Dataset Information

Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP

ABSTRACT: We have identified the plant biflavonoid hinokiflavone as an inhibitor of splicing in vitro and modulator of alternative splicing in cells. Chemical synthesis confirms hinokiflavone is the active molecule. Hinokiflavone inhibits splicing in vitro by blocking spliceosome assembly, preventing formation of the B complex. Cells treated with hinokiflavone show altered subnuclear organization specifically of splicing factors required for A complex formation, which relocalize together with SUMO1 and SUMO2 into enlarged nuclear speckles containing polyadenylated RNA. Hinokiflavone increases protein SUMOylation levels, both in in vitro splicing reactions and in cells. Hinokiflavone also inhibited a purified, E. coli expressed SUMO protease, SENP1, in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased in cells following hinokiflavone treatment, with the major targets including 6 proteins that are components of the U2 snRNP and required for A complex formation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Andrea Pawellek

LAB HEAD: Angus Iain Lamond

PROVIDER: PXD007629 | Pride | 2017-09-07

REPOSITORIES: Pride

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Publications

Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP.

Pawellek Andrea A Ryder Ursula U Tammsalu Triin T King Lewis J LJ Kreinin Helmi H Ly Tony T Hay Ronald T RT Hartley Richard C RC Lamond Angus I AI

eLife 20170908

We have identified the plant biflavonoid hinokiflavone as an inhibitor of splicing in vitro and modulator of alternative splicing in cells. Chemical synthesis confirms hinokiflavone is the active molecule. Hinokiflavone inhibits splicing in vitro by blocking spliceosome assembly, preventing formation of the B complex. Cells treated with hinokiflavone show altered subnuclear organization specifically of splicing factors required for A complex formation, which relocalize together with SUMO1 and SU ...[more]

PMID: 28884683

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Project description:Ubiquitination is a post-translational modification that has been shown to have a range of effects, such as regulating protein function, protein:protein interactions, protein localization, and protein abundance by targeting proteins for degradation by the 26S proteasome. We have previously shown that the muscle-specific ubiquitin E3 ligase, ASB2, is down-regulated in models of muscle growth and that overexpression ASB2 is sufficient to induce muscle atrophy. To gain insight into the effect of increased ASB2 expression on skeletal muscle mass and function, we used 2-dimensional nano-ultra-high-performance mass spectrometry to investigate ASB2-induced changes to the mouse skeletal muscle proteome, and whether these were associated with changes in protein ubiquitination. The results show that in vivo recombinant adeno-associated viral vector-mediated ASB2β overexpression induces impaired intrinsic force production and progressive muscle atrophy over 12 weeks, while ASB2 knockdown induces mild muscle hypertrophy. ASB2-induced muscle atrophy and dysfunction were associated with the early down regulation of mitochondrial and contractile proteins, and the upregulation of proteins involved in proteasome-mediated protein degradation, protein synthesis and the cytoskeleton/sarcomere. The overexpression ASB2β also resulted in marked changes in protein ubiquitination, however, there was no simple relationship between changes in ubiquitination status and protein abundance. To gain insights into proteins that interact with ASB2, and potential ASB2 targets, in muscle cells, flag-tagged wild type ASB2, and a mutant ASB2 with a deleted C-terminal SOCS box domain (dSOCS) were immunoprecipitated from C2C12 myotubes and subjected to label-free proteomic analysis to determine the ASB2 interactome. ASB2β was found to interact with a range of cytoskeletal and nuclear proteins. When combined with the in vivo ubiquitinomic data, we identified several novel potential ASB2β target substrates that await further investigation. Overall, this study reveals for the first time the complexity of changes to the proteome and ubiquitinome during E3 ligase-mediated skeletal muscle atrophy and dysfunction.

Dataset Information

Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP

Publications

Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP.

Similar Datasets

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