The Gemin5 Protein of the SMN Complex Identifies snRNAs

Slides:



Advertisements
Similar presentations
Volume 35, Issue 4, Pages (August 2009)
Advertisements

Volume 55, Issue 1, Pages (July 2014)
Volume 33, Issue 2, Pages (January 2009)
Volume 42, Issue 6, Pages (June 2011)
Volume 41, Issue 5, Pages (March 2011)
Volume 47, Issue 3, Pages (August 2012)
Volume 135, Issue 3, Pages (October 2008)
Nicolas Charlet-B, Gopal Singh, Thomas A. Cooper  Molecular Cell 
Volume 22, Issue 3, Pages (May 2006)
Volume 3, Issue 1, Pages (January 1999)
hnRNP A1 Proofreads 3′ Splice Site Recognition by U2AF
Utz Fischer, Qing Liu, Gideon Dreyfuss  Cell 
Volume 38, Issue 4, Pages (May 2010)
Volume 36, Issue 2, Pages (October 2009)
Transcriptional Activators Enhance Polyadenylation of mRNA Precursors
Volume 16, Issue 5, Pages (December 2004)
Human mRNA Export Machinery Recruited to the 5′ End of mRNA
Volume 64, Issue 3, Pages (November 2016)
Yingqun Huang, Renata Gattoni, James Stévenin, Joan A. Steitz 
Volume 68, Issue 2, Pages e6 (October 2017)
Volume 23, Issue 2, Pages (July 2006)
Volume 38, Issue 4, Pages (May 2010)
Ras Induces Mediator Complex Exchange on C/EBPβ
The Spinal Muscular Atrophy Disease Gene Product, SMN, and Its Associated Protein SIP1 Are in a Complex with Spliceosomal snRNP Proteins  Qing Liu, Utz.
Zbigniew Dominski, Xiao-cui Yang, William F. Marzluff  Cell 
Volume 5, Issue 6, Pages (June 2000)
DNA Methylation Mediated by a MicroRNA Pathway
PARP1 Represses PAP and Inhibits Polyadenylation during Heat Shock
PP1/PP2A Phosphatases Are Required for the Second Step of Pre-mRNA Splicing and Target Specific snRNP Proteins  Yongsheng Shi, Bharat Reddy, James L.
Volume 29, Issue 2, Pages (February 2008)
MAGE-RING Protein Complexes Comprise a Family of E3 Ubiquitin Ligases
Volume 123, Issue 2, Pages (October 2005)
Volume 15, Issue 6, Pages (September 2004)
Volume 38, Issue 3, Pages (May 2010)
Volume 66, Issue 4, Pages e5 (May 2017)
HDAC5, a Key Component in Temporal Regulation of p53-Mediated Transactivation in Response to Genotoxic Stress  Nirmalya Sen, Rajni Kumari, Manika Indrajit.
Multiple mRNA Decapping Enzymes in Mammalian Cells
Inactivation of the SMN Complex by Oxidative Stress
Expression of SYCE2 inhibits the interaction of HP1α with H3K9me3.
Volume 32, Issue 1, Pages (October 2008)
Per Stehmeier, Stefan Muller  Molecular Cell 
MyoD Targets TAF3/TRF3 to Activate Myogenin Transcription
The Mammalian RNA Polymerase II C-Terminal Domain Interacts with RNA to Suppress Transcription-Coupled 3′ End Formation  Syuzo Kaneko, James L. Manley 
c-Src Activates Endonuclease-Mediated mRNA Decay
Livio Pellizzoni, Naoyuki Kataoka, Bernard Charroux, Gideon Dreyfuss 
A Critical Role for Noncoding 5S rRNA in Regulating Mdmx Stability
Volume 11, Issue 24, Pages (December 2001)
Yi Tang, Jianyuan Luo, Wenzhu Zhang, Wei Gu  Molecular Cell 
Autoantigen La Promotes Efficient RNAi, Antiviral Response, and Transposon Silencing by Facilitating Multiple-Turnover RISC Catalysis  Ying Liu, Huiling.
Volume 133, Issue 4, Pages (May 2008)
Polypyrimidine Tract Binding Protein Blocks the 5′ Splice Site-Dependent Assembly of U2AF and the Prespliceosomal E Complex  Shalini Sharma, Arnold M.
Volume 29, Issue 1, Pages (January 2008)
tRNA Binds to Cytochrome c and Inhibits Caspase Activation
Exon Identity Established through Differential Antagonism between Exonic Splicing Silencer-Bound hnRNP A1 and Enhancer-Bound SR Proteins  Jun Zhu, Akila.
Volume 20, Issue 6, Pages (December 2005)
Volume 18, Issue 5, Pages (May 2005)
Rita Das, Zhaolan Zhou, Robin Reed  Molecular Cell 
Yap1 Phosphorylation by c-Abl Is a Critical Step in Selective Activation of Proapoptotic Genes in Response to DNA Damage  Dan Levy, Yaarit Adamovich,
Fan Yang, Huafeng Zhang, Yide Mei, Mian Wu  Molecular Cell 
Lin28 Mediates the Terminal Uridylation of let-7 Precursor MicroRNA
Regulation of Yeast mRNA 3′ End Processing by Phosphorylation
Volume 45, Issue 1, Pages (January 2012)
Volume 2, Issue 4, Pages (October 2012)
Volume 7, Issue 5, Pages (May 2001)
Volume 9, Issue 1, Pages (January 2002)
Volume 55, Issue 1, Pages (July 2014)
Volume 65, Issue 5, Pages e4 (March 2017)
Volume 41, Issue 4, Pages (February 2011)
Volume 3, Issue 1, Pages (January 1999)
Volume 123, Issue 2, Pages (October 2005)
Presentation transcript:

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