The N-terminal dimerization is required for TDP-43 splicing activity

Abstract

TDP-43 is a nuclear factor that functions in promoting pre-mRNA splicing. Deletion of the N-terminal domain (NTD) and nuclear localization signal (NLS) (i.e., TDP-35) results in mislocalization to cytoplasm and formation of inclusions. However, how the NTD functions in TDP-43 activity and proteinopathy remains largely unknown. Here, we studied the structure and function of the NTD in inclusion formation and pre-mRNA splicing of TDP-43 by using biochemical and biophysical approaches. We found that TDP-43 NTD forms a homodimer in solution in a concentration-dependent manner, and formation of intermolecular disulfide results in further tetramerization. Based on the NMR structure of TDP-43 NTD, the dimerization interface centered on Leu71 and Val72 around the β7-strand was defined by mutagenesis and size-exclusion chromatography. Cell experiments revealed that the N-terminal dimerization plays roles in protecting TDP-43 against formation of cytoplasmic inclusions and enhancing pre-mRNA splicing activity of TDP-43 in nucleus. This study may provide mechanistic insights into the physiological function of TDP-43 and its related proteinopathies.

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing

“…However, the importance of the details of the superhelical polymer is unknown as the functionally relevant range of polymer lengths populated in cells remains to be determined. The relatively weak NTD isodesmic K D we measure here, in general agreement with other NMR and biophysical studies (Jiang et al , ; Mompean et al , ), suggests that even at the high concentrations estimated for phase separated assemblies of TDP‐43 (1 mM ~ 50 mg/ml) (Schmidt & Rohatgi, ), the great majority of the NTD would be expected to be incorporated in oligomers < 10mer (Appendix Fig S1B). This size distribution favoring low‐order polymers is consistent with dynamic rearrangements expected for liquid‐like behavior that is observed for TDP‐43 assemblies by fluorescence recovery after photobleaching (FRAP) (Schmidt & Rohatgi, ).…”

“…The RNA‐binding‐deficient mutant F147/149L (Buratti & Baralle, ) used as control failed to rescue TDP‐43 activity. Compared to wild‐type, S48E showed approximately 40% reduction in regulatory function (Fig I), similar to other variants disrupting TDP‐43 NTD assembly, as recently demonstrated (Afroz et al , ; Jiang et al , ; Mompean et al , ). The corresponding Ala substitution (S48A) also decreased TDP‐43 splicing regulatory activity, by approximately 25%, consistent with decreased self‐association in vitro (see below).…”

Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2

“…Recent findings have shown that the dimerization of TDP-43 through the N-terminal domain helps block TDP-43 aggregation by separating the LCD from neighboring copies of the protein, thus preventing amyloid formation 60–62 . Additionally, the structure of the mouse RRM2 domain, which shares 88% sequence identity with the human RRM2, is folded and highly stable.…”

Isolation and characterization of soluble human full‐length TDP‐43 associated with neurodegeneration

“…In the various structural studies so far reported, the isolated NTD domain was found to adopt either a dimeric (34,39,55,56) or oligomeric (18,19,57,58) state. Moreover, it was suggested that the native (nonmisfolded) full-length protein might adopt a complex oligomeric state because of the ability of the CTD to increase the oligomerization state of the protein (18,19).…”