The Gemin5 Protein of the SMN Complex Identifies snRNAs Daniel J. Battle, Chi-Kong Lau, Lili Wan, Hongying Deng, Francesco Lotti, Gideon Dreyfuss  Molecular Cell  Volume 23, Issue 2, Pages 273-279 (July 2006) DOI: 10.1016/j.molcel.2006.05.036 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Gemin5 Binds to snRNAs (A) 32P labeled U4 snRNA was incubated in HeLa extract and irradiated at 254 nm to allow protein-RNA crosslinks to form. SMN, Gemin3, Gemin5, and Sm proteins were isolated by immunoprecipitation in 1% Empigen-BB detergent. Isolated crosslinked proteins were analyzed by 4%–12% SDS-PAGE and imaged by autoradiography. Control represents immunoprecipitation with mouse nonimmune antibody. Total represents HeLa extract containing 15 μg total protein prior to immunoprecipitation. (B) 32P labeled U4, U4ΔSm, and U6 snRNAs were added to HeLa extract and crosslinked as above and then immunoprecipitated with anti-Gemin5 antibody or nonimmune mouse antibody as a control. (C) UV crosslinking was performed as in (B) but using a cell line expressing Flag-Gemin2. Intact SMN complex was isolated by anti-Flag immunoprecipitation after UV crosslinking. (D) Western Blot analysis of anti-SMN, anti-Gemin3, anti-Gemin5, or anti-Sm immunoprecipitations performed from HeLa extract in the presence of 1% Empigen-BB detergent. (E) After immunoprecipitation as in (D), purified SMN/Gemin2, Gemin3, and Gemin5 were incubated with 32P labeled U4 snRNA. U6 snRNA was included as a negative control. Bound RNAs were isolated and analyzed by denaturing polyacrylamide gel electrophoresis and imaged by autoradiography. Input represents 5% of the total RNA in the reaction. (F) Silver stain of the immunopurified proteins used in the experiments. (h.c.) and (l.c.) denote the antibody heavy and light chains, respectively. Molecular Cell 2006 23, 273-279DOI: (10.1016/j.molcel.2006.05.036) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Gemin5 Specifically Binds to snRNAs (A) 32P labeled U1, U2, U4, and U5 snRNAs were incubated with Empigen-purified Gemin5 immobilized on protein G Sepharose beads. U6 snRNA was included as a negative control. Bound RNAs were isolated and analyzed by denaturing polyacrylamide gel electrophoresis and imaged by autoradiography. Input represents 5% of the total RNA in the reaction. Control immunoprecipitations were carried out with mouse nonimmune antibody. (B) Same as in (A) but with U7 snRNA. (C) In vitro binding experiment as in (A) but with an HSUR5 mutant with the 3′ stem loop deleted (Δ3′SL). (D) Single site point mutations were made in the Sm site of HSUR5min RNA and in vitro binding experiments were performed as in (A). HSUR5min represents HSUR5 nt 60-114 shown to be a minimal SMN complex binding domain. Molecular Cell 2006 23, 273-279DOI: (10.1016/j.molcel.2006.05.036) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Recombinant Gemin5 Specifically Binds to snRNA (A) Recombinant Gemin5 was expressed in E. coli and purified by immunoprecipitation with anti-Gemin5 antibodies. Shown is a silver-stained gel of the protein used in the experiments. An asterisk (∗) indicates a proteolytic fragment of Gemin5. (h.c.) and (l.c.) denote the antibody heavy and light chains, respectively. (B) Gemin5 immobilized on protein G Sepharose beads was incubated with 32P labeled U4, U4ΔSm, and U6 snRNAs. Bound RNAs were isolated and analyzed by denaturing polyacrylamide gel electrophoresis and imaged by autoradiography. Input represents 5% of the total RNA in the reaction. Control represents immunoprecipitation from E. coli lysate not expressing recombinant Gemin5. Molecular Cell 2006 23, 273-279DOI: (10.1016/j.molcel.2006.05.036) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Gemin5 Is Required for snRNP Assembly (A) SMN complex was isolated by immunoprecipitation using anti-SMN antibodies from HeLa cells stably expressing shRNAs against Gemin5 (Gemin5 RNAi) or nontargeting shRNAs (Control RNAi). Proteins were detected by Western blot. (B) SMN complexes isolated in (A) were incubated with 32P labeled U4 and U6 snRNAs. Bound RNAs were isolated and analyzed by denaturing polyacrylamide gel electrophoresis and imaged by autoradiography. Input represents 5% of the total RNA in the reaction. Control immunoprecipitations were performed with mouse nonimmune antibody. (C) Western blot of the total cell extracts from the stable RNAi cell lines used in the snRNP assembly assay shown in (D). JBP1 is shown as an internal control. (D) Total cell extracts of the stable RNAi cell lines were incubated with biotin-labeled U4 snRNA and assayed for Sm core assembly. Protein levels were determined by quantitative Western blot analysis. The errors are the standard deviation from the mean of three independent experiments. (E) MN-1 cells were transfected with siRNAs targeting Gemin5 or a negative control siRNA. Shown is a Western blot of the cell extracts used for snRNP assembly. Magoh is shown as an internal control. (F) Total cell extracts from the MN-1 cells shown in (E) were incubated with U4 snRNA and assayed for Sm core assembly. (G) 293T cells were transfected with siRNAs targeting Gemin5 or a negative control siRNA. Shown is a Western blot of the cell extracts used for snRNP assembly. Magoh is shown as an internal control. (H) Total cell extracts from the 293T cells shown in (G) were incubated with U4 snRNA and assayed for Sm core assembly. Molecular Cell 2006 23, 273-279DOI: (10.1016/j.molcel.2006.05.036) Copyright © 2006 Elsevier Inc. Terms and Conditions