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 six proteins that are components of the U2 snRNP and required for A complex formation.
Highlights
Pre-messenger RNA (mRNA) splicing is an essential step in gene expression in eukaryotes
We tested a set of bioflavonoids for a potential effect on pre-mRNA splicing in vitro, including amentoflavone, cupressuflavone, hinokiflavone and sciadopitysin (Figure 1A)
We screened each compound at a high final concentration (500 mM), for a potential inhibitory effect on splicing of the model Ad1 and HPV18 E6 pre-mRNAs, using HeLa nuclear extract in conjunction with a non-radioactive RT-PCR in vitro splicing assay
Summary
Pre-mRNA splicing is an essential step in gene expression in eukaryotes. Intron sequences are removed from nascent, pre-mRNA gene transcripts via two, sequential transesterification reactions, thereby joining exon sequences to generate messenger RNA (mRNA) for protein translation (reviewed in [Wahl et al, 2009; Papasaikas and Valcarcel, 2016; Lee and Rio, 2015]). The splicing of pre-mRNA takes place in the cell nucleus and is catalyzed by the large (>3 MDa), ribonucleoprotein spliceosome complex. Spliceosome complexes assemble at the splice sites in a pre-mRNA transcript, involving a stepwise assembly pathway of the U1, U2 and U4/5/6 snRNP spliceosome subunits, together with additional protein splicing factors. The core splicing machinery, spliceosome assembly pathway and reaction mechanism is highly conserved across eukaryotes. In mammalian cells the splicing machinery typically shows a punctate, or ‘speckled’ localisation pattern in the nucleus, with snRNPs located in bright nuclear foci, which are termed Cajal bodies (Lamond and Spector, 2003)
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