Abstract
526-residue FUS functions to self-assemble into reversible droplets/hydrogels, which could be further solidified into pathological fibrils. FUS is intrinsically prone to aggregation, composed of N-terminal low-sequence complexity (LC); RNA-recognition motif (RRM) and C-terminal LC domains. Intriguingly, previous in vivo studies revealed that its RRM is required for manifesting FUS cytotoxicity but the underlying mechanism remains unknown. Here, we characterized solution conformations of FUS and its five differentially dissected fragments, followed by detailed investigations on thermal unfolding, NMR dynamics and self-assembly of RRM. The results decipher: (1) the N- and C-terminal LC domains are intrinsically disordered, while RRM is folded. Intriguingly, well-dispersed HSQC peaks of RRM disappear in the full-length FUS, reminiscent of the previous observation on TDP-43. (2) FUS RRM is characteristic of irreversible unfolding. “Model-free” analysis of NMR relaxation data decodes that RRM has high ps-ns conformational dynamics even over some residues within secondary structure regions. (3) RRM spontaneously self-assembles into amyloid fibrils. Therefore, in addition to the well-established prion-like region, FUS RRM is also prone to self-assembly to form amyloid fibrils. Taken together, FUS RRM appears to play a crucial role in exaggerating the physiological/reversible self-assembly into pathological/irreversible fibrillization, thus contributing to manifestation of FUS cytotoxicity.
Highlights
Fused in Sarcoma/Translocated in Sarcoma (FUS) consisting 526 residues is encoded by a gene which was first identified as a fusion oncogene in human liposarcomas1, 2
As we previously observed on other aggregation-prone proteins21–24 such as TDP43 N-domain23 and C-terminal prion-like domain24, the purified full-length FUS and its low-sequence complexity (LC) domains were highly soluble and stable in Milli-Q water at pH 4.0, and this allowed us to prepare the protein samples in 1 mM phosphate buffers at pH 5.0 and 6.8 respectively by quickly diluting their stock samples in Milli-Q water to phosphate buffers as we used for studying TDP43 prion-like domain24
We acquired and analyzed the CPMG-based relaxation dispersion data, and we found no residue with significant relaxation dispersion, suggesting that significant μs-ms conformational exchanges are absent for the FUS RNA-recognition motif (RRM) domain, or/and that the chemical shift differences between exchanging states are too small to be detected by CPMG-based relaxation dispersion experiments31
Summary
Fused in Sarcoma/Translocated in Sarcoma (FUS) consisting 526 residues is encoded by a gene which was first identified as a fusion oncogene in human liposarcomas . Genetic variants in the FUS gene have been identified as causative or risk factors for ALS, essential tremor and rare forms of FTLD11–15. These findings suggest that FUS might have a general role in neurodegenerative diseases. FUS is a multi-domain protein intrinsically prone to aggregation, which is composed of an N-terminal low-sequence complexity (LC) domain [1–267] including a QGSY-rich prion-like region [1–165] and a G-rich region [166–267]; an RNA-recognition motif (RRM: 285–371) capable of binding a large array of RNA and DNA1, 16, 17; and C-terminal LC domain [371–526] including a RGG repeat region and a highly conserved nonclassical nuclear localization signal (Fig. 1A). Our study provides key biophysical insights into the role of RRM in solidifying the FUS self-assembly, which may rationalize its essentiality in manifesting FUS cytotoxicity [20]
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