Fragile X Mental Retardation Protein (FMRP) is an RNA-binding protein (RBP) known to control different steps of mRNA metabolism, even though its complete function is not fully understood yet. It is well established that FMRP has a multi-domain architecture, characterized by the presence of 2 Tudor Domains (TD), three K Homology (KH) Domains and an arginine glycine-rich (RGG) box, feature that allows this RBP to be engaged in a large interaction network with numerous proteins, mRNAs or non-coding RNAs. Lack or mutations of FMRP lead to Fragile X Syndrome (FXS). Especially, beside the most common form due to the expansion of a CGG triplet located in the 5¿UTR of the gene encoding for FMRP (FMR1), there are Small deletions and single-nucleotide polymorphisms (SNPs) in critical domains of the FMR1 gene that are reported to cause FXS-like phenotypes. Interestingly, three of the four clinically relevant reported mutations fall in the KH domains, suggesting a particular important role for these domains in the physiological function of the protein. In this project we aim at characterizing the three KH domains that constitute the FMRP protein and their pathological mutants. In particular our goal is two-fold: 1) to solve the crystal structure of the three KH domain both in isolation and linked together and to unveil the structural rearrangements leading to cooperative RNA binding. The possibility to solve the structure of the KH domains in complex with the target RNA will also be evaluated. 2) to study the folding mechanism of these KH domains both in isolation and linked together and to characterize the folding intermediates (if any) which may be relevant in the reported ability of the KH domains to form ordered aggregates such as beta-amyloids. We believe that this study will be useful to a better comprehension of the features of the FMRP protein and will pave the way to further studies aimed at fighting FXS and FXS correlated pathologies.
FMRP is a multitasking binding protein able to interact and mediate the action of a large number and typologies of nucleic acids (predominantly RNA) and proteins, determining the subsequent homeostasis of a huge amount of biological processes for examples in the neural cells of the brain [1,2].
It is therefore clear that the study of the functions, structure and the characterization of the binding partners of FMRP is important to allow a better comprehension of the phenomena in which FMRP is implicated, paving the way to the development of new strategies against FXS. Moreover, the discovery of patients with FXS-like phenotypes without the canonical CGG expansion in the FMR1 gene, but with single mutation in key domains, pointed out the possibility to the development of strategies aimed at rescuing the protein function. In fact, differently from the classic FXS, in this case the protein is present but not functional. Very interestingly, in the latter case three of the four mutation responsible to cause FXS-like phenotypes are placed in the KH domains [3]which are important RNA binding domains diffuse from prokaryote to eukaryote [4]. This evidence highlights the particular importance of the KH domains in the protein functions, in particular in the binding of some of its RNA partners as demonstrated in the case of I304N mutation that disrupt KH2 RNA-binding ability [5].
Surprisingly, to date there are no folding studies of the FMRP KH domains, but only analyses about the difference in stability of the pathological mutants. Moreover, there are no 3D structures of the FMRP KH domains linked together or in complex with one of their numerous binding partners.
Our aim is to fill this gap, providing a folding analysis of the FMRP KH domains and pathological mutants and attempting at solving their structures, also in complex with their RNA partners.
The folding analysis on the wild type and mutant FMRP KH domains could allow an overview of the folding mechanism that will be instrumental for better understanding of the binding mechanism of the KH domains with RNA.
Moreover, since it is reported that a region spanning from the N-terminal to the central domain of FMRP is able to form ordered aggregates [6] , our interest is also to unveil the specific ability of the KH domains to form ¿-fibrils. This feature, linked to the presence of mutations, could shed light on the pathogenesis of FXS-like phenotypes but also on other FXS correlated pathologies like FXTAS, that are in turn characterized by overexpression of FMRP.
On the other, solving the 3D structure of the three KH domain linked together or in complex with one of its RNA binding partner (i.e. kissing steep loop) could elucidate the specific binding mechanism of FMRP KH domains as well as understand how the domains arrange each other¿s in the space, information that, as already said, are central to a better comprehension of the FMRP physiological functions.
Taken together, we believe that this research will provide relevant information about FMRP features and functions paving the way to further studies aimed at the development of new strategies against FXS or FXS correlated pathologies.
References
[1] A. Banerjee, M.F. Ifrim, A.N. Valdez, N. Raj, G.J. Bassell, , Brain Res. 1693 (2018) 24¿36.
[2] T.C. Dockendorff, M. Labrador, Mol. Neurobiol. 56 (2019) 711¿721.
[3] A. Tekcan, In Silico Analysis of FMR1 Gene Missense SNPs, Cell Biochem. Biophys. 74 (2016) 109¿127.
[4] G. Nicastro, I.A. Taylor, A. Ramos, Curr. Opin. Struct. Biol. 30 (2015) 63¿70.
[5] J.C. Darnell, C.E. Fraser, O. Mostovetsky, G. Stefani, T.A. Jones, S.R. Eddy, R.B. Darnell, , Genes Dev. 19 (2005) 903¿918.
[6] L. Sjeklo¿a, K. Pauwels, A. Pastore, FEBS J. 278 (2011) 1912¿1921.