Titolo della tesi: The role of selected single-point mutations in the Fragile-X Messenger Ribonucleoprotein: from (mis)folding properties to Liquid-Liquid Phase Separation.
Fragile X Messenger Ribonucleoprotein (FMRP) is an RNA-binding protein implicated in cognitive development that plays a key role in RNA metabolism, and synaptic plasticity. FMRP is involved in Fragile X syndrome (FXS), a genetic disorder representing the most common inherited form of intellectual disability. FMRP has a complex architecture, consisting of a structured N-terminal domain (NTD) composed of two Tudor domains (TD), three K Homology (KH) domains, and an unstructured C-terminal arginine-glycine (RGG)-rich region. This multi-domain organization enables FMRP to be engaged in a wide range of interactions with numerous proteins and RNAs. Depletion or mutations in FMRP lead to fragile X syndrome (FXS). Usually, FXS results from the expansion of a CGG triplet in the 5' untranslated region (UTR) of the FMR1 gene, leading to its hypermethylation, transcriptional silencing, and thus loss of FMRP. However, cases of FXS-like phenotypes caused by small deletions and single nucleotide polymorphisms (SNPs) have also been reported. Notably, three clinically significant mutations (R138Q KH0, G266E in KH1, and I304N in KH2) reported so far occur within the KH domains, indicating their importance in the protein's physiological function.
This work focuses on characterizing the folding and aggregation properties of the isolated KH0 and KH1 domains. Moreover, the impact of the corresponding single-point mutation FXS-related (R138Q and G266E, respectively) on secondary structure and thermodynamics stability was analyzed.
Interestingly, the R138Q substitution does not alter the secondary structure of KH0 but affects its thermodynamic stability and increases its aggregation propensity. By contrast, a combination of Circular Dichroism, Small-angle X-ray scattering, and One-dimensional nuclear magnetic resonance techniques revealed that substituting glycine 266 with glutamic acid leads to the complete unfolding of the KH1 domain in solution.
Furthermore, considering FMRP's involvement in stress granules and liquid-liquid phase separation (LLPS), particularly due to the role of its unstructured C-terminal region, we demonstrate that its structured regions can also actively contribute to LLPS.
In conclusion, we show that FXS-related single-point mutations significantly affect the thermodynamics stability and the balance between phase separation and amyloid aggregation in the multidomain context of FMRP.
These insights provide a crucial basis for future investigations into how these mutations influence the behavior of the complete, multi-domain FMRP and its role in RNA granule dynamics.