Characterisation of the biflavonoid hinokiflavone as a premRNA splicing modulator that inhibits SENP

Andrea Pawellek, Ursula Ryder, Triin Tammsalu, Lewis J King, Helmi Kreinin, Tony Ly, Ronald T. Hay, Richard Hartley, Angus Lamond (Lead / Corresponding author)

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Abstract

Here, we identify the plant biflavanoid hinokiflavone as an inhibitor of splicing in vitro and modulater of alternative splicing in multiple human cell lines. Hinokiflavone inhibits splicing in vitro by blocking one or more early steps of spliceosome assembly, leading to accumulation of the A complex. Multiple human cell lines treated with hinokiflavone show changes in the alternative splicing of different pre-mRNA substrates, but little or no change in transcription. They also show altered subnuclear organization, specifically of splicing factors required for A complex formation, which relocalized together with SUMO1 and SUMO2 into enlarged nuclear speckles. While most cell lines treated with hinokiflavone showed cell cycle arrest and eventual cell death, dependent on time and concentration, the promyelocytic NB4 cell line, which expresses the SUMO target PML-RARalpha fusion protein, was exquisitely sensitive to apoptosis following hinokiflavone treatment. Hinokiflavone treatment increased protein SUMOylation levels, both in in vitro splicing reactions and in cells, with little or no effect on levels of ubiquitinylated proteins. Hinokiflavone also inhibited the catalytic activity of 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 following hinokiflavone treatment, with the major targets including 6 proteins that are associated with U2 snRNP and required for A complex formation. These data identify hinokiflavone as a SUMO protease inhibitor and indicate SUMOylation of splicing factors may be important for modulating splice site selection.
Original languageEnglish
Article numbere27402
JournaleLife
Volume6
Early online date8 Sep 2017
DOIs
Publication statusPublished - 8 Sep 2017

Fingerprint

Biflavonoids
Modulators
Sumoylation
Cells
Cell Line
Alternative Splicing
Proteins
U2 Small Nuclear Ribonucleoproteins
Spliceosomes
sulfoenolpyruvate
hinokiflavone
Site selection
RNA Precursors
Cell death
Transcription
Speckle
Cell Cycle Checkpoints
Protease Inhibitors
Proteomics
Escherichia coli

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Pawellek, Andrea ; Ryder, Ursula ; Tammsalu, Triin ; King, Lewis J ; Kreinin, Helmi ; Ly, Tony ; Hay, Ronald T. ; Hartley, Richard ; Lamond, Angus. / Characterisation of the biflavonoid hinokiflavone as a premRNA splicing modulator that inhibits SENP. In: eLife. 2017 ; Vol. 6.
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abstract = "Here, we identify the plant biflavanoid hinokiflavone as an inhibitor of splicing in vitro and modulater of alternative splicing in multiple human cell lines. Hinokiflavone inhibits splicing in vitro by blocking one or more early steps of spliceosome assembly, leading to accumulation of the A complex. Multiple human cell lines treated with hinokiflavone show changes in the alternative splicing of different pre-mRNA substrates, but little or no change in transcription. They also show altered subnuclear organization, specifically of splicing factors required for A complex formation, which relocalized together with SUMO1 and SUMO2 into enlarged nuclear speckles. While most cell lines treated with hinokiflavone showed cell cycle arrest and eventual cell death, dependent on time and concentration, the promyelocytic NB4 cell line, which expresses the SUMO target PML-RARalpha fusion protein, was exquisitely sensitive to apoptosis following hinokiflavone treatment. Hinokiflavone treatment increased protein SUMOylation levels, both in in vitro splicing reactions and in cells, with little or no effect on levels of ubiquitinylated proteins. Hinokiflavone also inhibited the catalytic activity of 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 following hinokiflavone treatment, with the major targets including 6 proteins that are associated with U2 snRNP and required for A complex formation. These data identify hinokiflavone as a SUMO protease inhibitor and indicate SUMOylation of splicing factors may be important for modulating splice site selection.",
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note = "This work was supported by a Wellcome Trust programme grant to AIL (073980/Z/03/B) and also supported by infrastructure funded by a Wellcome Trust Strategic award (097045/B/11/Z). R.T.H. is a Senior Investigator of the Wellcome Trust (grant 098391/Z/12/7) T.T. was funded by the EU Initial Training Network (ITN) ‘‘UPSTREAM’’ (PITN-GA-2011-290257) and an ISSF grant funded by the Wellcome Trust (105606/Z/14/Z).",
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Characterisation of the biflavonoid hinokiflavone as a premRNA splicing modulator that inhibits SENP. / Pawellek, Andrea; Ryder, Ursula; Tammsalu, Triin; King, Lewis J; Kreinin, Helmi ; Ly, Tony; Hay, Ronald T.; Hartley, Richard ; Lamond, Angus (Lead / Corresponding author).

