Potential RNA binding functions for Shadoo through identification and characterisation of an RGG box motif

Abstract

Shadoo (Sho) is a member of the prion protein (PrP) family, found mainly in the brain. PrP is well-known as the central agent involved in the prion diseases - a form of fatal neurodegenerative disease. Despite decades of intense research, the natural functions of PrP have not been clearly elucidated. The reasons for this are complex, but it is likely that there is a level of redundancy in the functions performed by PrP. In particular, it has been suggested that Sho may have overlapping functions with PrP and that both proteins can compensate for one another. Therefore, investigating the natural function/s of Sho may provide some insight into the roles that the non-pathogenic form of PrP may play in vivo. Although the natural function of Sho is currently unknown, recent studies point to it having a role in neural tube development and neuroprotection. Comparative genomics is a powerful technique for providing insight into functionally important protein domains. Comparing Sho protein sequences over the course of evolution highlighted a strongly conserved sequence at the beginning of the N-terminus. Literature-based data mining led me to hypothesize that it may constitute an RGG box, a known RNA binding motif. This hypothesis provides an interesting link to PrP, which can bind RNA, although an RNA binding function has not yet been identified. However, the RNA binding region of PrP, also located at the beginning of the N-terminal region, does not have the characteristics of an RGG box. This thesis reports the results of a series of studies designed to test the hypothesis that Sho has an RGG box and establish the plausibility that Sho may play a functional role as an RNA-binding protein. The RGG box of Sho has strong sequence similarity to the RGG box of the Fragile X Mental Retardation Protein (FMRP), which is known to bind a range of mRNAs and play an important role in neural plasticity. Comparison of the RGG boxes of Sho and FMRP reveals that, like many RNA binding domains, the RGG boxes of Sho and FMRP lie within disordered protein domains. This work examines the nature of these flexible protein domains using molecular dynamics (MD) simulations. The RGG box of FMRP has an affinity for G-quadruplex RNA, which is also the form of RNA that binds most strongly to PrP. Here, MD simulations and biophysical experiments are used to investigate whether Sho, too, binds G-quadruplex RNA. Binding of 5 different RNA transcripts to the RGG box regions of Sho and FMRP and the N-terminus RNA binding domain of PrP was explored through circular dichroism, fluorescence and surface plasmon resonance experiments. The results of both MD simulations and biophysical experiments show that a peptide derived from the RGG box region of Sho is capable of binding G-quadruplex RNA with physiologically relevant affinity. The Sho RGG box peptide binds to certain RNA transcripts with similar affinity to peptides comprising the FMRP RGG box and the RNA-binding region of PrP. However, some differences in RNA-binding affinities across all three peptides also indicate their ability to discriminate between different RNA targets. Overall, the findings reported here suggest that Sho is likely to function, in some capacity, as an RNA-binding protein. As Sho and PrP are capable of binding G-quadruplex RNA with similar affinities, it is possible that they share an RNA binding function. Given the primary location of Sho and PrP on the outer cell membrane it seems most plausible that they bind extracellular RNA in a signaling context. This finding is particularly interesting as recent research indicates that RNA may be a cofactor in the conversion of PrP to its disease-producing isoform. There is also growing interest in the role of extracellular RNA as a target for cell surface receptors. Future studies to identify likely RNA binding partners for Sho provide a promising avenue for elucidating the natural function of this protein.