Approaches to Understanding Protein-RNA Binding: TDP-43

Frontotemporal lobular dementia (FTLD) is one of the most prevalent neurodegenerative diseases today, and spontaneously occurs in individuals with no known discrimination for sex, age, or any other demographic category (Young et al., 2018).  Transactive response DNA Binding Protein 43 kDA (TDP-43) has been implicated in FTLD (Geser et al., 2009).  The current mechanism for this is currently unknown.  TDP-43 is capable of binding DNA and has been observed to participate in many aspects of RNA-metabolism, such as transcription, translation, mRNA transport and mRNA stabilization.  This ability to bind to nucleotide sequences is due to the presence of RNA-recognition motifs (RRMs), which are in-between the N-terminal and C-terminal domains (NTD and CTD respectively).  TDP-43 facilitates RNA-metabolism by binding to sequences rich in (UG)¬¬n and shuttling the RNA molecules (Lukavsky et al., 2013).  This is possible due to a nuclear localization signal (NLS) and a nuclear export signal (NES), in addition to the RRMs.  While structural data does exist of these domains, a native full-length protein has not been characterized. 

The difficulty of this characterization is partially due to the disorder the glycine-rich CTD, which aids in phase separation of the protein to allow inclusion in liquid-liquid phase separated droplets.  Very few studies have been focused on the computational analysis of the RRMs bound to RNA, leaving a gap in our understanding of RNA-protein binding outside of experimentation.  In this research, HDOCK, the AMBER Suite, Pymol and Chimera were utilized to model the binding of RNA sequences shown to bind to the RRMs of TDP-43 (Lukavsky et al., 2013).  Additionally, titration assays were performed to investigate the binding TDP-43 and RNA sequences.  To aid in the applicability of this research to biologically relevant studies, a procedure is being developed to the eGFP from our construct of TDP-43.