Nuclear egress of TDP-43 and FUS occurs independently of Exportin-1/CRM1

Abstract

TDP-43 and FUS are nuclear proteins with multiple functions in mRNA processing. They play key roles in ALS (amyotrophic lateral sclerosis) and FTD (frontotemporal dementia), where they are partially lost from the nucleus and aggregate in the cytoplasm of neurons and glial cells. Defects in nucleocytoplasmic transport contribute to this pathology, hence nuclear import of both proteins has been studied in detail. However, their nuclear export routes remain poorly characterized and it is unclear whether aberrant nuclear export contributes to TDP-43 or FUS pathology. Here we show that predicted nuclear export signals in TDP-43 and FUS are non-functional and that both proteins are exported independently of the export receptor CRM1/Exportin-1. Silencing of Exportin-5 or the mRNA export factor Aly/REF, as well as mutations that abrogate RNA-binding do not impair export of TDP-43 and FUS. However, artificially enlarging TDP-43 or FUS impairs their nuclear egress, suggesting that they could leave the nucleus by passive diffusion. Finally, we found that inhibition of transcription causes accelerated nuclear egress of TDP-43, suggesting that newly synthesized RNA retains TDP-43 in the nucleus, limiting its egress into the cytoplasm. Our findings implicate reduced nuclear retention as a possible factor contributing to mislocalization of TDP-43 in ALS/FTD.

FUS/TLS undergoes calcium-mediated nuclear egress during excitotoxic stress and is required for Gria2 mRNA processing

“…4B). Our KPT-330 studies suggest that Glu excito -induced FUS egress occurs through a mechanism other than active CRM1 export, and could entail passive diffusion 30 or alternative transport factors 57 . Selectivity of RBP egress following Glu excito may stem from differences in nucleocytoplasmic shuttling dynamics, which are influenced by multiple factors including binding interactions and post-translational modifications 58 .…”

“…To understand the mechanism(s) underlying endogenous FUS egress in response to Glu excito , we began with an examination of nucleocytoplasmic transport factors. FUS contains two predicted chromosome region maintenance 1 (CRM1)-dependent nuclear export sequences (NES) within the RNA-recognition motif 30 . CRM1 is a major protein export factor, although whether this receptor controls nuclear FUS export is controversial 30,31 .…”

“…FUS contains two predicted chromosome region maintenance 1 (CRM1)-dependent nuclear export sequences (NES) within the RNA-recognition motif 30 . CRM1 is a major protein export factor, although whether this receptor controls nuclear FUS export is controversial 30,31 . To determine if excitotoxic FUS egress is CRM1-dependent, we pretreated neurons with the CRM1 inhibitor, KPT-330, prior to treatment with Glu excito32 .…”

Comprehensive analysis of nuclear export of herpes simplex virus type 1 tegument proteins and their Epstein‐Barr virus orthologs

“…One should, however, keep in mind that mutating hydrophobic residues of a putative NES may disrupt the overall conformation of a given protein thereby disrupting various functions including the binding to a partner protein that may be critical for indirect nuclear export. To avoid the misidentification of NESs as previously reported, further validation is important. To this end, the ability of a peptide NES to mediate nuclear export of an unrelated protein, and to bind CRM1/Xpo1 in a Ran‐GTP‐dependent manner can be tested.…”

“…Here, a protein X to be tested for nuclear export is fused to EYFP for visualization, to the classical SV40 NLS for constitutive nuclear import and to the FK506‐Rapamycin (FR)‐binding domain (FRB) for dimerization. The EYFP‐NLS‐FRB‐Protein X in general exceeds the diffusion limit of the nuclear pore disabling its passive export . The reporter protein gM‐FKBP is based on a fusion of HSV1 glycoprotein M (gM), a resident integral membrane protein of the trans‐ Golgi network (TGN), and three tandem repeats of the immunophilin FK506‐binding protein‐12 (FKBP) exposed to the cytoplasm.…”