RESUMO
Splicing factor proline- and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associated-splicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.
Assuntos
Proteínas Associadas à Matriz Nuclear/genética , Fatores de Transcrição de Octâmero/genética , Splicing de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transcrição Gênica , Animais , Arginina/genética , Arginina/metabolismo , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Células HeLa , Humanos , Metilação , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas Associadas à Matriz Nuclear/química , Fatores de Transcrição de Octâmero/química , Fator de Processamento Associado a PTB , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/genética , Proteínas de Ligação a RNA/química , Proteínas Repressoras/genética , Ribonucleoproteínas/genéticaRESUMO
The 2-D peptide separations employing mixed mode reversed phase anion exchange (MM (RP-AX)) HPLC in the first dimension in conjunction with RP chromatography in the second dimension were developed and utilised for shotgun proteome analysis. Compared with strong cation exchange (SCX) typically employed for shotgun proteomic analysis, peptide separations using MM (RP-AX) revealed improved separation efficiency and increased peptide distribution across the elution gradient. In addition, improved sample handling, with no significant reduction in the orthogonality of the peptide separations was observed. The shotgun proteomic analysis of a mammalian nuclear cell lysate revealed additional proteome coverage (2818 versus 1125 unique peptides and 602 versus 238 proteins) using the MM (RP-AX) compared with the traditional SCX hyphenated to RP-LC-MS/MS. The MM analysis resulted in approximately 90% of the unique peptides identified present in only one fraction, with a heterogeneous peptide distribution across all fractions. No clustering of the predominant peptide charge states was observed during the gradient elution. The application of MM (RP-AX) for 2-D LC proteomic studies was also extended in the analysis of iTRAQ-labelled HeLa and cyanobacterial proteomes using nano-flow chromatography interfaced to the MS/MS. We demonstrate MM (RP-AX) HPLC as an alternative approach for shotgun proteomic studies that offers significant advantages over traditional SCX peptide separations.
Assuntos
Cromatografia de Fase Reversa/métodos , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/análise , Mapeamento de Peptídeos/métodos , Proteômica/métodos , Acetonitrilas , Cátions , Núcleo Celular/química , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Marcação por Isótopo , Proteínas Nucleares/análise , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteoma/química , Tripsina/metabolismoRESUMO
The structure of BPSL1549, a protein of unknown function from Burkholderia pseudomallei, reveals a similarity to Escherichia coli cytotoxic necrotizing factor 1. We found that BPSL1549 acted as a potent cytotoxin against eukaryotic cells and was lethal when administered to mice. Expression levels of bpsl1549 correlate with conditions expected to promote or suppress pathogenicity. BPSL1549 promotes deamidation of glutamine-339 of the translation initiation factor eIF4A, abolishing its helicase activity and inhibiting translation. We propose to name BPSL1549 Burkholderia lethal factor 1.