Pawellek, A. | bioRxiv Science

bioRxiv2025

Differential contribution of the RNA recognition motifs to SRSF1 protein interaction network

The protein SRSF1, initially identified as a splicing factor, is currently known to participate in a variety of cellular processes. However, its role and regulatory mechanisms in these processes remain incompletely understood. Using a SILAC proteomic approach, we have characterised the SRSF1 interactome, thereby identifying multiple diverse protein interaction partners of SRSF1 and the molecular complexes with which it is associated. Given that the RNA-binding ability of SRSF1 is necessary for many of its activities, we extended the analysis of the SRSF1 interactome to compare the wild type protein against two site-specific SRSF1 variants, bearing mutations in each of its two RNA recognition motifs (RRMs). The first mutation disrupted the canonical RRM (RRM1), while the second affected the pseudo-RRM (RRM2). Interestingly, the data show clear differences between the SRSF1 interacting proteins, depending on the functional status of each RRM domain. In particular, a functional RRM1 was critical for maintaining interactions with multiple partners, especially those involved in translation-related processes, showing a far greater loss of interactions upon mutation than RRM2. This study provides valuable new insights into the broader cellular roles and interaction landscape of SRSF1.

molecular biology↗

bioRxiv2017

Characterisation Of The Biflavonoid Hinokiflavone As A Pre-mRNA Splicing Modulator That Inhibits SENP

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.

biochemistry↗