Abstract
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting in severe muscle weakness and eventual death by respiratory failure. Although little is known about its pathogenesis, mutations in fused in sarcoma/translated in liposarcoma (FUS) are causative for familial ALS. FUS is a multifunctional protein that is involved in many aspects of RNA processing. To elucidate the role of FUS in ALS, we overexpressed wild-type and two mutant forms of FUS in HEK-293T cells, as well as knocked-down FUS expression. This was followed by RNA-Seq to identify genes which displayed differential expression or altered splicing patterns. Pathway analysis revealed that overexpression of wild-type FUS regulates ribosomal genes, whereas knock-down of FUS additionally affects expression of spliceosome related genes. Furthermore, cells expressing mutant FUS displayed global transcription patterns more similar to cells overexpressing wild-type FUS than to the knock-down condition. This observation suggests that FUS mutants do not contribute to the pathogenesis of ALS through a loss-of-function. Finally, our results demonstrate that the R521G and R522G mutations display differences in their influence on transcription and splicing. Taken together, these results provide additional insights into the function of FUS and how mutations contribute to the development of ALS.
Highlights
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting upper and lower motor neurons causing progressive muscle weakness
To investigate the global effects of wild-type and mutant fused in sarcoma/translated in liposarcoma (FUS) protein on cellular transcription and splicing, HEK-293T cells were transfected with expression vectors encoding wild-type FUS, two FUS mutants (R521G and R522G) or small interfering RNA (siRNA) directed against FUS
Quality control assessments revealed that .69.9% of reads uniquely mapped to the genome and less than 2.72% of reads were derived from ribosomal RNA (Table S1 in File S1)
Summary
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting upper and lower motor neurons causing progressive muscle weakness. Mutations in FUS are detected in ,4% of FALS patients and infrequently in sporadic ALS (SALS) cases [7,8]. The FUS protein is 526 amino acids long and contains an N-terminal serine, tyrosine, glycine and glutamine (SYGQ)-rich region, an RNA-recognition motif (RRM), a C2/C2 zinc finger motif, multiple arginine, glycine, and glycine (RGG)-repeat regions and a nuclear localization signal (NLS) at the extreme C-terminus [8,10]. Even though mutations in FUS account for only a small fraction of FALS and SALS patients, it has been suggested that FUS protein may be a common component of cellular inclusions in non-SOD1 ALS and other neurodegenerative conditions [13]. Cytosolic mislocalization of FUS has already been shown to kindle misfolding of wild-type SOD1 in non-SOD1 ALS, implying a shared pathogenic pathway underlying SALS, non-SOD1 FALS, ALS/FTD, and related disorders [13,14]
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