutathione-Sepharose beads. The proteins were incubated with [32P]UTP-labeled 7S RNA or U1 snRNA, in the constant presence of U6 snRNA, used as an internal negative control. The recombinant proteins used in the study have been analyzed by SDS–PAGE and coomassie staining. In panels B and C, bound RNAs were isolated after washing and analyzed by electrophoresis on denaturing polyacrylamide gels. Lanes labeled ‘Total’ represent 10% of the input.
	Figure 6. The SMN complex associates with SRP in HeLa cell extracts. IP experiments were carried out on HeLa cytoplasmic extracts using anti-SRP9 (A), anti-SRP54 (B), or anti-SMN 4B3 (C) antibodies bound to A-sepharose [in (A) and (B)] or G-dynabeads [in (C)]. The unrelated anti-Sox2 and anti-Lin28 antibodies were used as negative controls in (A) (Controls 1 and 2, respectively). A-sepharose alone was used as control in (B) and negative control mouse IgG (DAKO) in (C). The immunoprecipitated proteins were analyzed by SDS–PAGE and western-blotting with antibodies to the indicated proteins; 3% of the inputs are shown (Total). The immunoprecipitated RNAs were analyzed by RT–PCR in (C).
	Figure 7. Anti-SMN and anti-Gemin2 antibodies interfere with the binding of SRP54 on 7S RNA in the cytoplasm of X. laevis oocytes. (A) A mixture of [32P]UTP-labeled U1, U6, 7S and 7S 5′-3′-regions RNAs was injected into the nucleus of X. laevis oocytes. After incubation for 6 h, the oocytes were manually dissected into nuclear (N) and cytoplasmic (C) fractions. IPs were then carried out from both fractions with the anti-SMN (2B1), anti-Gemin2 (2E17), anti-SRP54 or control non-immune (SP2/O) antibodies. Bound RNAs were analyzed by electrophoresis on a denaturing polyacrylamide gel and autoradiography. About 10% of each fraction was loaded on the gel (Total). (B) Either non-immune (SP2/O), anti-SRP54, anti-Gemin2 (2E17) or anti-SMN (2B1) antibodies were injected into the cytoplasm of X. laevis oocytes. The same oocytes were nuclear injected 1 h later with a mixture of [32P]UTP-labeled U6 snRNA and 7S RNA. After 6 h, total cellular extracts were prepared from these oocytes. Immunoprecipitations were then carried out with the anti-Sm core (Y12) (IP Y12) or anti-SRP54 (IP SRP54) antibodies. RNAs were analyzed by electrophoresis on a denaturing polyacrylamide gel and autoradiography. About 10% of each injected RNA was loaded on the gel (Total).
	Figure 8. The SMN protein associates with the 7S RNA in vivo in fission yeast cells and is required for the stability of the SRP complex. (A) Quantitative RT–PCR experiments were performed for the indicated RNA species using RNA affinity-purified from extracts prepared from fission yeast cells carrying a TAP-SMN construct or an empty TAP vector as control. Experiments were done in duplicate. (B) Northern blot analysis of RNA isolated from wild-type and temperature-sensitive tdSMN cells before and after shift for 8 h at 37°C. The RNA was separated on 6% denaturing polyacrylamide gels, subjected to northern blot and hybridized with oligonucleotide probes for the indicated RNA. (C) Analysis of RNP complexes in wild-type and temperature-sensitive tdSMN cells by native gel electrophoresis. Extracts were prepared from cells grown before and after a 8-h shift at 37°C and 20 µg of extract were separated on 4% native gels. The RNA was subjected to northern blot analysis and hybridized with probes for the indicated RNAs. The arrow points to the position of the U2/U5/U6 tri-snRNP.
	Figure 9. Analysis of endogenous 7S RNA level in tissues of SMA mice. Total RNA from the spinal cord, brain and heart of post-natal Day 10 control and SMA mice were analyzed by real-time RT–PCR. The level of 7S RNA was monitored by five independent biological replicates (n = 5). The relative amount of 7S RNA in SMA mice tissues is plotted as percent of the controls. RPS29 mRNA was used to normalize the RNA input. The level of 7S RNA in spinal cord was significantly reduced in SMA mice, whereas its levels in brain and heart remain the same (P < 0.05).

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