Abstract
Amyotrophic lateral sclerosis (ALS) is associated with progressive degeneration of motor neurons. Several of the genes associated with this disease encode proteins involved in RNA processing, including fused-in-sarcoma/translocated-in-sarcoma (FUS/TLS). FUS is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins that bind thousands of pre-mRNAs and can regulate their splicing. Here, we have examined the possibility that FUS is also a component of the cellular response to DNA damage. We show that both GFP-tagged and endogenous FUS re-localize to sites of oxidative DNA damage induced by UVA laser, and that FUS recruitment is greatly reduced or ablated by an inhibitor of poly (ADP-ribose) polymerase activity. Consistent with this, we show that recombinant FUS binds directly to poly (ADP-ribose) in vitro, and that both GFP-tagged and endogenous FUS fail to accumulate at sites of UVA laser induced damage in cells lacking poly (ADP-ribose) polymerase-1. Finally, we show that GFP-FUSR521G, harbouring a mutation that is associated with ALS, exhibits reduced ability to accumulate at sites of UVA laser-induced DNA damage. Together, these data suggest that FUS is a component of the cellular response to DNA damage, and that defects in this response may contribute to ALS.
Highlights
Amyotrophic lateral sclerosis (ALS) is caused by progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex
We show that FUS is recruited to chromosomal sites of oxidative DNA damage, and that this recruitment is reduced by a mutation that is associated with ALS
A clone containing the human FUS open reading frame was obtained from Source Bioscience and this was subcloned into peGFP-C3 using polymerase chain reaction (PCR) primers tacgtcgactATGgcctcaaacgatt and cttggatccttTTAa tacggcctctc (SalI and BamHI sites underlined, start and stop codons in upper case)
Summary
Amyotrophic lateral sclerosis (ALS) is caused by progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. The mutated proteins in ALS may aggregate and sequester normal proteins, or may inactivate protein complexes in which they are involved, reducing and/or disrupting critical cellular processes necessary for neural function and maintenance. Consistent with this idea, it has emerged recently that several of the genes associated with ALS encode RNA binding proteins involved in mRNA splicing, polyadenylation and stability [4,5]. FUS depletion generates elevated levels of RNAP II harbouring a Ser2-phosphorylated C-terminal domain at transcription start sites [10] This posttranslational modification is associated with elongating RNAP II and is enriched at the 30-termini of genes
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