“IGF2BP1 phosphorylation regulates ribonucleoprotein condensate formation by impairing low-affinity protein and RNA interactions”

Abstract

The insulin-like growth factor 2 mRNA binding protein (IGF2BP1) is a conserved RNA-binding protein that regulates RNA stability, localization, and translation. IGF2BP1 is part of various ribonucleoprotein (RNP) condensates regulating RNA outputs. However, the mechanism that regulates its assembly into condensates remains unknown. Here we found, using proteomics, that IGF2BP1 phosphorylation at S181 in a disordered linker is regulated in a stress-dependent manner. Phosphomimetic mutations in two disordered linkers, S181E and Y396E, modulated RNP condensate formation by IGF2BP1 without impacting its binding affinity for RNA. Intriguingly, the S181E mutant, which lies in linker 1, impaired IGF2BP1 condensate formationin vitroand in cells, whereas a Y396E mutant in the second linker increased condensate size and dynamics. Structural approaches showed that the first linker binds RNAs nonspecifically through its RGG/RG motif, an interaction weakened in the S181E mutant. Notably, linker 2 interacts with IGF2BP1’s folded domains and these interactions were partially impaired in the Y396E mutant. Our data reveal how phosphorylation modulates low affinity interaction networks in disordered linkers to regulate RNP condensate formation.

Structural insights into IMP2 dimerization and RNA binding

“…Our data suggest that protein-protein and protein-RNA interactions occur among both IMP2 structured domains and unstructured linker regions. Further biological relevance and importance of these linker regions is exemplified by studies demonstrating how condensate formation is impacted by as little as a single amino acid modification positioned in these unstructured stretches (31). Especially in the context of IGF2BPs, these unstructured protein elements appear to be critical for RNA binding specificity, protein domain organization and multimeric state interactions (5, 32).…”

“…Differential HDX-MS experiments were conducted as previously described with a few modifications (31). Peptides were identified using tandem MS (MS/MS) with an Orbitrap mass spectrometer (Q Exactive, ThermoFisher).…”