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Research Training Group RTG 2467 „Intrinsically Disordered Proteins – Molecular Principles, Cellular Functions, and Diseases“
Deutsche Forschungsgemeinschaft (DFG) ;
Project II.2: Marcel Köhn - The regulation of SRSF1 function by disordered RS-domains and ncRNA-binding
SR-proteins constitute a family of RNA-binding proteins, which mainly regulate constitutive and alternative splicing. They localize primarily to the nucleus and are enriched in so called splicing speckles. In addition to RNA recognition motifs (RRMs) the main characteristic of SR-proteins are disordered arginine and serine (RS) repeat regions. The intrinsically disordered RS-regions are thought to serve key roles in orchestrating self-organizing splicing networks within the nucleus. Our preliminary results suggest that SRSF1 (serine and arginine-rich splicing factor 1) and presumably other SR-proteins associate with non-coding (nc) RNAs, the highly structured 4.5SH RNA and Y RNAs. In contrast to Y RNAs the 4.5SH RNA is enriched in splicing speckles, suggesting that these ncRNAs modulate the association of SR-proteins within splicing speckles as well as other nuclear functions. We hypothesize that ncRNAs tether SR-proteins in splicing speckles, competing with their role in mRNA splicing. SRSF1 is upregulated in a variety of cancers, promoting and inhibiting pro-proliferative versus anti-apoptotic mRNA splice variants, respectively. This suggests that ncRNAs counteract the oncogenic functions of SR-proteins especially those of SRSF1. Aiming to unravel the function of SRSF1’s IDRs in ncRNA-dependent localization, mRNA/ncRNA association, and oncogenic potential, we will investigate: 1) RS-/RNA-dependent subcellular localization of SRSF1 by fluorescence microscopy including both, indirect immunostaining and fluorescence-recovery after photobleaching (FRAP); 2) RS-/RNA-dependent association of proteins by immunoprecipitation and mass spectrometry (MS); 3) RS-/RRM-dependent mRNA and ncRNA association by Cross-link RNA-immunoprecipitation (RIP/CLIP); 4) functional role of RS-/ncRNA-association in pre-mRNA splicing using RT-qPCR and RNA-sequencing; 5) role of RS-/ncRNA-association in determining SRSF1’s oncogenic potential by determining RS-/ncRNA-dependent regulation of apoptosis versus proliferation.
Sinz A.: Analysis of SRSF1 associated proteins by MS
Hüttelmaier S.: Analysis of the oncogenic potential of SR-proteins by e.g. migration, invasion and proliferation assays. Bioinformatics support in RIP/CLIP-studies and RNA-sequencing (splicing regulation).
Bley N.: Immunofluorescence, FRAP analyses and fluorescence in situ hybridization (FISH) experiments.