Arginine methylation by PRMT1 regulates nuclear-cytoplasmic localization and toxicity of FUS/TLS harbouring ALS-linked mutations

Abstract

Mutations in FUS/TLS (fused in sarcoma/translated in liposarcoma) cause an inheritable form of amyotrophic lateral sclerosis (ALS6). In contrast to FUS(WT), which is concentrated in the nucleus, these mutants are abnormally distributed in the cytoplasm where they form inclusions and associate with stress granules. The data reported herein demonstrate the importance of protein arginine methylation in nuclear-cytoplasmic shuttling of FUS and abnormalities of ALS-causing mutants. Depletion of protein arginine methyltransferase 1 (PRMT1; the enzyme that methylates FUS) in mouse embryonic fibroblasts by gene knockout, or in human HEK293 cells by siRNA knockdown, diminished the ability of ALS-linked FUS mutants to localize to the cytoplasm and form inclusions. To examine properties of FUS mutants in the context of neurons vulnerable to the disease, FUS(WT) and ALS-linked FUS mutants were expressed in motor neurons of dissociated murine spinal cord cultures. In motor neurons, shRNA-mediated PRMT1 knockdown concomitant with the expression of FUS actually accentuated the shift in distribution of ALS-linked FUS mutants from the nucleus to the cytoplasm. However, when PRMT1 was inhibited prior to expression of ALS-linked FUS mutants, by pretreatment with a global methyltransferase inhibitor, ALS-linked FUS mutants were sequestered in the nucleus and cytoplasmic inclusions were reduced, as in the cell lines. Mitochondria were significantly shorter in neurons with cytoplasmic ALS-linked FUS mutants, a factor that could contribute to toxicity. We propose that arginine methylation by PRMT1 participates in the nuclear-cytoplasmic shuttling of FUS, particularly of ALS6-associated mutants, and thus contributes to the toxic gain of function conferred by these disease-causing mutations.

Methylation of the nuclear poly(A)-binding protein by type I protein arginine methyltransferases – how and why

“…As in the case of PABPN1, the binding of FUS to transportin was reduced by arginine methylation (Fronz et al , 2011 ;Dormann et al , 2012 ). The inhibition of methylation had no detectable effect on the nuclear import of wild-type FUS (Dormann et al , 2012 ;Tradewell et al , 2012 ). Thus, like the data on S. pombe Pab2, the results suggest that the concentration of transportin in the cytoplasm is sufficiently high that arginine methylation has little effect on nuclear import unless a cargo mutation weakens the affinity for transportin.…”

“…FUS is known to carry asymmetrically dimethylated arginine residues; in contrast to PABPN1, these are not in the canonical motifs of the transportin-binding site but in an adjacent RGG domain (Rappsilber et al , 2003 ). The inhibition of arginine methylation by the use of the general inhibitor adenosine dialdehyde, knockdown of PRMT1, or deletion of the PRMT1 gene rescued the nuclear import of FUS when it was compromised by mutations in its PY-NLS (Dormann et al , 2012 ;Tradewell et al , 2012 ). As in the case of PABPN1, the binding of FUS to transportin was reduced by arginine methylation (Fronz et al , 2011 ;Dormann et al , 2012 ).…”

“…For this and other reasons, it is unknown whether the effect of methylation is direct or indirect. In this context, it should be pointed out that even the data on the effect of arginine methylation on the localization of FUS may not be as clear-cut as it may seem: although there is agreement that the introduction of FUS mutated in its PY-NLS into cells incapable of arginine methylation rescues the protein ' s nuclear localization, one study reported that the introduction of mutant FUS into cells simultaneously with an shRNA-depleting PRMT1 had the opposite effect, leading to enhanced cytoplasmic localization (Tradewell et al , 2012 ). This puzzling observation has not been further explored and remains unexplained.…”

The FUS about arginine methylation in ALS and FTLD

“…In recent times, it has been established that this arginine methylation can affect their nuclear localization (Araya et al, 2005;Jobert et al, 2009;Tradewell et al, 2012). In this issue, Dormann et al (2012) sought to delve further into the mechanism by which this occurs.…”

Phosphorylation of C-terminal tyrosine 526 in FUS impairs its nuclear import

“…It has been previously reported that FUS undergoes arginine methylation in arginine-glycine-glycine (RGG) domains near to the NLS that impairs TNPO1 binding to RGG domains, inhibiting nuclear import (Rappsilber et al, 2003;Hung et al, 2009;Dormann et al, 2012;Tradewell et al, 2012;Yamaguchi and Kitajo, 2012;Scaramuzzino et al, 2013). Finally, phosphorylation of the C-terminal Y656 residue within the NLS of the EWSR1 protein has been shown to inhibit nuclear import (Leemann-Zakaryan et al, 2011).…”