Understanding how multi-domain RNA binding proteins discriminate unspecific and specific targets to regulate alternative splicing using integrative structural cell biology

• Applicants: Professorin Michaela Müller-McNicoll; Professor Dr. Andreas Schlundt
• Subject Area: Biochemistry ; Biophysics ; Structural Biology ; Cell Biology
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 583887849

In humans, the majority of genes undergo alternative splicing, which is crucial for cellular identity and function. In a fine-tuned but incompletely understood process, splice sites are selected by serine/arginine-rich splicing factors (SRSFs), a family of essential multifunctional RNA binding proteins. Splice site mutations or misregulation of SRSF levels disrupts correct splicing patterns, which is associated with a plethora of diseases including multiple cancers. The SRSF family comprises 12 canonical members (SRSF1-12), each harboring one or two RNA recognition motifs (RRMs), a glycine/arginine-rich linker region, and a C-terminal unstructured region rich in arginine-serine repeats (RS domain); a target of complex regulatory phosphorylation. In this proposal we address two central open questions in the field: 1) How can SRSFs bind specific targets and promote alternative splicing despite their common domain structure, similar RNA binding preferences and abundant presence in the same cellular compartment. And 2) How can the same SRSF promote constitutive splicing of most exons in a partially redundant fashion with other SRSFs and regulate alternative splicing of a small number of targets at the same time? We speculate that differential contributions of individual protein domains of SRSFs that induce subtle changes in RNA binding specificities contribute to this phenomenon, but also differences in their cellular mobility and activity play an important role, but are usually overlooked. To address these questions our groups will combine structural and cell biology methods to determine the mechanisms of RNA binding and function of the essential splicing factor SRSF6. We will provide the molecular and structural basis for its RNA specificity, delineate the specific contributions of its two RRMs, the inter-domain linker and length/composition of the unstructured phosphorylated RS domain in vitro and in vivo, and probe the role of SRSF6 nuclear mobility in alternative splicing decisions. We will address apparent discrepancies from in vitro vs. in vivo derived RNA binding data and include the systematic comparison of SRSF6 with SRSF1, because we expect a competition between both proteins. With this combined approach, we will decipher the code of target specificity of SRSF6 and its functionality in alternative splicing, which is not understood. Moreover, through the comparison with SRSF1 we will derive general rules for the entire SR protein family.

• DFG Programme: Research Grants