Author ORCID Identifier
Year of Publication
Doctor of Philosophy (PhD)
Toxicology and Cancer Biology
Dr. Haining Zhu
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron death and subsequent muscle atrophy. Approximately 15% of ALS cases are inheritable, and mutations in the Fused in Sarcoma (FUS) gene contribute to approximately 5% of these cases, as well as about 2% of sporadic cases. FUS performs a diverse set of cellular functions, including being a major regulator of RNA metabolism. FUS undergoes liquid- liquid phase transition in vitro, allowing for its participation in stress granules and RNA transport granules. Phase transition also contributes to the formation of cytoplasmic inclusions found in the cell bodies of FUS ALS patients motor neurons. The nature of these inclusions has remained elusive, as the proteins localized to them have not been identified. Additionally, the functional consequence of the accumulation of cytoplasmic FUS inclusions has not been established, nor is it understood how they contribute to selective motor neuron death.
We carried out two related, but independent studies to characterize the proteins that may be included in FUS-positive inclusions. In this first study, we utilized immunoprecipitation of wild-type and mutant FUS in the presence and absence of RNase, followed by LC MS/MS. The identified proteins represent those that directly or indirectly interact with FUS, with relatively high affinity that can be pulled down with immunoprecipitation. A wide variety of interacting proteins were identified and they are involved in a multitude of pathways including: chromosomal organization, transcription, RNA splicing, RNA transport, localized translation, and stress response. Their interaction with FUS varied greatly in their requirements for RNA. Most notably, FUS interacted with hnRNPA1 and Matrin-3, proteins also known to cause familial ALS. Immunofluorescent staining of proteins interacting with mutant FUS were localized to cytoplasmic inclusions. We concluded that mis-localization of these proteins potentially lead to their dysregulation or loss of function, thus contributing to FUS pathogenesis.
In the second study, we developed a protocol to isolate dynamic FUS inclusions and employed LC MS/MS to identify all proteins associated with FUS inclusions. We identified a cohort of proteins involved in translation, splicing, and RNA export to be associated with the FUS inclusions. Further pathway and disease association analysis suggested that proteins associated with translation and RNA quality control pathways may be the most significant. Protein translation assays using both N2A and ALS patient fibroblasts demonstrated suppression of protein biosynthesis in mutant FUS expressing cells. However, translation initiation was not impaired. To understand how protein synthesis is suppressed by mutant FUS mediated defects in RNA metabolism, we examined changes in a well conserved RNA turnover pathway namely: nonsense mediated decay (NMD). We found that NMD is hyperactivated in cells expressing mutant FUS, likely due to chronic suppression of protein translation shifting the pathways autoregulatory circuit to allow for hyperactivation. We concluded that mutant FUS suppresses protein biosynthesis and disrupts NMD regulation. These defects together likely contribute to motor neuron death.
Digital Object Identifier (DOI)
The Department of Toxicology and Cancer Biology’s T32 training grant, the College of Medicine’s Fellowship for Excellence in Graduate Research Award, the Lyman T. Johnson Fellowship, National Institute of Neurological Disorder, Stroke (NINDS) and Amyotrophic Lateral Sclerosis (ALS) Association, and the Veterans Association (VA).
Kamelgarn, Marisa Elizabeth, "MUTATIONS OF FUS CAUSE AGGREGATION OF RNA BINDING PROTEINS, DISRUPTIONS IN PROTEIN SYNTHESIS, AND DYSREGULATION OF NONSENSE MEDIATED DECAY" (2019). Theses and Dissertations--Toxicology and Cancer Biology. 27.
Supplemental Table 3.1. The identified non-ribosomal FUS interacting partners.
Supplemental Table 3.2. The identified ribosomal FUS interacting partners.pdf (130 kB)
Supplemental Table 3.2. The identified ribosomal FUS interacting partners.
Supplemental Table 4.1. FUS WT Protemics.pdf (1150 kB)
Supplemental Table 4.1. FUS WT Proteomics.
Supplemental Table 4.2. FUS R495X Proteomics.pdf (1090 kB)
Supplemental Table 4.2. FUS R495X Proteomics
Supplemental Table 4.3. FUS P525L Proteomics.pdf (1078 kB)
Supplemental Table 4.3. FUS P525L Proteomics
Supplemental Table A. List of Primers.pdf (150 kB)
Supplemental Table A. List of Primers.