MassIVE MSV000079597

Partial Public PXD003832

Novel spliceosomal SF3B component in SAGA functions in transcriptional coactivation

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Description

MudPIT analyses of novel spliceosomal SF3B component in SAGA complex Stable Drosophila S2 cell lines expressing SF3B5 (CG11985, NP_652189.1), U2B (SNF; CG4528, NP_511045.1), Ada2b-PB (CG9638, NP_001027151.1), Spt3 (CG3169, NP_650146.1), Spt20 (CG17689, NP_648659.2), ATXN7 (CG9866, NP_722803.1), Ada1 (CG31866, NP_723703.1), SAF6 (CG3883, NP_608545.1), Sgf29 (CG30390, NP_726051.1), and WDA (CG4448, NP_651136.1) in the pRmHa3-CHA2FL2 vector were generated as described (Guelman S, et al., Mol Cell Biol. 2006;26:7178-89). Protein complexes were purified by tandem FLAG-HA affinity purification as described (Weake VM, et al. Genes Dev. 2009;23:2818-23). Two negative controls were purified in parallel from S2 cells without any vectors and from S2 cells expressing CG6459. Where indicated, 250 ug/mL RNAseA was added to soluble nuclear extract from WDA-expressing cells prior to immunoprecipitation. Proteins were digested with endoproteinase LysC followed by trypsin. The resulting peptide mixtures were analyzed by Multidimensional Protein Identification Technology (MudPIT) on an LTQ ion trap mass spectrometer as described (Florens L, Washburn MP. Methods Mol Biol 2006;328:159-75). RAW files were extracted into .ms2 file format using RawDistiller v. 1.0 (in-house developed software). The MS/MS spectra were searched using SEQUEST v.27 (rev.9) with a peptide mass tolerance of 3 amu and of +/- 0.5 amu for fragment ions. No enzyme specificity was imposed during the SEQUEST searches against a protein database containing 18425 non-redundant Drosophila melanogaster proteins (NCBI 2011-04-11 release), as well as 177 usual contaminants such as human keratins, IgGs and proteolytic enzymes. To estimate false discovery rates (FDR), each protein sequence was randomized (keeping the same amino acid composition and length) and the resulting "shuffled" sequences were added to the database used for the SEQUEST searches, for a total search space of 37204 amino acid sequences. To account for alkylation by CAM, 57.02146 Da were added statically to cysteine residues for all searches. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Chromatin ; SAGA complex ; Splicing factors ; SF3B3 ; SF3B5 ; Affinity Purification

Contact

Principal Investigators:
(in alphabetical order) 		Laurence Florens
Submitting User: laflorens

Publications

Stegeman R, Spreacker PJ, Swanson SK, Stephenson R, Florens L, Washburn MP, Weake VM.
The Spliceosomal Protein SF3B5 is a Novel Component of Drosophila SAGA that Functions in Gene Expression Independent of Splicing.
J. Mol. Biol. Epub 2016 May 13.

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