Date of Award
Doctor of Philosophy (PhD)
Tin Tin Su
Tar-DNA Binding Protein 43 (TDP-43) was first discovered as a negative regulator of HIV transcription. Subsequently, TDP-43 was found as the major protein component of ubiquitin positive aggregates in a variety of neurodegenerative diseases, including ALS. Intensive study of TDP-43 function indicates that this protein plays a direct or indirect role in a large number of RNA-mediated processes. However, the basic function of TDP-43 and its connection to neurodegeneration remains elusive. In this thesis, I have undertaken the goal of discovering the basic biological function of TDP-43. I show that one of the basic functions of TDP-43 is to limit the accumulation of double stranded RNA (dsRNA). TDP-43 is required to maintain the abundance of transcripts with potential dsRNA structure and in the absence of TDP-43 nuclear dsRNA accumulates. I show that the dsRNA structure/stability of multiple transcript types is controlled by TDP-43 and that TDP-43 associates with these transcripts cotranscriptionally.
Using this knowledge, I then go on to characterize the molecular and phenotypic consequences of dsRNA accumulation in TDP-43 knockdown/deletion. I find that TDP-43 deletion in worms results in a severe chemotaxis defect, which is due to processes that metabolize dsRNA. Utilizing published data sets of TDP-43 knockdown in mice, I show that deletion of this protein in mammals results in the up-regulation of type I interferon response genes, which is a major consequence of increased dsRNA in mammals.
I also show that TDP-43 deletion in worms results in an increased level of A-to-I RNA editing, likely as a result of increased dsRNA structure/stability. Preliminary data indicates that mammalian TDP-43 also controls A-to-I RNA editing, but that this could be due in part to TDP-43 controlling the processing of the ADAR transcripts. Finally, I show evidence suggesting that TDP-43 may associate with inosine-containing RNA.
I also discover that TDP-43 deletion worms are differentially sensitive to RNA interference and that this sensitivity is due to a heightened response to nuclear RNAi or Transcriptional Gene Silencing (TGS). I show that worm TDP-43 associates in the TGS complex in an RNA-dependent manner. In the absence of worm TDP-43, the abundance of small interfering RNAs (siRNA) increases, but these siRNA are likely nonfunctional as the abundance of the transcripts they target is also increased in TDP-43 mutants. Importantly, I show that many repeat regions of the genome that fail to undergo TGS in TDP-43 mutants have expanded the DNA.
Finally, I show that worm TDP-43 maintains snoRNA/rRNA processing. In the absence of TDP-43 function, an extended form of U3 snoRNA as well as rRNA processing intermediates/defects accumulate. I provide suggestive evidence that TDP-43 may control rRNA processing by association with the nuclear exosome component, RRP6 and/or by limiting the stability of snoRNA/rRNA interactions. Consistent with the observed processing defect in rRNA, I show that TDP-43 deletion animals are sensitive to 5-florouracil (5-FU), a chemical that confers differential sensitivity in nuclear exosome mutants. As rRNA is a major component of ribosomes, I also investigated the effect of worm TDP-43 deletion and associated rRNA processing defects on protein aggregation, a hallmark of ALS. I find that both 5-FU treatment and worm TDP-43 deletion result in increased aggregation of a poly(Q) containing protein, potentially linking defects in rRNA processing with defective ribosomes/translation.
Saldi, Tassa, "Fundamental Roles of the Neurodegeneration-Associated Protein TDP-43, in RNA Metabolism" (2013). Molecular, Cellular, and Developmental Biology Graduate Theses & Dissertations. 20.