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In a new publication by Ly et. al. (eLife, 2017), the Lamond group report a workflow combining FACS and MS-based proteomics to analyse protein abundance and phosphorylation changes, proteome-wide, across the mitotic cell division cycle, including resolution of mitotic subphases.
This approach, called ‘PRIMMUS’, i.e. proteomics of intracellular immunolabelled cell subsets, combines intracellular immunostaining, FACS and mass spectrometry to isolate and analyse cell subsets defined by intracellular markers, which in this study was used to study cell cycle progression. Cytometric separation avoids potential artefacts associated with cell cycle synchronization, e.g. as induced by either metabolic arrest, or via specific and non-specific effects of small molecule kinase inhibitors, and/or spindle poisons. Key findings in the new study include the identification of a set of protein phosphorylation sites, called ‘early risers’, that increase during G2 phase and the identification of ~100 proteins, including RRM2, that show decreased abundances during early mitosis.
The functional significance of some of these new data were further corroborated with follow-on cell biology experiments, performed in collaboration with Pat Wadsworth at University of Massachusetts, Amherst, and Emma Lundberg at the Human Protein Atlas. The PRIMMUS method enabled here the first deep analysis of proteome variation in cells at different mitotic substages and has many potential future applications in other areas of cell biology where it is important to isolate and characterise specific cell types/responders in heterogeneous populations.
November 6th 2017
A new article from the Lamond group, published in eLife, reports the identification of the plant biflavone, hinokiflavone, as a pre-mRNA splicing modulator (Pawellek et al., 2017). In collaboration with Ron Hay’s group in GRE and the group of Richard Hartley, (University of Glasgow), they show that both natural and synthetic hinokiflavone inhibits splicing in vitro by blocking spliceosome assembly, specifically preventing progression from the spliceosome complex A to complex B. Further, they find that hinokiflavone binds to and inhibits the SUMO protease, SENP1, in vitro and causes an increase in the levels of SUMO2-modified proteins in cellulo. Using an unbiased, SILAC MS-based proteomics assay, the major protein targets that are hyper-SUMOylated in cells treated with hinokiflavone were shown to include six proteins that are all components of the U2 snRNP spliceosome subunit that is required for A complex formation. This study identifies hinokiflavone as a potential novel cancer therapeutic and points to a role for protein SUMOylation in regulating spliceosome formation and alternative splicing.