In: eLife, Vol. 6, e27402, 08.09.2017.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Characterisation of the biflavonoid hinokiflavone as a premRNA splicing modulator that inhibits SENP

AU - Pawellek, Andrea

AU - Ryder, Ursula

AU - Tammsalu, Triin

AU - King, Lewis J

AU - Kreinin, Helmi

AU - Ly, Tony

AU - Hay, Ronald T.

AU - Hartley, Richard

AU - Lamond, Angus

N1 - This work was supported by a Wellcome Trust programme grant to AIL (073980/Z/03/B) and also supported by infrastructure funded by a Wellcome Trust Strategic award (097045/B/11/Z). R.T.H. is a Senior Investigator of the Wellcome Trust (grant 098391/Z/12/7) T.T. was funded by the EU Initial Training Network (ITN) ‘‘UPSTREAM’’ (PITN-GA-2011-290257) and an ISSF grant funded by the Wellcome Trust (105606/Z/14/Z).

PY - 2017/9/8

Y1 - 2017/9/8

N2 - Here, we identify the plant biflavanoid hinokiflavone as an inhibitor of splicing in vitro and modulater of alternative splicing in multiple human cell lines. Hinokiflavone inhibits splicing in vitro by blocking one or more early steps of spliceosome assembly, leading to accumulation of the A complex. Multiple human cell lines treated with hinokiflavone show changes in the alternative splicing of different pre-mRNA substrates, but little or no change in transcription. They also show altered subnuclear organization, specifically of splicing factors required for A complex formation, which relocalized together with SUMO1 and SUMO2 into enlarged nuclear speckles. While most cell lines treated with hinokiflavone showed cell cycle arrest and eventual cell death, dependent on time and concentration, the promyelocytic NB4 cell line, which expresses the SUMO target PML-RARalpha fusion protein, was exquisitely sensitive to apoptosis following hinokiflavone treatment. Hinokiflavone treatment increased protein SUMOylation levels, both in in vitro splicing reactions and in cells, with little or no effect on levels of ubiquitinylated proteins. Hinokiflavone also inhibited the catalytic activity of 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 following hinokiflavone treatment, with the major targets including 6 proteins that are associated with U2 snRNP and required for A complex formation. These data identify hinokiflavone as a SUMO protease inhibitor and indicate SUMOylation of splicing factors may be important for modulating splice site selection.

AB - Here, we identify the plant biflavanoid hinokiflavone as an inhibitor of splicing in vitro and modulater of alternative splicing in multiple human cell lines. Hinokiflavone inhibits splicing in vitro by blocking one or more early steps of spliceosome assembly, leading to accumulation of the A complex. Multiple human cell lines treated with hinokiflavone show changes in the alternative splicing of different pre-mRNA substrates, but little or no change in transcription. They also show altered subnuclear organization, specifically of splicing factors required for A complex formation, which relocalized together with SUMO1 and SUMO2 into enlarged nuclear speckles. While most cell lines treated with hinokiflavone showed cell cycle arrest and eventual cell death, dependent on time and concentration, the promyelocytic NB4 cell line, which expresses the SUMO target PML-RARalpha fusion protein, was exquisitely sensitive to apoptosis following hinokiflavone treatment. Hinokiflavone treatment increased protein SUMOylation levels, both in in vitro splicing reactions and in cells, with little or no effect on levels of ubiquitinylated proteins. Hinokiflavone also inhibited the catalytic activity of 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 following hinokiflavone treatment, with the major targets including 6 proteins that are associated with U2 snRNP and required for A complex formation. These data identify hinokiflavone as a SUMO protease inhibitor and indicate SUMOylation of splicing factors may be important for modulating splice site selection.

U2 - 10.7554/eLife.27402

DO - 10.7554/eLife.27402

M3 - Article

VL - 6

JO - eLife

JF - eLife

SN - 2050-084X

M1 - e27402

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