TDP-43 and FUS–structural insights into RNA recognition and self-association
Introduction
The life cycle of RNA is finely orchestrated by RNA-binding proteins which coat nascent RNAs and regulate their processing, localization, functions and, ultimately, their degradation. TAR DNA-binding protein 43 (TDP-43) and Fused in Sarcoma (FUS) are multifunctional RNA-binding proteins with essential functions in post-transcriptional gene expression and the ability to contribute to RNP granule formation via an RNA-dependant self-association [1]. RNA granules such as P-bodies, stress granules and paraspeckles (also known as ‘membraneless organelles’) are formed in part through liquid–liquid phase separation (LLPS), a process similar to the de-mixing of oil and water and are vital for RNA metabolism [2••]. Numerous mutations in each of TDP-43 and FUS have been identified in cases of Amyotrophic Lateral Sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). These proteins are also present in mutually exclusive aggregates found in both familial and sporadic disease cases [3]. Although the molecular mechanisms of neurotoxicity are poorly understood, high protein concentrations within RNA granules are widely hypothesized to promote the formation of irreversible pathological aggregates [1]. Thus, the ability of both TDP-43 and FUS proteins to undergo self-association and the role of RNA in modulating this phenomenon potentially links essential biological functions with dysregulation of RNA metabolism and pathological aggregate formation in ALS and FTLD [2••].
TDP-43 and FUS are often described as similar proteins—with commonalities in their domain composition, GU-rich RNA-binding preferences and association with ALS and FTLD. Each protein possesses a Prion-like domain (PrLD)—an intrinsically disordered region with amino acid composition similar to yeast prion proteins and an RNA recognition motif (RRM). Distinctively, TDP-43 also possesses a globular oligomerization N-terminal domain (NTD) whereas FUS possesses an RNA-binding zinc finger (ZnF) and three intrinsically disordered Arg–Gly-rich domains (RGGs) which bind both RNA, protein partners and contribute to self-association (Figure 1). Recent structural analyses of these proteins provide extensive inroads towards understanding both common and distinctive molecular mechanisms of RNA binding, LLPS and labile cross-β sheet mediated aggregation. These lay the foundation for a better molecular understanding of both their function and malfunction underlying disease (Table 1).
Section snippets
The structural basis of RNA recognition by TDP-43
TDP-43 is involved in multiple steps of RNA processing, for which RNA binding is essential [1]. For example, TDP-43 has a well-established role in regulating alternative splicing of cystic fibrosis transmembrane regulator (CFTR) exon9 [4] and tightly regulates its own expression levels through binding to its own 3′UTR [5]. Genome wide studies show that TDP-43 binds thousands of genes and regulates alternative splicing of many of these targets, binding long UG tracts in the order of ∼30–100
Biological oligomerization by the NTD of TDP-43
TDP-43 is well known as an aggregation prone protein frequently observed in neurodegenerative pathologies. More recently the mechanism and potential functions of a native oligomerization domain have been elucidated. The N-terminal DIX-like domain [25, 26, 27] mediates a native nuclear oligomeric state of TDP-43 in a head to tail fashion which is distinct from that observed in pathological cytoplasmic aggregates (Figure 3a). The interface is largely polar with a low micromolar affinity of
Role of RNA in the self-association of FUS and TDP-43
Finally—cellular RNA that regulate the formation and identity of many RNA granules also modulates the self-association of TDP-43 and FUS. In vitro, substoichiometric amounts of RNA can promote LLPS of FUS acting as a seed, whereas stoichiometric and excess RNA stabilizes both FUS and TDP-43, potentially due to a structural rearrangement [12,30,35]. The same trends are observed in vivo, in which the high RNA content of the nucleus solubilizes FUS, whereas in the cytoplasm FUS easily forms
Conclusion and outlook
The RNA-binding proteins TDP-43 and FUS have many similarities, yet a detailed understanding of their molecular mechanisms in RNA binding and self-association reveals unique characteristics. RNA recognition is achieved by RRM domains of TDP-43 that cooperate to recognize short stretches of GU rich sequences with high affinity and specificity. In contrast, RRM, ZnF and RGG domains of FUS recognize bi-partite RNA sites through a combination of sequence and shape recognition, in which
Conflict of interest statement
Nothing declared.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgement
This work was supported by National Health and Medical Research Council, Australia NHMRC project grant AP1105801 awarded to JA Wilce.
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2022, Neurobiology of DiseaseCitation Excerpt :Thus, the effect of ALS-associated TDP-43 mutations on SGs may be the key to understanding the role of TDP-43 in ALS. FUS is a member of the FET family of proteins, which included FUS, EWSR1, and TAF15 (Law et al., 2006; Loughlin and Wilce, 2019). The domains described in Part 2.3 are necessary to mediate the interaction between FUS and DNA, RNA, or protein (Deng et al., 2014; Guo and Shorter, 2015; Murray et al., 2017).