Requirements for Stress Granule Recruitment of Fused in Sarcoma (FUS) and TAR DNA-binding Protein of 43 kDa (TDP-43)

Eva Bentmann,Manuela Neumann,Sabina Tahirovic,Ramona Rodde,Dorothee Dormann,Christian Haass

doi:10.1074/jbc.m111.328757

Abstract

Cytoplasmic inclusions containing TAR DNA-binding protein of 43 kDa (TDP-43) or Fused in sarcoma (FUS) are a hallmark of amyotrophic lateral sclerosis (ALS) and several subtypes of frontotemporal lobar degeneration (FTLD). FUS-positive inclusions in FTLD and ALS patients are consistently co-labeled with stress granule (SG) marker proteins. Whether TDP-43 inclusions contain SG markers is currently still debated. We determined the requirements for SG recruitment of FUS and TDP-43 and found that cytoplasmic mislocalization is a common prerequisite for SG recruitment of FUS and TDP-43. For FUS, the arginine-glycine-glycine zinc finger domain, which is the protein's main RNA binding domain, is most important for SG recruitment, whereas the glycine-rich domain and RNA recognition motif (RRM) domain have a minor contribution and the glutamine-rich domain is dispensable. For TDP-43, both the RRM1 and the C-terminal glycine-rich domain are required for SG localization. ALS-associated point mutations located in the glycine-rich domain of TDP-43 do not affect SG recruitment. Interestingly, a 25-kDa C-terminal fragment of TDP-43, which is enriched in FTLD/ALS cortical inclusions but not spinal cord inclusions, fails to be recruited into SG. Consistently, inclusions in the cortex of FTLD patients, which are enriched for C-terminal fragments, are not co-labeled with the SG marker poly(A)-binding protein 1 (PABP-1), whereas inclusions in spinal cord, which contain full-length TDP-43, are frequently positive for this marker protein.

Highlights

Stress granules (SG) have been implicated in the formation of pathological Fused in sarcoma (FUS) and TAR DNA-binding protein of 43 kDa (TDP-43) inclusions
We investigated a variety of stress conditions such as heat shock (44 °C), oxidative stress caused by sodium arsenite treatment, and mitochondrial stress caused by clotrimazole treatment for their ability to induce SG formation
RNA binding of TDP-43 depends on the N-terminal RRM1 domain but not the C-terminal glycine-rich domain. Because both domains are required for SG recruitment of TDP-43, we suggest that RNA binding plus additional features encoded in the C-terminal glycine-rich domain such as protein-protein interactions might contribute to SG recruitment of TDP-43

Summary

Background

Stress granules (SG) have been implicated in the formation of pathological FUS and TDP-43 inclusions. TDP-43 contains a C-terminal glycine-rich domain that mediates interactions with other heterogeneous nuclear ribonucleoproteins and is required for splicing regulation (13) This domain is highly aggregation-prone (14 –18) and due to its amino acid composition has been suggested to have prion-like properties (19 –22). Even though it is still unclear how reduced nuclear import of FUS leads to neurodegeneration, it has been shown that blockade of Transportin-mediated nuclear import or FUS mutations leads to recruitment of FUS into stress granules (SG), implicating SG and reduced nuclear transport in disease pathogenesis (33, 34, 36, 40, 41) This is supported by the presence of SG markers in inclusions in ALS/FTLD-FUS patients (33, 42). We analyzed the effect of various forms of ALS-associated TARDBP mutations on SG recruitment of TDP-43 and further investigated the presence of SG marker proteins in TDP-43 inclusions in ALS/FTLD-TDP cortex and spinal cord

EXPERIMENTAL PROCEDURES

RESULTS

NLSmut RRM1

DISCUSSION