The Truncated C-terminal RNA Recognition Motif of TDP-43 Protein Plays a Key Role in Forming Proteinaceous Aggregates

Yi-Ting Wang‡1, Pan-Hsien Kuo‡1, Chien-Hao Chiang‡§, Jhe-Ruei Liang‡¶, Yun-Ru Chen, Shuying Wang**‡‡,
James C. K. Shen‡, and Hanna S. Yuan‡§§2

From the ‡Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, the §Institute of Bioinformatics and Structural
Biology, National Tsing Hua University, Hsin Chu 30013, Taiwan, the ¶Department of Life Sciences, Institute of Genome Sciences,
National Yang-Ming University, Taipei 11221, Taiwan, the Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan, the
**Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan 70457, Taiwan, the
‡‡Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan 70457, Taiwan, and the
§§Graduate Institute of Biochemistry and Molecular Biology, National Taiwan University, Taipei 10048, Taiwan


* This work was supported by research grants from Academia Sinica (to H. Y. and J. C. K. S.) and the National Science Council (Taiwan) (to H. Y. and J. C. K. S.).
Author’s Choice—Final version full access.
□S This article contains supplemental Fig. 1.
1 Both authors contributed equally to this work.
2 To whom correspondence should be addressed: Inst. of Molecular Biology,
Academia Sinica, Taipei 11529, Taiwan. Tel.: 886-2-27884151; Fax: 886-2-
27826085; E-mail: hanna@sinica.edu.tw.
3 The abbreviations used are: CTF, C-terminal fragment; NTD, N-terminal domain; RRM, RNA recognition motif; SAXS, small angle x-ray scattering; PDB, Protein Data Bank; ThT, thioflavin T; tRRM2, truncated RRM
2

TDP-43 is the major pathological protein identified in the cellular inclusions in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The pathogenic forms of TDP-43 are processed C-terminal fragments containing a truncate RNA-recognition motif (RRM2) and a glycine-rich region. Although extensive studies have focused on this protein, it remains unclear how the dimeric full-length TDP-43 is folded and assembled and how the processed C-terminal fragments are misfolded and aggregated. Here, using size-exclusion chromatography, pulldown assays, and small angle x-ray scattering, we show that the C-terminal-deleted TDP-43 without the glycinerich tail is sufficient to form a head-to-head homodimer primarily via its N-terminal domain. The truncated RRM2, as well as two beta-strands within the RRM2, form fibrils in vitro with a similar amyloid-negative staining property to those of TDP-43 pathogenic fibrils in diseases. In addition to the glycine-rich region, the truncated RRM2, but not the intact RRM2, plays a key role in forming cytoplasmic inclusions in neuronal cells. Our data thus suggest that the process that disrupts the dimeric structure, such as the proteolytic cleavage of TDP-43 within the RRM2 that removes the N-terminal dimerization domain, may produce unassembled truncated RRM2 fragments with abnormally exposed beta-strands, which can oligomerize into high-order inclusions.

JBC_2013


Misfolding of TDP-43 by the processing within the RRM2 domain. A, the proteolytic processing within the RRM2 disrupts the dimeric structure of TDP-43 and produces truncated RRM2 fragments that can oligomerize into high-order inclusions. B, the truncated RRM2 fragments without the beta1 strand located in the center of the beta-sheet are misfolded with abnormally exposed beta-3 and beta-5 strands and Gly-rich region that may be further assembled intooligomers.
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